Red blood cells as oxygen carrier during normothermic machine perfusion of kidney grafts: Friend or foe?

Renal ex vivo normothermic machine perfusion (NMP) is under development as an assessment tool for high-risk kidney grafts and as a means of achieving more physiologically accurate organ preservation. On-going hemolysis has been reported during NMP, as this technique relies on red blood cells for oxygen delivery. In this study, we confirm the occurrence of progressive hemolysis during 6-hour kidney NMP. NMP-associated erythrostasis in the glomeruli and in peri-glomerular vascular networks points to an interaction between the red blood cells and the graft. Continuous hemolysis resulted in prooxidative changes in the perfusate, which could be quenched by addition of fresh frozen plasma. In a cell-based system, this hemolysis induced redox stress and exhibited toxic effects at high concentrations. These findings highlight the need for a more refined oxygen carrier in the context of renal NMP.

Toward Uniform Case Identification Criteria in Observational Studies on Peripheral Arterial Disease: A Scoping Review.

BACKGROUND: The diagnosis of peripheral arterial disease (PAD) is commonly applied for symptoms related to atherosclerotic obstructions in the lower extremity, though its clinical manifestations range from an abnormal ankle-brachial index to critical limb ischemia. Subsequently, management and prognosis of PAD vary widely with the disease stage. A critical aspect is how this variation is addressed in administrative database-based studies that rely on diagnosis codes for case identification. The objective of this scoping review is to inventory the identification strategies used in studies on PAD that rely on administrative databases, to map the pros and cons of the International Classification of Diseases (ICD) codes applied, and to propose a first outline for a consensus framework for case identification in administrative databases. METHODS: Registry-based reports published between 2010 and 2021 were identified through a systematic PubMed search. Studies were subcategorized on the basis of the expressed study focus: claudication, critical limb ischemia, or general peripheral arterial disease, and the ICD code(s) applied for case identification mapped. RESULTS: Ninety studies were identified, of which 36 (40%) did not specify the grade of PAD studied. Forty-nine (54%) articles specified PAD grade studied. Five (6%) articles specified different PAD subgroups in methods and baseline demographics, but not in further analyses. Mapping of the ICD codes applied for case identification for studies that specified the PAD grade studied indicated a remarkable heterogeneity, overlap, and inconsistency. CONCLUSIONS: A large proportion of registry-based studies on PAD fail to define the study focus. In addition, inconsistent strategies are used for PAD case identification in studies that report a focus. These findings challenge study validity and interfere with inter-study comparison. This scoping review provides a first initiative for a consensus framework for standardized case selection in administrative studies on PAD. It is anticipated that more uniform coding will improve study validity and facilitate inter-study comparisons.

Persistent High Long-term Excess Mortality After Elective AAA Repair Especially in Women: A Large Population-based Study.

OBJECTIVE: The aim of this time-trend analysis is to estimate long-term excess mortality and associated cardiovascular risk for abdominal aortic aneurysm (AAA) patients after elective repair while addressing the changes in AAA management and patient selection over time. BACKGROUND: Despite the intensification of endovascular aneurysm repair and cardiovascular risk management, Swedish population data suggest that AAA patients retain a persistently high long-term mortality after elective repair. The question is whether this reflects suboptimal treatment, a changing patient population over time, or a national phenomenon. METHODS: Nationwide time-trend analysis including 40,730 patients (87% men) following elective AAA repair between 1995 and 2017. Three timeframes were compared, each reflecting changes in the use of endovascular aneurysm repair and intensification of cardiovascular risk management. Relative survival analyses were used to estimate disease-specific excess mortality. Competing risk of death analysis evaluated the risk of cardiovascular versus noncardiovascular death. Sensitivity analysis evaluated the impact of changes in patient selection over time. RESULTS: Short-term excess mortality significantly improved over time. Long-term excess mortality remained high with a doubled mortality risk for women (relative excess risk=1.87, 95% CI: 1.73-2.02). Excess mortality did not differ between age categories. The risk of cardiovascular versus noncardiovascular death remained similar over time, with a higher risk of cardiovascular death for women. Changes in patient population (ie, older and more comorbid patients in the latter period) marginally impacted excess mortality (2%). CONCLUSIONS: Despite changes in AAA care, patients retain a high long-term excess mortality after elective repair with a persistent high cardiovascular mortality risk. In this, a clear sex - but no age - disparity stands out.

