Development of a bioresorbable self-expanding microstent for interventional applications - an innovative approach for stent-assisted coiling.

OBJECTIVES: Stent-assisted coiling prevents coil migration in broad-based intracranial aneurysms. So far, only permanent metal stents are approved for intracranial use. Bioresorbable stents allow a new therapeutic approach that may prevent the need for lifelong anticoagulation. We developed a neurovascular bioresorbable microstent (NBRS) and compared it in vitro to the commercial Neuroform EZ stent. MATERIALS AND METHODS: The self-expanding NBRS design is oriented on the Neuroform EZ stent. Poly L-lactic acid (PLLA) was used to manufacture semi-finished products in a dipping process. For the compensation of the inferior material properties of PLLA, design adjustments were made. The NBRS were cut by means of femtosecond (fs) laser and were morphologically and mechanically compared in vitro to the Neuroform EZ stent. In vitro implantation of an NBRS was performed using a complex patient-specific 3D-printed aneurysm model. In addition, an in vitro coiling procedure to assess the stent's ability to support a coil package was conducted. RESULTS: The NBRS could be reproducibly manufactured and had high quality regarding surface morphology. The radial force at the indicated vessel diameter of 3.0 mm was slightly higher for the Neuroform EZ stent compared to the NBRS. The self-expansion ability of the NBRS could be proven. The kink behavior of the NBRS was comparable to that of the Neuroform EZ stent, so no vessel lumen size reduction is expected. The stents showed identical deformation under local compression of 25 % based on the initial diameter, resulting in maximum forces of 24 ±â€Š5 mN (Neuroform EZ) and 8 ±â€Š2 mN (NBRS). The implanted NBRS expanded uniformly, and proper vessel wall adaptation was observed. The NBRS has the ability to retain a coil package. CONCLUSION: This study reported a reproducible manufacturing process for the developed NBRS as well as mechanical and morphological in vitro tests. Furthermore, successful NBRS implantation into a complex patient-specific vessel model was presented as proof of concept. The promising results of this study, also considering the commercial Neuroform EZ stent, support the idea of fully biodegradable microstents for intracranial aneurysm treatment. KEY POINTS: · High-performance polymer-based self-expanding neurovascular microstents were manufactured with good reproducibility.. · The bioresorbable microstent meets the requirements to pass through narrow radii.. · Implantability in a patient-specific and close-to-physiology vascular in vitro model was proven..

Revisiting SFA stent technology: an updated overview on mechanical stent performance.

OBJECTIVES: The study investigated mechanical parameters of stent systems indicated for treatment of femoropopliteal (FP) arterial disease to support interpretation of clinical results and the related causalities. METHODS: Eight stent system types of same dimensions were investigated (n=2). Parameters were the profile of stent delivery system (SDS), radiopacity, trackability and pushability, bending stiffness (flexibility) and axial stiffness of expanded stents, length change during expansion, radial force, crush resistance, strut thickness and general surface condition. RESULTS: The trackability ranged from 0.237 to 0.920 N and the pushability was 47.9-67.6 %. The bending stiffness of SDS was between 108.42 and 412.68 N mm2. The length change during stent release to 5 mm was low, with one exception. The bending stiffness of the expanded stents was 2.73-41.67 N mm2. The normalized radial forces at 5 mm diameter ranged from 0.133 N/mm to 0.503 N/mm. During non-radial compression by 50 %, the forces were 3.07-8.42 N, with one exception (58.7 N). The strut thickness was 153-231 µm. CONCLUSIONS: Large differences occurred for flexibility, radial force and length change during expansion. The data should be used when choosing the proper device for restoring vascular function.

Initial Clinical Results and In Vitro Testing of the New CGuard MicroNet-Covered "One-Size-Fits-All" Carotid Stent.

