Long-term outcome upon treatment of calcified lesions of the lower limb using scoring angioplasty balloon (AngioSculpt™).

AIMS: In peripheral artery disease (PAD), endovascular treatment success of heavily calcified lesions is often compromised by a number of vascular complications, such as recoils, dissections and need for target vessel re-interventions. The increasing use of scoring balloon techniques has raised the hope for better periprocedural outcomes; however, the knowledge regarding the actual benefits of the scoring balloon technique in comparison to standard therapy is still limited. Thus, the aim of the current study was to determine the safety and effectiveness of scoring balloon angioplasty in a real-life patients' collective with PAD. METHODS AND RESULTS: A total of 425 patients with moderate to severely calcified femoropopliteal lesions received interventional treatment between 2011 and 2018 at the single center; 230 received a treatment with a scoring balloon (AngioSculpt™), and 195 received a plain procedure without AngioSculpt™. Key questions of this analysis were: (1) whether AngioSculpt™ can be used as a safe and effective stand-alone treatment in heavily calcified lesions in a 24-month follow-up, as well as (2) whether target lesion preparation with scoring balloon bears additional benefits to standard treatment (PTA ± stent implantation). In terms of freedom from target lesion revascularization there were no significant differences between AngioSculpt™ and standard procedure (82.3% vs. 78.1%, P > 0.05). Vessel preparation with balloon angioplasty had no additional effects on survival and amputation rates in comparison to standard treatment without AngioSculpt™ (P > 0.05). The deployment of a scoring balloon did not reduce the subsequent need for additional stent implantations (32.6%, and 32.3%, P > 0.05). CONCLUSION: Lesion preparation with AngioSculpt™ scoring balloon represents a safe and effective tool in the treatment of complex femoropopliteal lesions. In this retrospective analysis, AngioSculpt™ scoring balloon angioplasty did not significantly improve vessel patency- both when used as an adjunctive in preparation for stenting and as stand-alone treatment. A prospective study is needed to further investigate the scoring balloon treatment options.

The cytochrome ba3 oxidase from Thermus thermophilus does not generate a tryptophan radical during turnover: Implications for the mechanism of proton pumping.

Oxygen reduction by cytochrome ba3 oxidase from Thermus thermophilus was studied by stopped-flow and microsecond freeze-hyperquenching analyzed with UV-Vis and EPR spectroscopy. In the initial phase, the low-spin heme b560 is rapidly and almost completely oxidized (kobs>33,000s(-1)) whereas CuA remains nearly fully reduced. The internal equilibrium between CuA and heme b560 with forward and reverse rate constants of 4621s(-1) and 3466s(-1), respectively, indicates a ~7.5mV lower midpoint potential for CuA compared to heme b560. The formation of the oxidized enzyme is relatively slow (693s(-1)). In contrast to the Paracoccus denitrificans cytochrome aa3 oxidase, where in the last phase of the oxidative half cycle a radical from the strictly conserved Trp272 is formed, no radical is formed in the cytochrome ba3 oxidase. Mutation of the Trp229, the cytochrome ba3 oxidase homologue to the Trp272, did not abolish the activity, again in contrast to the Paracoccus cytochrome aa3 oxidase. Differences in the proton pumping mechanisms of Type A and Type B oxidases are discussed in view of the proposed role of the strictly conserved tryptophan residue in the mechanism of redox-linked proton pumping in Type A oxidases. In spite of the differences between the Type A and Type B oxidases, we conclude that protonation of the proton-loading site constitutes the major rate-limiting step in both catalytic cycles.

Electrochemical and infrared spectroscopic analysis of the interaction of the Cu(A) domain and cytochrome c(552) from Thermus thermophilus.

The hydrophobically guided complex formation between the Cu(A) fragment from Thermus thermophilus ba(3) terminal oxidase and its electron transfer substrate, cytochrome c(552), was investigated electrochemically. In the presence of the purified Cu(A) fragment, a clear downshift of the c(552) redox potential from 171 to 111mV±10mV vs SHE' was found. Interestingly, this potential change fully matches complex formation with this electron acceptor site in other oxidases guided by electrostatic or covalent interactions. Redox induced FTIR difference spectra revealed conformational changes associated with complex formation and indicated the involvement of heme propionates. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).

A novel heme a insertion factor gene cotranscribes with the Thermus thermophilus cytochrome ba3 oxidase locus.

Studying the biogenesis of the Thermus thermophilus cytochrome ba(3) oxidase, we analyze heme a cofactor insertion into this membrane protein complex. Only three proteins linked to oxidase maturation have been described for this extreme thermophile, and in particular, no evidence for a canonical Surf1 homologue, required for heme a insertion, is available from genome sequence data. Here, we characterize the product of an open reading frame, cbaX, in the operon encoding subunits of the ba(3)-type cytochrome c oxidase. CbaX shares no sequence identity with any known oxidase biogenesis factor, and CbaX homologues are found only in the Thermaceae group. In a series of cbaX deletion and complementation experiments, we demonstrate that the resulting ba(3) oxidase complexes, affinity purified via an internally inserted His tag located in subunit I, are severely affected in their enzymatic activities and heme compositions in both the low- and high-spin sites. Thus, CbaX displays typical features of a generic Surf1 factor essential for binding and positioning the heme a moiety for correct assembly into the protein scaffold of oxidase subunit I.

Biogenesis of cytochrome c oxidase--in vitro approaches to study cofactor insertion into a bacterial subunit I.

Biogenesis of cytochrome c oxidase is a complex process involving more than 30 known accessory proteins in yeast for the regulation of transcription and translation, membrane insertion and protein processing, cofactor insertion, and subunit assembly. Here, we focus on the process of cofactor insertion into subunit I of cytochrome c oxidase using the soil bacterium Paracoccus denitrificans as a model organism. The use of bacterial systems facilitates biogenesis studies, as the number of required assembly factors is reduced to a minimum. Both, co- and posttranslational cofactor insertion scenarios are discussed, and several approaches to shed light on this aspect of biogenesis are presented. CtaG, the Paracoccus homolog of yeast Cox11 which is involved in copper delivery to the Cu(B) center, has been purified and characterized spectroscopically. A previously unreported signal at 358 nm allows monitoring copper transfer from copper-loaded CtaG to an acceptor. Both CtaG and apo-subunit I were purified after expression in Escherichia coli to develop an in vitro copper transfer system, probing the posttranslational insertion hypothesis. To mimic a potential cotranslational insertion process, cell-free expression systems using E. coli and P. denitrificans extracts have been established. Expression of subunit I in the presence of the detergent Brij-35 produces high amounts of "solubilized" subunit I which can be purified in good yield. With this system it may be feasible to trap and purify assembly intermediates after adding free cofactors, purified assembly proteins, or P. denitrificans membranes.