Plaque Evaluation by Ultrasound and Transcriptomics Reveals BCLAF1 as a Regulator of Smooth Muscle Cell Lipid Transdifferentiation in Atherosclerosis.

BACKGROUND: Understanding the processes behind carotid plaque instability is necessary to develop methods for identification of patients and lesions with stroke risk. Here, we investigated molecular signatures in human plaques stratified by echogenicity as assessed by duplex ultrasound. METHODS: Lesion echogenicity was correlated to microarray gene expression profiles from carotid endarterectomies (n=96). The findings were extended into studies of human and mouse atherosclerotic lesions in situ, followed by functional investigations in vitro in human carotid smooth muscle cells (SMCs). RESULTS: Pathway analyses highlighted muscle differentiation, iron homeostasis, calcification, matrix organization, cell survival balance, and BCLAF1 (BCL2 [B-cell lymphoma 2]-associated transcription factor 1) as the most significant signatures. BCLAF1 was downregulated in echolucent plaques, positively correlated to proliferation and negatively to apoptosis. By immunohistochemistry, BCLAF1 was found in normal medial SMCs. It was repressed early during atherogenesis but reappeared in CD68+ cells in advanced plaques and interacted with BCL2 by proximity ligation assay. In cultured SMCs, BCLAF1 was induced by differentiation factors and mitogens and suppressed by macrophage-conditioned medium. BCLAF1 silencing led to downregulation of BCL2 and SMC markers, reduced proliferation, and increased apoptosis. Transdifferentiation of SMCs by oxLDL (oxidized low-denisty lipoprotein) was accompanied by upregulation of BCLAF1, CD36, and CD68, while oxLDL exposure with BCLAF1 silencing preserved MYH (myosin heavy chain) 11 expression and prevented transdifferentiation. BCLAF1 was associated with expression of cell differentiation, contractility, viability, and inflammatory genes, as well as the scavenger receptors CD36 and CD68. BCLAF1 expression in CD68+/BCL2+ cells of SMC origin was verified in plaques from MYH11 lineage-tracing atherosclerotic mice. Moreover, BCLAF1 downregulation associated with vulnerability parameters and cardiovascular risk in patients with carotid atherosclerosis. CONCLUSIONS: Plaque echogenicity correlated with enrichment of distinct molecular pathways and identified BCLAF1, previously not described in atherosclerosis, as the most significant gene. Functionally, BCLAF1 seems necessary for survival and transdifferentiation of SMCs into a macrophage-like phenotype. The role of BCLAF1 in plaque vulnerability should be further evaluated.

Thoracic aortic atherosclerosis in patients with a bicuspid aortic valve; a case-control study.

INTRODUCTION: Bicuspid aortic valve (BAV) patients have an increased risk to develop thoracic aortic complications. Little is known about the prevalence and severity of atherosclerosis in the BAV ascending aortic wall. This study evaluates and compares the prevalence of thoracic aortic atherosclerosis in BAV and tricuspid aortic valve (TAV) patients. METHODS: Atherosclerosis was objectified using three diagnostic modalities in two separate BAV patient cohorts (with and without an aortic dilatation). Within the first group, atherosclerosis was graded histopathologically according to the modified AHA classification scheme proposed by Virmani et al. In the second group, the calcific load of the ascending aorta and coronary arteries, coronary angiographies and cardiovascular risk factors were studied. Patients were selected from a surgical database (treated between 2006-2020), resulting in a total of 128 inclusions. RESULTS: Histopathology showed atherosclerotic lesions to be more prevalent and severe in all TAV as compared to all BAV patients (OR 1.49 (95%CI 1.14 - 1.94); p = 0.003). Computed tomography showed no significant differences in ascending aortic wall calcification between all BAV and all TAV patients, although a tendency of lower calcific load in favor of BAV was seen. Coronary calcification was higher in all TAV as compared to all BAV (OR 1.30 (95%CI 1.06 - 1.61); p = 0.014). CONCLUSION: Ascending aortic atherosclerotic plaques were histologically more pronounced in TAV as compared to the BAV patients, while CT scans revealed equal amounts of calcific depositions within the ascending aortic wall. This study confirms less atherosclerosis in the ascending aortic wall and coronary arteries of BAV patients as compared to TAV patients. These results were not affected by the presence of a thoracic aortic aneurysm.