Purpose: To evaluate a MicroNet-covered stent designed for the carotid artery with the new ability to adjust to different vessel diameters. Materials and Methods: Thirty consecutive patients (mean age 72.1±7.7 years; 26 men) with symptomatic stenosis (86.3%±6.4%) of the internal carotid artery were treated with the new self-adjusting nitinol stent, which has a self-expanding, open-cell design covered by an outer conformable layer (MicroNet). The only stent used was the "One-Size-Fits-All" CGuard stent with lengths of 30 or 40 mm. In bench testing, the chronic outward force of the One-Size-Fits-All stent was determined with a segmented head radial force test device. The stent was deployed directly into the test device at a diameter of 5.0 mm, and the chronic outward force was measured up to 10.0 mm, the maximum expansion of the stent. Results: The stent was successfully implanted in all 30 patients without periprocedural complications, including no neurological events within 30 days. The chronic outward force normalized by stent length demonstrated a near-equivalent radial force outcome: The stent displayed only a minor difference between the minimal radial force at 9.0 mm (0.195 N/mm) and the maximal radial force at 5.5 mm (0.330 N/mm). Conclusion: The new self-adjusting, MicroNet-covered stent has high conformability combined with an almost equivalent radial force at expansion diameters ranging from 5.5 to 9.0 mm. The first clinical results demonstrate that the new One-Size-Fits-All stent can be safely implanted in internal carotid arteries with reference diameters within this range.

Mechanical behavior of in vivo degraded second generation resorbable magnesium scaffolds (RMS).

Resorbable magnesium scaffolds are used for the treatment of atherosclerotic coronary vascular disease and furthermore, for vascular restoration therapy. Recently, the first-in-man clinical studies with Magmaris showed promising results regarding the target lesion failure as well as vasomotion properties after 12 and 24 month. The consistency of in vivo degraded magnesium alloys in a cardiovascular environment is qualitatively described in literature, but only little has been disclosed about the actual change in mechanical properties and the behavior of the magnesium alloy degradation products. In the present study, uncoated magnesium scaffolds 3.0 × 20 mm were implanted in coronary arteries of two healthy Goetinnger mini-swine. The scaffolds were explanted to evaluate the mechanical properties of the degraded magnesium scaffolds after 180 days in vivo. Ex vivo sample preparation and test conditions were adapted to a customized compression test setup which was developed to investigate the micro-scale scaffold fragments (width 225 ±â€¯75 µm, thickness 150 µm). As reference bare undegraded magnesium scaffold fragments were tested. Mechanical parameters relating to force as a function of displacement were determined for both sample groups. The undegraded samples showed no fracturing at the maximum applied force of 8 N, whereas the in vivo degraded test samples showed forces of 0.411 ±â€¯0.197 N at the first fracturing and a maximum force of 0.956 ±â€¯0.525 N. The deformation work, calculated as area beneath the force-displacement curve, of the in vivo degraded test samples was reduced by approximately 87-88% compared to the undegraded samples (5.20 mN mm and 40.79 mN mm, both at 7.5% deformation). The indication for a complete loss of structural integrity through a reduction of mechanical properties after a certain degradation time increases the chance to restore vascular function and physiological vasomotion in the stented vessel compartment.

Direct comparison of coronary bare metal vs. drug-eluting stents: same platform, different mechanics?

BACKGROUND: Drug-eluting stents (DES) compared to bare metal stents (BMS) have shown superior clinical performance, but are considered less suitable in complex cases. Most studies do not distinguish between DES and BMS with respect to their mechanical performance. The objective was to obtain mechanical parameters for direct comparison of BMS and DES. METHODS: In vitro bench tests evaluated crimped stent profiles, crossability in stenosis models, elastic recoil, bending stiffness (crimped and expanded), and scaffolding properties. The study included five pairs of BMS and DES each with the same stent platforms (all n = 5; PRO-Kinetic Energy, Orsiro: BIOTRONIK AG, Bülach, Switzerland; MULTI-LINK 8, XIENCE Xpedition: Abbott Vascular, Temecula, CA; REBEL Monorail, Promus PREMIER, Boston Scientific, Marlborough, MA; Integrity, Resolute Integrity, Medtronic, Minneapolis, MN; Kaname, Ultimaster: Terumo Corporation, Tokyo, Japan). Statistical analysis used pooled variance t tests for pairwise comparison of BMS with DES. RESULTS: Crimped profiles in BMS groups ranged from 0.97 ± 0.01 mm (PRO-Kinetic Energy) to 1.13 ± 0.01 mm (Kaname) and in DES groups from 1.02 ± 0.01 mm (Orsiro) to 1.13 ± 0.01 mm (Ultimaster). Crossability was best for low profile stent systems. Elastic recoil ranged from 4.07 ± 0.22% (Orsiro) to 5.87 ± 0.54% (REBEL Monorail) including both BMS and DES. The bending stiffness of crimped and expanded stents showed no systematic differences between BMS and DES neither did the scaffolding. CONCLUSIONS: Based on in vitro measurements BMS appear superior to DES in some aspects of mechanical performance, yet the differences are small and not class uniform. The data provide assistance in selecting the optimal system for treatment and assessment of new generations of bioresorbable scaffolds. TRIAL REGISTRATION: not applicable.

