Functional, morphologic, and molecular characterization of cold storage injury

ObjectiveCold storage is used to preserve tissue for later transplantation. There is particular interest in prolonging cold storage time for transplantation purposes. To date, the mechanisms that contribute to vascular dysfunction in response to cold storage are poorly understood. The present study aims to characterize cold storage injury of blood vessels on functional and molecular levels.MethodsTo assess vessel function of mouse aorta, isometric force measurements were performed in a Mulvany myograph after cold storage at 4°C for various intervals. Morphologic changes were judged by histologic analysis of aortic cross-sections. To characterize cold storage-induced alterations on RNA levels, microarray analysis with subsequent polymerase chain reaction analysis was performed.ResultsCold storage for 2 days revealed significant impairment of vessel function with respect to potassium-induced vessel tone development and acetylcholine-induced vessel relaxation. Detailed analysis of acetylcholine-mediated vascular response using specific pharmacologic blockers revealed that calcium-activated potassium channels seem to be impaired after 2 days of cold storage. At this point, no severe histologic changes (eg, elastic fiber disruption) were visible. RNA expression of 24 genes was significantly changed due to cold storage even after 2 hours. These include genes associated with vessel tone development (prostaglandin E3 receptor), cardiovascular function (adiponectin), electron transport chain (uncoupling protein 1), or calcium signaling (protein kinase A regulatory subunit 2b).ConclusionsLong-term cold storage impairs vascular function, especially with respect to potassium signaling by calcium-dependent potassium channels. Microarray analysis confirmed impairment of pathways that are involved in calcium signaling and vascular function. Furthermore, various genes were significantly altered even after 2 hours, significantly before functional impairment was observed.Clinical RelevanceIn bypass surgery, vessels are stored ex situ for <2 hours and for vessel banking for up to 48 hours during decontamination before cryopreservation. We investigated the onset of storage-induced alterations. The experiments reveal various genes were altered significantly, even within the first 2 hours of cold storage. This is of major importance, because of the time delay between regulation of messenger RNA and protein level and functional consequences. The alterations described here on the molecular level occur before any alterations on morphologic or functional levels are obvious. These molecular alterations, however, may affect later graft function

Primary fibroblast co-culture stimulates growth and metabolism in Sdhb-impaired mouse pheochromocytoma MTT cells

Pheochromocytomas and paragangliomas (PGLs) due to mutations of succinate dehydrogenase (SDH) B, a subunit of the SDH complex with a role in the Krebs cycle and the respiratory chain, tend to be larger at diagnosis and more prone to metastatic disease than other tumors. This presentation contrasts with the behavior of some cell line models of SDHB impairment, which show reduced growth compared to wild type. We hypothesize that reduced growth of SDHB-impaired monolayer culture models might reflect lack of support from sources within the tumor microenvironment. The present study therefore investigates how the microenvironment, modeled here by fibroblast co-culture, modulates cell metabolism, growth and invasion in an Sdhb-impaired mouse pheochromocytoma cell line. We employed two different constructs of short hairpin RNA to knockdown Sdhb and compared growth in a monolayer with and without fibroblast co-culture. Sdhb-silenced cells showed functional impairment of SDH with elevated succinate to fumarate ratio and decreased oxidative capacity. Cell growth was delayed with an increase in doubling time of 2&nbsp;h or 20&nbsp;h. Clonogenic cell survival and viability, on the other hand, were either unchanged or increased compared to control. In standard monolayer culture, no differences in pro-metastatic features were present. Co-culture with primary mouse fibroblast reversed the difference of proliferation between control and Sdhb knockdown but was unable to significantly influence invasiveness under these culture conditions. Metabolic studies identified that lactate secreted by fibroblasts was taken up preferentially by Sdhb-silenced cells. In summary, the present study identified a potential role for the tumor microenvironment in influencing phenotypic features of SDHB-mutated PGLs, providing a basis for the use of therapies targeted towards the tumor microenvironment

An epidemic CC1-MRSA-IV clone yields false-negative test results in molecular MRSA identification assays: a note of caution, Austria, Germany, Ireland, 2020

We investigated why a clinical meticillin-resistant Staphylococcus aureus (MRSA) isolate yielded false-negative results with some commercial PCR tests for MRSA detection. We found that an epidemic European CC1-MRSA-IV clone generally exhibits this behaviour. The failure of the assays was attributable to a large insertion in the orfX/SCCmec integration site. To ensure the reliability of molecular MRSA tests, it is vital to monitor emergence of new SCCmec types and junction sites

MicroRNA-23a mediates post-transcriptional regulation of CXCL12 in bone marrow stromal cells