Cytoskeletal protein degradation in brain death donor kidneys associates with adverse posttransplant outcomes.

In brain death, cerebral injury contributes to systemic biological dysregulation, causing significant cellular stress in donor kidneys adversely impacting the quality of grafts. Here, we hypothesized that donation after brain death (DBD) kidneys undergo proteolytic processes that may deem grafts susceptible to posttransplant dysfunction. Using mass spectrometry and immunoblotting, we mapped degradation profiles of cytoskeletal proteins in deceased and living donor kidney biopsies. We found that key cytoskeletal proteins in DBD kidneys were proteolytically cleaved, generating peptide fragments, predominantly in grafts with suboptimal posttransplant function. Interestingly, α-actinin-4 and talin-1 proteolytic fragments were detected in brain death but not in circulatory death or living donor kidneys with similar donor characteristics. As talin-1 is a specific proteolytic target of calpain-1, we investigated a potential trigger of calpain activation and talin-1 degradation using human ex vivo precision-cut kidney slices and in vitro podocytes. Notably, we showed that activation of calpain-1 by transforming growth factor-ß generated proteolytic fragments of talin-1 that matched the degradation fragments detected in DBD preimplantation kidneys, also causing dysregulation of the actin cytoskeleton in human podocytes; events that were reversed by calpain-1 inhibition. Our data provide initial evidence that brain death donor kidneys are more susceptible to cytoskeletal protein degradation. Correlation to posttransplant outcomes may be established by future studies.

Preclinical models versus clinical renal ischemia reperfusion injury: A systematic review based on metabolic signatures.

Despite decennia of research and numerous successful interventions in the preclinical setting, renal ischemia reperfusion (IR) injury remains a major problem in clinical practice, pointing toward a translational gap. Recently, two clinical studies on renal IR injury (manifested either as acute kidney injury or as delayed graft function) identified metabolic derailment as a key driver of renal IR injury. It was reasoned that these unambiguous metabolic findings enable direct alignment of clinical with preclinical data, thereby providing the opportunity to elaborate potential translational hurdles between preclinical research and the clinical context. A systematic review of studies that reported metabolic data in the context of renal IR was performed according to the PRISMA guidelines. The search (December 2020) identified 35 heterogeneous preclinical studies. The applied methodologies were compared, and metabolic outcomes were semi-quantified and aligned with the clinical data. This review identifies profound methodological challenges, such as the definition of IR injury, the follow-up time, and sampling techniques, as well as shortcomings in the reported metabolic information. In light of these findings, recommendations are provided in order to improve the translatability of preclinical models of renal IR injury.

Athero-occlusive Disease Appears to be Associated with Slower Abdominal Aortic Aneurysm Growth: An Exploratory Analysis of the TEDY Trial.

OBJECTIVE: The role of atherosclerosis in abdominal aortic aneurysm (AAA) pathogenesis is controversial. The aim of this study was to compare AAA growth in patients who did and did not have concurrent athero-occlusive disease (AOD). METHODS: Patients with an AAA measuring 35 - 49 mm in maximum diameter were recruited as part of the TElmisartan in the management of abdominal aortic aneurysm (TEDY) trial. TEDY participants who had infrarenal aortic volume and orthogonal diameter assessed by computed tomography at entry and at least one other time point during the trial (12 and/or 24 months) were included. AOD was defined by prior diagnoses of coronary heart disease, stroke, or peripheral arterial disease or an ankle brachial pressure index < 0.90. The increase in AAA volume and diameter from entry for participants who did and did not have AOD was assessed using linear mixed effects models; 131 of the 210 participants recruited to TEDY were included. RESULTS: In an unadjusted analysis, the mean (95% confidence interval) annual increases in AAA volume and diameter for participants with AOD were 3.26 (0.82 - 5.70) cm3 and 0.70 (0.19 - 1.22) mm slower than those without AOD, p = .008 and .007 respectively. The association between AOD and significantly slower AAA growth was maintained after adjusting for risk factors and medications, significantly unequally distributed between participants with and without an AOD diagnosis. CONCLUSION: In an exploratory analysis of a selective cohort from the TEDY trial, AOD was associated with slower AAA growth. Validation of these findings in other cohorts is needed.