Molecular basis for asymmetry sensing of siRNAs by the Drosophila Loqs-PD/Dcr-2 complex in RNA interference.

RNA interference defends against RNA viruses and retro-elements within an organism's genome. It is triggered by duplex siRNAs, of which one strand is selected to confer sequence-specificity to the RNA induced silencing complex (RISC). In Drosophila, Dicer-2 (Dcr-2) and the double-stranded RNA binding domain (dsRBD) protein R2D2 form the RISC loading complex (RLC) and select one strand of exogenous siRNAs according to the relative thermodynamic stability of base-pairing at either end. Through genome editing we demonstrate that Loqs-PD, the Drosophila homolog of human TAR RNA binding protein (TRBP) and a paralog of R2D2, forms an alternative RLC with Dcr-2 that is required for strand choice of endogenous siRNAs in S2 cells. Two canonical dsRBDs in Loqs-PD bind to siRNAs with enhanced affinity compared to miRNA/miRNA* duplexes. Structural analysis, NMR and biophysical experiments indicate that the Loqs-PD dsRBDs can slide along the RNA duplex to the ends of the siRNA. A moderate but notable binding preference for the thermodynamically more stable siRNA end by Loqs-PD alone is greatly amplified in complex with Dcr-2 to initiate strand discrimination by asymmetry sensing in the RLC.

RNA binding and chaperone activity of the E. coli cold-shock protein CspA.

Ensuring the correct folding of RNA molecules in the cell is of major importance for a large variety of biological functions. Therefore, chaperone proteins that assist RNA in adopting their functionally active states are abundant in all living organisms. An important feature of RNA chaperone proteins is that they do not require an external energy source to perform their activity, and that they interact transiently and non-specifically with their RNA targets. So far, little is known about the mechanistic details of the RNA chaperone activity of these proteins. Prominent examples of RNA chaperones are bacterial cold shock proteins (Csp) that have been reported to bind single-stranded RNA and DNA. Here, we have used advanced NMR spectroscopy techniques to investigate at atomic resolution the RNA-melting activity of CspA, the major cold shock protein of Escherichia coli, upon binding to different RNA hairpins. Real-time NMR provides detailed information on the folding kinetics and folding pathways. Finally, comparison of wild-type CspA with single-point mutants and small peptides yields insights into the complementary roles of aromatic and positively charged amino-acid side chains for the RNA chaperone activity of the protein.

A Stably Protonated Adenine Nucleotide with a Highly Shifted pKa Value Stabilizes the Tertiary Structure of a GTP-Binding RNA Aptamer.

RNA tertiary structure motifs are stabilized by a wide variety of hydrogen-bonding interactions. Protonated A and C nucleotides are normally not considered to be suitable building blocks for such motifs since their pKa values are far from physiological pH. Here, we report the NMR solution structure of an in vitro selected GTP-binding RNA aptamer bound to GTP with an intricate tertiary structure. It contains a novel kind of base quartet stabilized by a protonated A residue. Owing to its unique structural environment in the base quartet, the pKa value for the protonation of this A residue in the complex is shifted by more than 5 pH units compared to the pKa for A nucleotides in single-stranded RNA. This is the largest pKa shift for an A residue in structured nucleic acids reported so far, and similar in size to the largest pKa shifts observed for amino acid side chains in proteins. Both RNA pre-folding and ligand binding contribute to the pKa shift.

Clinical Results and Mechanical Properties of the Carotid CGUARD Double-Layered Embolic Prevention Stent.

