Chronic kidney disease (CKD) is a global health issue, affecting more than 10% of the worldwide population. The current approach for formal diagnosis and prognostication of CKD typically relies on non-invasive serum and urine biomarkers such as serum creatinine and albuminuria. However, histological evidence of tubulointerstitial fibrosis is the ‘gold standard’ marker of the likelihood of disease progression. The development of novel biomedical technologies to evaluate exfoliated kidney cells from urine for non-invasive diagnosis and prognostication of CKD presents opportunities to avoid kidney biopsy for the purpose of prognostication. Efforts to apply these technologies more widely in clinical practice are encouraged, given their potential as a cost-effective approach, and no risk of post-biopsy complications such as bleeding, pain and hospitalization. The identification of biomarkers in exfoliated kidney cells from urine via western blotting, enzyme-linked immunosorbent assay (ELISA), immunofluorescence techniques, measurement of cell and protein-specific messenger ribonucleic acid (mRNA)/micro-RNA and other techniques have been reported. Recent innovations such as multispectral autofluorescence imaging and single-cell RNA sequencing (scRNA-seq) have brought additional dimensions to the clinical application of exfoliated kidney cells from urine. In this review, we discuss the current evidence regarding the utility of exfoliated proximal tubule cells (PTC), podocytes, mesangial cells, extracellular vesicles and stem/progenitor cells as surrogate markers for the early diagnosis and prognostication of CKD. Future directions for development within this research area are also identified. CKD is 3 months . The ide3 months . Early d3 months ,12.Histopathological examination of the kidney is the gold standard for the diagnosis and prognostication of CKD . HoweverA reliable non-invasive method to detect early kidney fibrosis in CKD, and to predict the trajectory of CKD progression remains desirable. As a disease marker, microalbuminuria has long been considered to be the potential solution, but it has since demonstrated to be non-specific in the detection of early CKD . FollowiDifficulties in the maintenance of exfoliated kidney cells from urine are acknowledged, as mature exfoliated cells have a short lifespan in culture . ImmortaOur review evaluates the various applications of exfoliated proximal tubule cells (PTC), podocytes and extracellular vesicles (EV) from urine in CKD. The potential application of exfoliated stem/progenitor kidney cells from urine is also discussed.Urinary exfoliated podocytes and podocyte-specific markers have demonstrated value for the early diagnosis of CKD and prognosticating CKD progression . DiabetiThe role of exfoliated podocytes from urine as markers to prognosticate the progression of mesangial diseases such as IgA nephropathy has been explored. Previous studies noted that urinary podocyte counts correlated with serum creatinine and proteinuria in IgA nephropathy . Those wUtilizing urinary podocytes as markers of disease activity in lupus nephritis has been evaluated. Studies have noted that the majority of urinary podocytes in patients with lupus nephritis are viable but dedifferentiated, with a greater proportion of apoptotic urinary podocytes being much lower in patients without kidney disease . UrinaryThere is minimal data regarding the use of exfoliated podocytes from urine for anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis. It was previously found that the rate of podocyte detachment to urine predicted kidney function loss in ANCA-associated vasculitis . ReportsUltimately, there are urinary podocyte-specific biomarkers which have shown universal prognostic value for all forms of CKD. Urine synaptopodin levels are described as a generic marker of podocyte damage. Synaptopodin protein expression, determined by Western blot, has demonstrated significant correlations with kidney function in CKD, regardless of the degree of albuminuria . UrinaryThe recent development of single-cell RNA sequencing (scRNA-seq) is a revolutionary technique in providing an unbiased genome-wide characterization of individual exfoliated cells from urine at scale . Both anThe utility of exfoliated PTCs from urine has proven to be clinically valuable in prognosticating the degree of disease for various tubular diseases that are associated with CKD, including conditions with a genetic predisposition ,50,51. I2; impaired kidney function was defined by eGFR <60 mL/min/1.73 m2) with a receiver operating characteristic area under the curve (AUC) of 0.99 [Differential multispectral autofluorescence imaging is an innovative technique that may have the potential to non-invasively diagnose CKD from an early stage and to prognosticate CKD progression. Cell autofluorescence originates from native fluorophores  that play important roles in cell and tissue metabolism ,58. The  of 0.99 . Further of 0.99 . FurtherAnother method involving the use of exfoliated PTCs from urine as a risk stratification tool in CKD is through proximal tubule-specific DNA methylation patterns that are identified from compartment-specific methylome analysis . These DThe application of scRNA-seq in exfoliated PTCs from urine has shown potential utility as a non-invasive technique for early diagnosis and risk stratification in CKD . The comThere are promising urinary biomarkers  which reflect proximal tubule injury, inflammation and fibrosis in CKD, although it remains uncertain whether these biomarkers could be reliably expressed in urinary exfoliated PTCs ,66. FurtMarkers that are derived from the EVs of urinary exfoliated kidney cells have demonstrated diagnostic and prognostic value for various etiologies of CKD . GSK-3β An advancement in our understanding of podocyte development has increased interest in the potential clinical applications of stem/progenitor cells in adult kidneys, though few studies have explicitly explored the marker utility of stem/progenitor cells exfoliated from human urine for the early diagnosis