Metabolic needs of the kidney graft undergoing normothermic machine perfusion.

Normothermic machine perfusion (NMP) is emerging as a novel preservation strategy. During NMP, the organ is maintained in a metabolically active state that may not only provide superior organ preservation, but that also facilitates viability testing before transplantation, and ex situ resuscitation of marginal kidney grafts. Although the prevailing perfusion protocols for renal NMP are refined from initial pioneering studies concerning short periods of NMP, it could be argued that these protocols are not optimally tailored to address the putatively compromised metabolic plasticity of marginal donor grafts (i.e., in the context of viability testing and/or preservation), or to meet the metabolic prerequisites associated with prolonged perfusions and the required anabolic state in the context of organ regeneration. Herein, we provide a theoretical framework for the metabolic requirements for renal NMP. Aspects are discussed along the lines of carbohydrates, fatty acids, amino acids, and micronutrients required for optimal NMP of an isolated kidney. In addition, considerations for monitoring aspects of metabolic status during NMP are discussed.

Towards Patient Centred Outcomes for Elective Abdominal Aortic Aneurysm Repair: A Scoping Review of Quality of Life Scales.

OBJECTIVE: In order to better incorporate the patient's perspective in medical decision making, core outcome sets (COS) are being defined. In the field of abdominal aortic aneurysm (AAA), efforts to capture the patient's perspective focus on generic quantitative quality of life (QoL) scales. The question arises whether these quantitative scales adequately reflect the patient's perspective on QoL, and whether they can be included in the QoL aspect of COS. A scoping review of QoL assessment in the context of elective AAA repair was undertaken. DATA SOURCES: PubMed, Embase, Web of Science, and the Cochrane Library. REVIEW METHODS: A scoping review was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Extension for Scoping Reviews (PRISMA-ScR) guidelines. Articles reporting QoL assessment in the context of elective AAA repair were identified. Quantitative studies (i.e., traditional QoL scales) were aligned (triangulation approach) with qualitative studies (i.e., patient perspective) to identify parallels and discrepancies. Mean Short Form 36 item survey (SF-36) scores were pooled using a random effects model to evaluate sensitivity to change. RESULTS: Thirty-three studies were identified, of which 29 (88%) were quantitative and four (12%) qualitative. The 33 studies reported a total of 54 quantitative QoL scales; the most frequently used were the generic SF-36 (16 studies) and five dimension EuroQol (EQ-5D; eight studies). Aneurysm specific scales were reported by one study. The generic quantitative scales showed poor alignment with the patient's perspective. The aneurysm specific scales better aligned but missed "concerns regarding symptoms" and "the impact of possible outcomes/complications". "Self control and decision making", which was brought forward by patients in qualitative studies, was not captured in any of the current scales. CONCLUSION: There is no established tool that fully captures all aspects of the patient's perspective appropriate for a COS for elective AAA repair. In order to fulfil the need for a COS for the management of, AAA disease, a more comprehensive overview of the patient's perspective is required.

Long-term Prognosis After Elective Abdominal Aortic Aneurysm Repair is Poor in Women and Men: The Challenges Remain.