PURPOSE: To report early clinical outcomes with a novel double-layer stent for the internal carotid artery (ICA) and the in vitro investigation of the stent's mechanical properties. METHODS: A prospective single-center study enrolled 30 consecutive patients (mean age 73.1±6.3 years; 21 men) with symptomatic (n=25) or high-grade (n=5) ICA stenosis treated with the new double-layer carotid CGUARD Embolic Prevention System (EPS) stent, which has an inner open-cell nitinol design with an outer closed-cell polyethylene terephthalate layer. The average stenosis of the treated arteries was 84.1%±7.9% with a mean lesion length of 16.6±2.1 mm. In the laboratory, 8×40-mm stents where tested in vitro with respect to their radial force during expansion, the bending stiffness of the stent system and the expanded stent, as well as the collapse pressure in a thin and flexible sheath. The wall adaptation was assessed using fluoroscopy after stent release in step and curved vessel models. RESULTS: The stent was successfully implanted in all patients. No peri- or postprocedural complications occurred; no minor or major stroke was observed in the 6-month follow-up. The bending stiffness of the expanded stent was 63.1 N·mm2 and (not unexpectedly) was clearly lower than that of the stent system (601.5 N·mm2). The normalized radial force during expansion of the stent to 7.0 mm, consistent with in vivo sizing, was relatively high (0.056 N/mm), which correlates well with the collapse pressure of 0.17 bars. Vessel wall adaptation was harmonic and caused no straightening of the vessel after clinical application. CONCLUSION: Because of its structure, the novel CGUARD EPS stent is characterized by a high flexibility combined with a high radial force and very good plaque coverage. These first clinical results demonstrate a very safe implantation behavior without any stroke up to 6 months after the procedure.

Measurement of Ligand-Target Residence Times by 1H Relaxation Dispersion NMR Spectroscopy.

A ligand-observed 1H NMR relaxation experiment is introduced for measuring the binding kinetics of low-molecular-weight compounds to their biomolecular targets. We show that this approach, which does not require any isotope labeling, is applicable to ligand-target systems involving proteins and nucleic acids of variable molecular size. The experiment is particularly useful for the systematic investigation of low affinity molecules with residence times in the micro- to millisecond time regime.

Excited States of Nucleic Acids Probed by Proton Relaxation Dispersion NMR Spectroscopy.

In this work an improved stable isotope labeling protocol for nucleic acids is introduced. The novel building blocks eliminate/minimize homonuclear (13) C and (1) H scalar couplings thus allowing proton relaxation dispersion (RD) experiments to report accurately on the chemical exchange of nucleic acids. Using site-specific (2) H and (13) C labeling, spin topologies are introduced into DNA and RNA that make (1) H relaxation dispersion experiments applicable in a straightforward manner. The novel RNA/DNA building blocks were successfully incorporated into two nucleic acids. The A-site RNA was previously shown to undergo a two site exchange process in the micro- to millisecond time regime. Using proton relaxation dispersion experiments the exchange parameters determined earlier could be recapitulated, thus validating the proposed approach. We further investigated the dynamics of the cTAR DNA, a DNA transcript that is involved in the viral replication cycle of HIV-1. Again, an exchange process could be characterized and quantified. This shows the general applicablility of the novel labeling scheme for (1) H RD experiments of nucleic acids.

m(1)A and m(1)G disrupt A-RNA structure through the intrinsic instability of Hoogsteen base pairs.

The B-DNA double helix can dynamically accommodate G-C and A-T base pairs in either Watson-Crick or Hoogsteen configurations. Here, we show that G-C(+) (in which + indicates protonation) and A-U Hoogsteen base pairs are strongly disfavored in A-RNA. As a result,N(1)-methyladenosine and N(1)-methylguanosine, which occur in DNA as a form of alkylation damage and in RNA as post-transcriptional modifications, have dramatically different consequences. Whereas they create G-C(+) and A-T Hoogsteen base pairs in duplex DNA, thereby maintaining the structural integrity of the double helix, they block base-pairing and induce local duplex melting in RNA. These observations provide a mechanism for disrupting RNA structure through post-transcriptional modifications. The different propensities to form Hoogsteen base pairs in B-DNA and A-RNA may help cells meet the opposing requirements of maintaining genome stability, on the one hand, and of dynamically modulating the structure of the epitranscriptome, on the other.

In vitro performance investigation of bioresorbable scaffolds - Standard tests for vascular stents and beyond.