and risk stratification of CKD. Urinary stem/progenitor kidney cells have been quantified in genetic kidney disease studies, appearing with increased numbers over time in conditions such as cystinosis, mainly in the presence of proteinuria ,88,89. TRecent developments in novel technologies to utilize the clinical application of exfoliated kidney cells from urine have been encouraging. There is increased evidence to support the use of these non-invasive techniques for the early diagnosis and prognostication of CKD. Future directions in which to expand research on this topic are exciting .Whilst there are growing data to support the clinical use of exfoliated PTCs, podocytes, extracellular vesicles and stem/progenitor cells for the early diagnosis and prognostication of CKD, such data have been mostly absent for other structures in the nephron, such as mesangial cells, distal tubule, loop of Henle and collecting duct cells. ScRNA-seq techniques suggest that these cells can be exfoliated from urine, and EV biomarkers have been found in these cells with kidney disease ,91,92,93


It is well-established that child maltreatment practiced by parents is associated with adolescent aggression. Emerging evidence has suggested that higher levels of mindful parenting are associated with fewer negative parenting practices. However, the relationships among mindful parenting, child maltreatment, and adolescent aggression remain unclear.To examine the association between mindful parenting, child maltreatment, and adolescent aggressive behavior among Chinese parent-adolescent dyads.Survey data from 554 Chinese parent-adolescent dyads were used for the analysis. Parents reported mindful parenting, and adolescents reported three forms of child maltreatment  by their parents and aggressive behavior. Path models were used to analyze whether higher levels of mindful parenting were associated with decreased likelihood of parent-to-adolescent maltreatment that were further related to lower levels of adolescent aggression.interaction with full attention and compassion and acceptance) were associated with lower likelihood of physical abuse and psychological aggression, which were related to decreased levels of adolescent aggressive behavior. Stratified analyses by parent gender showed that the path from interaction with full attention to adolescent aggression through psychological aggression was also significant or marginally significant in both mother-adolescent and father-adolescent dyads. Stratified analyses by adolescent gender demonstrated that the paths from interaction with full attention to physical abuse and psychological aggression were significant, which were also significantly correlated with adolescent aggression among male adolescents, whereas the mindful parenting-child maltreatment-adolescent aggression paths were not significant among female adolescents.Mindful parenting and its two factors (i.e., The findings contributed to the existing literature by assessing mindful parenting as a multifaceted construct and exploring the gender differences in the relationships. Gender-tailored interventions to improve mindful parenting, specifically focusing on the parents’ ability of interacting with adolescents providing full attention, compassion, and acceptance may work towards preventing child maltreatment and promoting adolescent behavioral health.The online version contains supplementary material available at 10.1186/s13034-022-00507-5. Aggression is one of the most prevalent and destructive behaviors among adolescents . AdolescMindful parenting can be an important parental factor that may be protective against child maltreatment. As a construct of growing interest, mindful parenting is defined as the parenting style that involves paying full attention in parent–child interactions, adopting an attitude of non-judgmental acceptance and compassion toward the self as a parent and the child, developing emotional awareness of the self and the child, and exerting self-regulation in the parenting relationship , 13. MinIn line with this, Duncan et al. proposed a theoretical model of mindful parenting’s effects, which depicted that mindful parenting can promote parents to adopt reasonable parenting practices and establish positive parent–child interactions, and in turn, such positive parent–child interactions ultimately decrease negative outcomes for children . SimilarTo our best knowledge, two empirical studies tested this relationship empirically. Both studies reported that higher mindful parenting was associated with fewer negative parenting practices , which were related to lower levels of internalizing and externalizing symptoms among adolescents , 18. HowBased on the existing theoretical models of mindful parenting , 17, theIn addition, considering that mothers and fathers exhibited different mean levels of mindfulness in parenting , and thaThe study used data from a large survey conducted in 2018, which purposively recruited adolescents from a local vocational secondary school located in a disadvantaged county in eastern China to investigate the relationship between childhood adversities and adolescent mental and behavioral health and its possible psychosocial mechanisms. According to the local and national statistical yearbook of 2018 , 30, thet =  − 6.20, p < 0.001), more likely to be female  and only child , less likely to experience paternal physical abuse , and exhibit a lower level of aggression , and were included in the analysis. Of the 554 dyads, 228 were mother-adolescent and 326 were father-adolescent dyads. A comparison between the adolescents included in the study and those excluded showed that the included adolescents were older .Adolescents reported their aggressive behavior using the Aggressive Behavior Subscale of the Youth Self Report . The 17-item Aggressive Behavior Subscale was rated on a 3-point scale . Higher scores indicate more aggressive behavior. The YSR has been validated in Chinese adolescents, showing acceptable to good reliability , 33. In interaction with full attention , compassion