OBJECTIVE: To evaluate the impact of changes in elective Abdominal Aortic Aneurysm (AAA) management on life-expectancy of AAA patients. BACKGROUND: Over the past decades AAA repair underwent substantial changes, that is, the introduction of EVAR and implementation of intensified cardiovascular risk management. The question rises to what extent these changes improved longevity of AAA patients. METHODS: National evaluation including all 12.907 (82.7% male) patients who underwent elective AAA repair between 2001 and 2015 in Sweden. The impact of changes in AAA management was established by a time-resolved analysis based on 3 timeframes: open repair dominated period (2001-2004, n = 2483), transition period (2005-2011, n = 6230), and EVAR-first strategy period (2012-2015, n = 4194). Relative survival was used to quantify AAA-associated mortality, and to adjust for changes in life-expectancy. RESULTS: Relative survival of electively treated AAA patients was stable and persistently compromised [4-year relative survival and 95% confidence interval: 0.87 (0.85-0.89), 0.87 (0.86-0.88), 0.89 (0.86-0.91) for the 3 periods, respectively]. Particularly alarming is the severely compromised survival of female patients (4-year relative survival females 0.78, 0.80, 0.70 vs males 0.89, 0.89, 0.91, respectively). Cardiovascular mortality remained the main cause of death (51.0%, 47.2%, 47.9%) and the proportion cardiovascular disease over non-cardiovascular disease death was stable over time. CONCLUSIONS: Changes in elective AAA management reduced short-term mortality, but failed to improve the profound long-term survival disadvantage of AAA patients. The persistent high (cardiovascular) mortality calls for further intensification of cardiovascular risk management, and a critical appraisal of the basis for the excess mortality of AAA patients.

Editor's Choice - A Systemic Evaluation of the Costs of Elective EVAR and Open Abdominal Aortic Aneurysm Repair Implies Cost Equivalence.

OBJECTIVE: The suggested high costs of endovascular aneurysm repair (EVAR) hamper the choice of insurance companies and financial regulators for EVAR as the primary option for elective abdominal aortic aneurysm (AAA) repair. However, arguments used in this debate are impeded by time related aspects such as effect modification and the introduction of confounding by indication, and by asymmetric evaluation of outcomes. Therefore, a re-evaluation minimising the impact of these interferences was considered. METHODS: A comparative analysis was performed evaluating a period of exclusive open repair (OR; 1998-2000) and a period of established EVAR (2010-2012). Data from four hospitals in The Netherlands were collected to estimate resource use. Actual costs were estimated by benchmark cost prices and a literature review. Costs are reported at 2019 prices. A break even approach, defining the costs for an endovascular device at which cost equivalence for EVAR and OR is achieved, was applied to cope with the large variation in endovascular device costs. RESULTS: One hundred and eighty-six patients who underwent elective AAA repair between 1998 and 2000 (OR period) and 195 patients between 2010 and 2012 (EVAR period) were compared. Cost equivalence for OR and EVAR was reached at a break even price for an endovascular device of €13 190. The main cost difference reflected the longer duration of hospital stay (ward and Intensive Care Unit) of OR (€11 644). Re-intervention rates were similar for OR (24.2%) and EVAR (24.6%) (p = .92). CONCLUSION: Cost equivalence for EVAR and OR occurs at a device cost of €13 000 for EVAR. Hence, for most routine repairs, EVAR is not costlier than OR until at least the five year follow up.

No Clinical Benefit of Intramuscular Delivery of Bone Marrow-derived Mononuclear Cells in Nonreconstructable Peripheral Arterial Disease: Results of a Phase-III Randomized-controlled Trial.

BACKGROUND AND AIMS: Prospects for no-option, end-stage peripheral artery disease (PAD) patients remain poor. Although results from open and semiblinded studies fuel hope for cell-based strategies in no-option patients, so far conclusions from the available placebo-controlled studies are not supportive. With the intention to end the remaining controversy with regard to cell therapy for PAD we conducted a confirmatory, double-blinded randomized placebo-controlled phase 3 trial. STUDY DESIGN: This randomized controlled trial was registered (NCT00539266). Inclusion criteria included stable or progressive disabling PAD, no imminent need for amputation, absent accepted options for revascularization. Diabetic disease was an exclusion criterion. Bone marrow (500-700 mL) was harvested and bone marrow-derived mononuclear cells were concentrated to 40 mL. Concentrated cells or placebo (diluted blood) were intramuscularly injected at 40 locations of the calf muscle. RESULTS: Fifty-four patients (mean (sd) age 58.2 (14.2) yrs, 58% males) were randomized. Twenty-eight patients received BM-MNCs, 26 placebo. Baseline criteria were similar in the 2 groups. No significant differences were observed for the primary (number of amputations, (pain free) walking distance) and secondary outcome parameters (ankle brachial index, pain scores, quality of life (SF-36)). DISCUSSION: This fully blinded replication trial of autologous BM-MNC fails to confirm a benefit for cell therapy in no-option PAD patients, consequently BM-MNC therapy should not be offered as a clinical treatment. Apparent contrasting conclusions from open and controlled studies underscore the importance of a controlled trial design in evaluating cell-based interventions in PAD.