BACKGROUND/PURPOSE: Biodegradable polymers are the main materials for coronary scaffolds. Magnesium has been investigated as a potential alternative and was successfully tested in human clinical trials. However, it is still challenging to achieve mechanical parameters comparative to permanent bare metal (BMS) and drug-eluting stents (DES). As such, in vitro tests are required to assess mechanical parameters correlated to the safety and efficacy of the device. METHODS/MATERIALS: In vitro bench tests evaluate scaffold profiles, length, deliverability, expansion behavior including acute elastic and time-dependent recoil, bending stiffness and radial strength. The Absorb GT1 (Abbott Vascular, Temecula, CA), DESolve (Elixir Medical Corporation, Sunnyvale, CA) and the Magmaris (BIOTRONIK AG, Bülach, Switzerland) that was previously tested in the BIOSOLVE II study, were tested. RESULTS: Crimped profiles were 1.38±0.01mm (Absorb GT1), 1.39±0.01mm (DESolve) and 1.44±0.00mm (Magmaris) enabling 6F compatibility. Trackability was measured depending on stiffness and force transmission (pushability). Acute elastic recoil was measured at free expansion and within a mock vessel, respectively, yielding results of 5.86±0.76 and 5.22±0.38% (Absorb), 7.85±3.45 and 9.42±0.21% (DESolve) and 5.57±0.72 and 4.94±0.31% (Magmaris). Time-dependent recoil (after 1h) was observed for the Absorb and DESolve scaffolds but not for the Magmaris. The self-correcting wall apposition behavior of the DESolve did not prevent time-dependent recoil under vessel loading. CONCLUSIONS: The results of the suggested test methods allow assessment of technical feasibility based on objective mechanical data and highlight the main differences between polymeric and metallic bioresorbable scaffolds.

NMR resonance assignments for the class II GTP binding RNA aptamer in complex with GTP.

The structures of RNA-aptamer-ligand complexes solved in the last two decades were instrumental in realizing the amazing potential of RNA for forming complex tertiary structures and for molecular recognition of small molecules. For GTP as ligand the sequences and secondary structures for multiple families of aptamers were reported which differ widely in their structural complexity, ligand affinity and ligand functional groups involved in RNA-binding. However, for only one of these families the structure of the GTP-RNA complex was solved. In order to gain further insights into the variability of ligand recognition modes we are currently determining the structure of another GTP-aptamer--the so-called class II aptamer--bound to GTP using NMR-spectroscopy in solution. As a prerequisite for a full structure determination, we report here (1)H, (13)C, (15)N and partial (31)P-NMR resonance assignments for the class II GTP-aptamer bound to GTP.

Chemo-enzymatic synthesis of site-specific isotopically labeled nucleotides for use in NMR resonance assignment, dynamics and structural characterizations.

Stable isotope labeling is central to NMR studies of nucleic acids. Development of methods that incorporate labels at specific atomic positions within each nucleotide promises to expand the size range of RNAs that can be studied by NMR. Using recombinantly expressed enzymes and chemically synthesized ribose and nucleobase, we have developed an inexpensive, rapid chemo-enzymatic method to label ATP and GTP site specifically and in high yields of up to 90%. We incorporated these nucleotides into RNAs with sizes ranging from 27 to 59 nucleotides using in vitro transcription: A-Site (27 nt), the iron responsive elements (29 nt), a fluoride riboswitch from Bacillus anthracis(48 nt), and a frame-shifting element from a human corona virus (59 nt). Finally, we showcase the improvement in spectral quality arising from reduced crowding and narrowed linewidths, and accurate analysis of NMR relaxation dispersion (CPMG) and TROSY-based CEST experiments to measure µs-ms time scale motions, and an improved NOESY strategy for resonance assignment. Applications of this selective labeling technology promises to reduce difficulties associated with chemical shift overlap and rapid signal decay that have made it challenging to study the structure and dynamics of large RNAs beyond the 50 nt median size found in the PDB.

Stable isotope-labeled RNA phosphoramidites to facilitate dynamics by NMR.