and acceptance , self-regulation , and emotional awareness of child , which were different from the factor structure of the original 31-item IM-P. This was because during the validation process by Pan et al., one item was deleted since it was easily misunderstood with Chinese parents, and six items mainly referring to compassion and acceptance of self were further deleted due to poor corrected item-total correlation and item discriminability : t =  −3.00, p = 0.003; father-adolescent dyads [FA]: t = − 2.92, p = 0.004) or psychological aggression  reported higher levels of aggressive behavior regardless of perpetrator. Aggression score did not differ significantly among adolescents with and without neglect. Parents perpetrating physical abuse reported lower scores on the total scale of mindful parenting , the compassion and acceptance subscale , and the interaction with full attention subscale . Parents perpetrating psychological aggression reported lower scores on the interaction with full attention subscale  than their counterparts. Neglectful parents did not differ from non-neglectful parents on mindful parenting and its factors.Compared to non-maltreated counterparts, adolescents who experienced physical abuse . Analyses among the father-adolescent dyads revealed that physically abusive fathers exhibited low levels of mindful parenting , interaction with full attention , and compassion and acceptance , and psychologically aggressive fathers showed lower levels of interaction with full attention . See Table Analyses among the mother-adolescent dyads showed that physically abusive mothers had lower levels of interaction with full attention subscale score , whereas its correlations with the total score for mindful parenting and the other two subscale scores were not statistically significant .As presented in Table OR = 0.59; 95% CI = ; p = 0.012], which was related to low levels of adolescent aggressive behavior  = 0.54, p = 0.030). Repetitive models using scores of different factors of mindful parenting as the exogenous variable showed that higher interaction with full attention subscale score was negatively associated with both psychological aggression  and physical abuse , which were positively related to lower levels of adolescent aggression . Additionally, higher compassion and acceptance subscale score was related to low levels of adolescent aggression through decreased likelihood of physical abuse , and psychological aggression . In addition, among the father-adolescent dyads, compassion and acceptance also related to adolescent aggression through psychological aggression . See Table Among both the mother-adolescent dyads and father-adolescent dyads, the path from interaction with full attention score was related to lower risks of physical abuse  and psychological aggression  and further lower levels of adolescent aggression  among male adolescents. The mindful parenting-child maltreatment-adolescent aggression paths were not significant among female adolescents. See Additional file High parental interaction with full attention and compassion and acceptance factors were negatively associated with physical abuse and psychological aggression, which were positively related to adolescent aggression,. The pathway from higher interaction with full attention to lower levels of adolescent aggression through reduced risks of psychological aggression was also evident in both mother-adolescent and father-adolescent dyads. In addition, among father-adolescent dyads, compassion and acceptance was related to adolescent aggression through psychological aggression. Stratified analyses by adolescent gender demonstrated that the relationship between interaction with full attention and adolescent aggression was mediated by physical abuse and psychological aggression among male adolescents, whereas the mindful parenting-child maltreatment-adolescent aggression paths were not significant among female adolescents. To the best of our knowledge, this is one of the first studies to examine the relationship of mindful parenting and its factors with child maltreatment and adolescent aggression, using data from parent-adolescent dyads.This study revealed that after adjusting adolescent sociodemographic characteristics, mindful parenting, especially its interaction with full attention factor was significantly and negatively correlated with aggression. Further path analysis also found that after adjusting adolescent sociodemographic covariates, parents with higher levels of mindful parenting, especially interaction with full attention and compassion and acceptance, were less likely to engaged in physical abuse and psychological aggression towards adolescents. Furthermore, non-maltreated adolescents exhibited low levels of aggressive behavior, which supported the models developed by Duncan et al. [Although we did not find significant correlation between the total score of mindful parenting and aggression in the zero-order correlation analysis, the n et al.  and Ahemn et al. , and wasn et al. , 18. When et al. , which mn et al. . Furthern et al. , which fn et al. . In addin et al. , and, heNevertheless, mindful parenting and its different factors were not significantly associated with neglect. In this study, the classification of neglect was driven by the item “showing or telling me that he/she loves me” . Culturally, Chinese parents tend to refrain from showing direct affection for their children, and choose to express their affect through providing instrumental support and making sacrifices , 46. Expself-regulation factor of mindful parenting and child maltreatment, indicating that not all mindful parenting factors are equally important in reducing child maltreatment. This is, to some extent, consistent with previous findings that different factors of mindful parenting were differentially associated with parenting practices [We did not find a significant relationship between the ractices , 23. Forinteraction with full attention on psychological aggression and adolescent aggression was evident in both mother-adolescent and