The chemokine CXCL12 regulates the interaction between hematopoietic stem and progenitor cells and bone marrow stromal cells. Although its relevance in the bone marrow niche is well recognized, the regulation of CXCL12 by microRNA is not completely understood. We transfected a library of 486 microRNA in the bone marrow stromal cell line SCP-1 and studied the expression of CXCL12. Twenty-seven microRNA were shown to downregulate expression of CXCL12. Eight microRNA (miR-23a, 130b, 135, 200b, 200c, 216, 222, and 602) interacted directly with the 3'UTR of CXCL12. Next, we determined that only miR-23a is predicted to bind to the 3'UTR and is strongly expressed in primary bone marrow stromal cells. Modulation of miR-23a changes the migratory potential of hematopoietic progenitor cells in co-culture experiments. We discovered that TGFB1 mediates its inhibitory effect on CXCL12 levels by upregulation of miR-23a. This process was partly reversed by miR-23a molecules. Finally, we determined an inverse expression of CXCL12 and miR-23a in stromal cells from patients with myelodysplastic syndrome indicating that the interaction has a pathophysiological role. Here, we show for the first time that CXCL12-targeting miR23a regulates the functional properties of the hematopoietic niche

Mass spectrometry reveals misdiagnosis of primary aldosteronism with scheduling for adrenalectomy due to immunoassay interference

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Influence of caveolin-1 and endothelial nitric oxide synthase on adventitial inflammation in aortic transplants

Background: Restenosis after endovascular interventions is a clinically relevant process that is directly associated with increased morbidity. Thereby, an increased migration and proliferation of vascular smooth muscle cells (VSMCs) is mainly responsible for recurrent lumen narrowing. Previously, we showed that caveolin‑1 (Cav‑1) and endothelial nitric oxide synthase (eNOS) were directly involved in neointimal proliferation. Aims: In the current study, we investigated the impact of Cav‑1 and eNOS on adventitial processes in a murine model. Methods: Denuded aortas from C57Bl6n (wild‑type [WT]), Cav‑1-/, eNOS-/, and Cav‑1-//eNOS-/ mice were transplanted into common carotid arteries of WT mice. The explantation was performed after 6 weeks, followed by Elastica van Gieson staining and immunohistochemistry. Results: The Cav‑1-/ and the eNOS-/ aortas showed an increase in the adventitial content of macrophages, whereas their combined knockout did not lead to additive effects. Differences were observed despite the same acceptor, suggesting the local origin of inflammatory cells. Furthermore, the WT transplants exhibited the highest content of vascular endothelial growth factor A (VEGF‑A) despite the lowest macrophage content. In contrast, the knockout aortas showed a decreased content of VEGF‑A as well as decreased expression of α-smooth muscle actin (α­‑SMA) in the tunica media, suggesting induced VSMC migration. Moreover, the WT aortas exhibited increased neovessel formation. Conclusions: Cav‑1 and eNOS inhibit adventitial macrophage‑derived inflammation and modulate its cellular function. The knockout of Cav‑1 and eNOS leads to a decreased expression of VEGF-A, with decreased neovessel formation and increased migration of VSMCs, which promote a proatherogenic phenotype.

Simple periprocedural precautions to reduce Doppler microembolic signals during AF ablation

Background!#!Doppler microembolic signals (MES) occur during atrial fibrillation ablation despite of permanent flushed transseptal sheaths, frequent controls of periprocedural coagulation status and the use of irrigated ablation catheters PURPOSE: To investigate the number and type of MES depending on the procedure time, prespecified procedure steps, the activated clotting time (ACT) during the ablation procedure and the catheter contact force.!##!Methods!#!In a prospective trial, 53 consecutive atrial fibrillation patients underwent pulmonary vein isolation by super-irrigated 'point-by-point' ablation. All patients underwent a periinterventional, continuous transcranial Doppler examination (TCD) of the bilateral middle cerebral arteries during the complete ablation procedure.!##!Results!#!An average of 686±226 microembolic signals were detected by permanent transcranial Doppler. Thereby, 569±208 signals were differentiated as gaseous and 117±31 as solid MES. The number of MES with regard to defined procedure steps were as follows: gaseous: [transseptal puncture, 26 ± 28; sheath flushing, 24±12; catheter change, 21±11; angiography, 101±28; mapping, 9±9; ablation, 439±192; protamine administration, 0±0]; solid: [transseptal puncture, 8±8; sheath flushing, 9±5; catheter replacement, 6±6; angiography, not measurable; mapping, 2±5; ablation, 41±22; protamine administration, 0±0]. Significantly less MES occurred with shorter procedure time, higher ACT and the use of tissue contact force monitoring.!##!Conclusion!#!The current study demonstrates that during atrial fibrillation ablation using irrigated, 'point-by-point' RF ablation, masses of microembolic signals are detected in transcranial ultrasound especially in the period of RF current application. The number of MES depends on the total procedure time and the reached ACT during ablation. The use of contact force monitoring might reduce MES during RF ablation