Given that Ribonucleic acids (RNAs) are a central hub of various cellular processes, methods to synthesize these RNAs for biophysical studies are much needed. Here, we showcase the applicability of 6-(13)C-pyrimidine phosphoramidites to introduce isolated (13)C-(1)H spin pairs into RNAs up to 40 nucleotides long. The method allows the incorporation of 6-(13)C-uridine and -cytidine residues at any desired position within a target RNA. By site-specific positioning of the (13)C-label using RNA solid phase synthesis, these stable isotope-labeling patterns are especially well suited to resolve resonance assignment ambiguities. Of even greater importance, the labeling pattern affords accurate quantification of important functional transitions of biologically relevant RNAs (e.g., riboswitch aptamer domains, viral RNAs, or ribozymes) in the µs- to ms time regime and beyond without complications of one bond carbon scalar couplings. We outline the chemical synthesis of the 6-(13)C-pyrimidine building blocks and their use in RNA solid phase synthesis and demonstrate their utility in Carr Purcell Meiboom Gill relaxation dispersion, ZZ exchange, and chemical exchange saturation transfer NMR experiments.

A Mini-Twister Variant and Impact of Residues/Cations on the Phosphodiester Cleavage of this Ribozyme Class.

Nucleolytic ribozymes catalyze site-specific cleavage of their phosphodiester backbones. A minimal version of the twister ribozyme is reported that lacks the phylogenetically conserved stem P1 while retaining wild-type activity. Atomic mutagenesis revealed that nitrogen atoms N1 and N3 of the adenine-6 at the cleavage site are indispensable for cleavage. By NMR spectroscopy, a pKa value of 5.1 was determined for a (13) C2-labeled adenine at this position in the twister ribozyme, which is significantly shifted compared to the pKa of the same adenine in the substrate alone. This finding pinpoints at a potential role for adenine-6 in the catalytic mechanism besides the previously identified invariant guanine-48 and a Mg(2+) ion, both of which are directly coordinated to the non-bridging oxygen atoms of the scissile phosphate; for the latter, additional evidence stems from the observation that Mn(2+) or Cd(2+) accelerated cleavage of phosphorothioate substrates. The relevance of this metal ion binding site is further emphasized by a new 2.6 ŠX-ray structure of a 2'-OCH3 -U5 modified twister ribozyme.

Ligand-detected relaxation dispersion NMR spectroscopy: dynamics of preQ1-RNA binding.

An NMR-based approach to characterizing the binding kinetics of ligand molecules to biomolecules, like RNA or proteins, by ligand-detected Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments is described. A (15)N-modified preQ1 ligand is used to acquire relaxation dispersion experiments in the presence of low amounts of the Fsu class I preQ1 aptamer RNA, and increasing ligand concentrations to probe the RNA small molecule interaction. Our experimental data strongly support the conformational selection mechanism postulated. The approach gives direct access to two parameters of a ligand-receptor interaction: the off rate and the population of the small molecule-receptor complex. A detailed description of the kinetics underlying the ligand binding process is of crucial importance to fully understanding a riboswitch's function and to evaluate potential new antibiotics candidates targeting the noncoding RNA species. Ligand-detected NMR relaxation dispersion experiments represent a valuable diagnostic tool for the characterization of binding mechanisms.

NMR resonance assignments of the archaeal ribosomal protein L7Ae in the apo form and bound to a 25 nt RNA.

The archaeal protein L7Ae forms part of a protein complex in the ribosome that specifically recognizes and binds to kink-turn RNA. In this complex, L7Ae directly interacts with the oligonucleotide and creates a functional arrangement for site-specific 2'-O-methylation. We report the solution NMR backbone assignment of Methanocaldococcus jannaschii L7Ae (117 residues, 12.7 kDa) in the ligand-free state and when bound to a 25 nucleotide C/D box kink-turn mimic RNA.

Magnetic resonance access to transiently formed protein complexes.

Protein-protein interactions are of utmost importance to an understanding of biological phenomena since non-covalent and therefore reversible couplings between basic proteins leads to the formation of complex regulatory and adaptive molecular systems. Such systems are capable of maintaining their integrity and respond to external stimuli, processes intimately related to living organisms. These interactions, however, span a wide range of dissociation constants, from sub-nanomolar affinities in tight complexes to high-micromolar or even millimolar affinities in weak, transiently formed protein complexes. Herein, we demonstrate how novel NMR and EPR techniques can be used for the characterization of weak protein-protein (ligand) complexes. Applications to intrinsically disordered proteins and transiently formed protein complexes illustrate the potential of these novel techniques to study hitherto unobserved (and unobservable) higher-order structures of proteins.