A registry-based rationale for discrete intervention thresholds for open and endovascular elective abdominal aortic aneurysm repair in female patients.

OBJECTIVE: An evidence-based consensus for a female-specific intervention threshold for abdominal aortic aneurysms (AAAs) is missing. This study aims to analyze sex-related differences in the epidemiology of ruptured AAA to establish an intervention threshold for women. METHODS: The Dutch Surgical Aneurysm Audit (DSAA) is a compulsory, nation-wide registry of AAA repairs in The Netherlands. All patients with emergency or elective AAA repair between January 1, 2013, and December 31, 2015, were included in the analysis. The main outcomes were age, sex, AAA diameter at time of rupture, and 30-day postoperative mortality. RESULTS: A total of 1561 ruptured AAA repairs (14.7% women) and 7063 cases of elective AAA repair (13.7% women) were included in the analysis. Women had significantly smaller mean ± standard deviation AAA diameter at time of rupture than men; 70.5 ± 14.4 mm and 78.6 ± 17.5 mm, respectively. In male patients, 8% of ruptures occurred at diameters below the 55 mm intervention threshold. The female equivalent of this eighth percentile is 52 mm. Female patients had significantly higher 30-day mortality after emergency repair, namely, 33% for women versus 24.2% for men, but were also significantly older, mean ± standard deviation age 76.7 ± 7.1 years and 73.9 ± 8.3 years for women and men, respectively. Correcting for age reduced the 30-day mortality risk for women after ruptured AAA repair from 1.53 (95% confidence interval, 1.14-2.04) to 1.27 (95% confidence interval, 0.92-1.73). Outcome after open elective repair was significantly worse for women compared with men, with a 30-day mortality of 7.97% 30 for women and 4.27% for men (P < .01). CONCLUSIONS: The equivalent of the 55-mm intervention threshold for elective endovascular AAA repair in men is 52 mm in women. The almost doubled mortality risk for elective open repair in women implies that the optimal point for open repair is at higher diameters, very possibly at least 55 mm.

Adventitial adipogenic degeneration is an unidentified contributor to aortic wall weakening in the abdominal aortic aneurysm.

OBJECTIVE: The processes driving human abdominal aortic aneurysm (AAA) progression are not fully understood. Although antiinflammatory and proteolytic strategies effectively quench aneurysm progression in preclinical models, so far all clinical interventions failed. These observations hint at an incomplete understanding of the processes involved in AAA progression and rupture. Interestingly, strong clinical and molecular associations exist between popliteal artery aneurysms (PAAs) and AAAs; however, PAAs have an extremely low propensity to rupture. We thus reasoned that differences between these aneurysms may provide clues toward (auxiliary) processes involved in AAA-related wall debilitation. A better understanding of the pathophysiologic processes driving AAA growth can contribute to pharmaceutical treatments in the future. METHODS: Aneurysmal wall samples were collected during open elective and emergency repair. Control perirenal aorta was obtained during kidney transplantation, and reference popliteal tissue obtained from the anatomy department. This study incorporates various techniques including (immuno)histochemistry, Western Blot, quantitative polymerase chain reaction, microarray, and cell culture. RESULTS: Histologic evaluation of AAAs, PAAs, and control aorta shows extensive medial (PAA) and transmural fibrosis (AAA), and reveals abundant adventitial adipocytes aggregates as an exclusive phenomenon of AAAs (P < .001). Quantitative polymerase chain reaction, immunohistochemistry, Western blotting, and microarray analysis showed enrichment of adipogenic mediators (C/EBP family P = .027; KLF5 P < .000; and peroxisome proliferator activated receptor-γ, P = .032) in AAA tissue. In vitro differentiation tests indicated a sharply increased adipogenic potential of AAA adventitial mesenchymal cells (P < .0001). Observed enrichment of adipocyte-related genes and pathways in ruptured AAA (P < .0003) supports an association between the extent of fatty degeneration and rupture. CONCLUSIONS: This translational study identifies extensive adventitial fatty degeneration as an ignored and distinctive feature of AAA disease. Enrichment of adipocyte genesis and adipocyte-related genes in ruptured AAA point to an association between the extent of fatty degeneration and rupture. This observation may (partly) explain the failure of medical therapy and could provide a lead for pharmaceutical alleviation of AAA progression.

CXCL8 hyper-signaling in the aortic abdominal aneurysm.

There are indications for elevated CXCL8 levels in abdominal aortic aneurysm disease (AAA). CXCL8 is concurrently involved in neutrophil-mediated inflammation and angiogenesis, two prominent and distinctive characteristics of AAA. As such we considered an evaluation of a role for CXCL8 in AAA progression relevant. ELISA's, real time PCR and array analysis were used to explore CXCL8 signaling in AAA wall samples. A role for CXCL8 in AAA disease was tested through the oral CXCR1/2 antagonist DF2156A in the elastase model of AAA disease. There is an extreme disparity in aortic wall CXCL8 content between AAA and aortic atherosclerotic disease (median [IQR] aortic wall CXCL8 content: 425 [141-1261] (AAA) vs. 23 [2.8-89] (atherosclerotic aorta) µg/g protein (P < 1 ·â€¯10-14)), and abundant expression of the CXCR1 and 2 receptors in AAA. Array analysis followed by pathway analysis showed that CXCL8 hyper-expression in AAA is followed increased by IL-8 signaling (Z-score for AAA vs. atherosclerotic control: 2.97, p < 0.0001). Interference with CXCL8 signaling through DF2156A fully abrogated AAA formation and prevented matrix degradation in the murine elastase model of AAA disease (p < 0.001). CXCL8-signaling is a prominent and distinctive feature of AAA, interference with the pathway constitutes a promising target for medical stabilization of AAA.

Wnt Signaling Pathway Inhibitor Sclerostin Inhibits Angiotensin II-Induced Aortic Aneurysm and Atherosclerosis.

OBJECTIVE: Sclerostin (SOST) has been identified as an important regulator of bone formation; however, it has not been previously implicated in arterial disease. The aim of this study was to assess the role of SOST in aortic aneurysm (AA) and atherosclerosis using human samples, a mouse model, and in vitro investigations. APPROACH AND RESULTS: SOST protein was downregulated in human and mouse AA samples compared with controls. Transgenic introduction of human SOST in apolipoprotein E-deficient (ApoE-/-) mice (SOSTTg .ApoE-/-) and administration of recombinant mouse Sost inhibited angiotensin II-induced AA and atherosclerosis. Serum concentrations of several proinflammatory cytokines were significantly reduced in SOSTTg .ApoE-/- mice. Compared with controls, the aortas of mice receiving recombinant mouse Sost and SOSTTg .ApoE-/- mice showed reduced matrix degradation, reduced elastin breaks, and preserved collagen. Decreased inflammatory cell infiltration and a reduction in the expression of wingless-type mouse mammary virus integration site/ß-catenin responsive genes, including matrix metalloproteinase-9, osteoprotegerin, and osteopontin, were observed in the aortas of SOSTTg .ApoE-/- mice. SOST expression was downregulated and the wingless-type mouse mammary virus integration site/ß-catenin pathway was activated in human AA samples. The cytosine-phosphate-guanine islands in the SOST gene promoter showed significantly higher methylation in human AA samples compared with controls. Incubation of vascular smooth muscle cells with the demethylating agent 5-azacytidine resulted in upregulation of SOST, suggesting that SOST is epigenetically regulated. CONCLUSIONS: This study identifies that SOST is expressed in the aorta and downregulated in human AA possibly because of epigenetic silencing. Upregulating SOST inhibits AA and atherosclerosis development, with potential important implications for treating these vascular diseases.

The hypoxanthine-xanthine oxidase axis is not involved in the initial phase of clinical transplantation-related ischemia-reperfusion injury.

The hypoxanthine-xanthine oxidase (XO) axis is considered to be a key driver of transplantation-related ischemia-reperfusion (I/R) injury. Whereas interference with this axis effectively quenches I/R injury in preclinical models, there is limited efficacy of XO inhibitors in clinical trials. In this context, we considered clinical evaluation of a role for the hypoxanthine-XO axis in human I/R to be relevant. Patients undergoing renal allograft transplantation were included (n = 40) and classified based on duration of ischemia (short, intermediate, and prolonged). Purine metabolites excreted by the reperfused kidney (arteriovenous differences) were analyzed by the ultra performance liquid chromatography-tandem mass spectrometer (UPLCMS/MS) method and tissue XO activity was assessed by in situ enzymography. We confirmed progressive hypoxanthine accumulation (P < 0.006) during ischemia, using kidney transplantation as a clinical model of I/R. Yet, arteriovenous concentration differences of uric acid and in situ enzymography of XO did not indicate significant XO activity in ischemic and reperfused kidney grafts. Furthermore, we tested a putative association between hypoxanthine accumulation and renal oxidative stress by assessing renal malondialdehyde and isoprostane levels and allantoin formation during the reperfusion period. Absent release of these markers is not consistent with an association between ischemic hypoxanthine accumulation and postreperfusion oxidative stress. On basis of these data for the human kidney we hypothesize that the role for the hypoxanthine-XO axis in clinical I/R injury is less than commonly thought, and as such the data provide an explanation for the apparent limited clinical efficacy of XO inhibitors.

Phenotypic Modulation of Smooth Muscle Cells in Atherosclerosis Is Associated With Downregulation of LMOD1, SYNPO2, PDLIM7, PLN, and SYNM.

OBJECTIVE: Key augmented processes in atherosclerosis have been identified, whereas less is known about downregulated pathways. Here, we applied a systems biology approach to examine suppressed molecular signatures, with the hypothesis that they may provide insight into mechanisms contributing to plaque stability. APPROACH AND RESULTS: Muscle contraction, muscle development, and actin cytoskeleton were the most downregulated pathways (false discovery rate=6.99e-21, 1.66e-6, 2.54e-10, respectively) in microarrays from human carotid plaques (n=177) versus healthy arteries (n=15). In addition to typical smooth muscle cell (SMC) markers, these pathways also encompassed cytoskeleton-related genes previously not associated with atherosclerosis. SYNPO2, SYNM, LMOD1, PDLIM7, and PLN expression positively correlated to typical SMC markers in plaques (Pearson r>0.6, P<0.0001) and in rat intimal hyperplasia (r>0.8, P<0.0001). By immunohistochemistry, the proteins were expressed in SMCs in normal vessels, but largely absent in human plaques and intimal hyperplasia. Subcellularly, most proteins localized to the cytoskeleton in cultured SMCs and were regulated by active enhancer histone modification H3K27ac by chromatin immunoprecipitation-sequencing. Functionally, the genes were downregulated by PDGFB (platelet-derived growth factor beta) and IFNg (interferron gamma), exposure to shear flow stress, and oxLDL (oxidized low-density lipoprotein) loading. Genetic variants in PDLIM7, PLN, and SYNPO2 loci associated with progression of carotid intima-media thickness in high-risk subjects without symptoms of cardiovascular disease (n=3378). By eQTL (expression quantitative trait locus), rs11746443 also associated with PDLIM7 expression in plaques. Mechanistically, silencing of PDLIM7 in vitro led to downregulation of SMC markers and disruption of the actin cytoskeleton, decreased cell spreading, and increased proliferation. CONCLUSIONS: We identified a panel of genes that reflect the altered phenotype of SMCs in vascular disease and could be early sensitive markers of SMC dedifferentiation.