father-adolescent dyads, while the protective role of compassion and acceptance was only evident in the father-adolescent dyads. Furthermore, the paths from interaction with full attention to adolescent aggression through physical abuse and psychological aggression were significant in male adolescents, but not in female adolescents. This is inconsistent with the findings from a previous study showing that the protective role of mindful parenting on parenting practices and child externalizing problems was equivalent for mothers and fathers, and for boys and girls [interaction with full attention and compassion and acceptance components of mindful parenting, and male adolescents are more likely to benefit from increasing parental ability of interacting with children with full attention.Stratified analysis by parent and adolescent gender showed that the protective role of nd girls . The incnd girls , culturand girls , and thend girls . Taken tThe findings should be interpreted cautiously because of limitations. First, the cross-sectional design did not allow the establishment of causal links between the variables. The direction of the association between parenting practices and adolescent behavioral development remained inconsistent, including both reciprocal and unidirectional links , 50. Futinteraction with full attention and compassion and acceptance factors, were associated with decreased likelihood of physical abuse and psychological aggression, and further related to low levels of adolescent aggression. The protective role of interaction with full attention against psychological aggression and adolescent aggression was evident in both mother-adolescent and father-adolescent dyads, while the protective role of compassion and acceptance was only evident in the father-adolescent dyads. These findings should be interpreted with caution given the unadjusted covariates. Despite this, the findings contributed to the existing literature by assessing mindful parenting as a multifaceted construct and exploring the gender differences in the relationships. In addition, the findings shed light on the importance of gender-tailored interventions on increasing parents’ ability of interacting with adolescents with full attention, compassion, and acceptance, in preventing child maltreatment and promoting adolescent behavioral health.This study revealed the protective role of mindful parenting and its factors against child maltreatment and adolescent aggressive behavior in a sample of Chinese parent-adolescent dyads. More specifically, high levels of mindful parenting, specifically its Additional file 1: Table S1. Comparisons between adolescents included in the study and those excluded. Table S2. Comparisons between the mothers/fathers included in the study and those excluded. Table S3. The paths from mindful parenting and its different factors to adolescent aggressive behavior through child maltreatment among female adolescents and male adolescents, respectively.


An endovascular arteriovenous fistula is a recent technological advancement in hemodialysis vascular access. This systematic review and meta-analysis aimed to investigate the efficacy and safety of endovascular arteriovenous fistula (eAVF) creation compared with surgical arteriovenous fistula (sAVF) creation among patients with chronic kidney disease.Trials.gov, and the WHO International Clinical Trials Registry Platform until May 2021 to perform meta-analyses using random-effects models. Pre-specified primary outcomes were fistula maturation, procedure-related complications, and patient satisfaction. Secondary outcomes were procedural technical success, procedure time, all adverse events, and medical expenditure. The risk of bias in non-randomized studies of the interventions assessment tool, and the Grading of Recommendation, Assessment, Development, and Evaluation (GRADE) approach were used to assess the quality of individual studies and the body of evidence, respectively.We searched Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, EMBASE, Clinical In seven studies including 860 patients, endovascular arteriovenous fistula creation had little to no effect on fistula maturation . Meta-analysis could not be performed for procedure-related complications and patient satisfaction due to insufficient data. For secondary outcomes, endovascular arteriovenous fistula resulted in a slight to no difference in procedural technical success  and all adverse events . Endovascular fistula creation incurred less medical expenditure than sAVF . Meta-analysis for procedure time was not performed because one of the studies had a critical risk of bias. All of these outcomes were of low certainty of evidence or very low certainty of evidence.There was limited evidence for supporting endovascular arteriovenous fistula creation over conventional surgical arteriovenous fistula creation for patients with chronic kidney disease. Multicenter randomized controlled trials are needed to confirm the efficacy and safety of eAVFs in selected populations. Chronic kidney disease (CKD) is a major health problem affecting approximately 700 million people worldwide . The numMaterials and methodsCompliance With Reporting Guidelinesprotocols.iohttps://protocols.io/view/efficacy-and-safety-of-endovascular-arteriovenous-bu95nz86), we conducted a systematic review of the relevant literature in accordance with the recommendations listed in the Cochrane Handbook and in the PRISMA guidelines [Using a prespecified protocol (idelines . We confEligibility CriteriaWe included: (ⅰ) all published articles  that assessed the efficacy and safety of eAVF creation, (ⅱ) abstracts from conferences and letters, (ⅲ) studies of any CKD etiology, follow-up duration, publication year, and country of origin, (ⅳ) patients of any age, sex, and ethnicity, (ⅴ) studies in which patients had undergone AV graft placement, and (ⅵ) studies in terms of varying methods used for anesthesia  or in relation to varying levels of expertise and clinical experience of the operators. We excluded: (ⅰ) case reports, case series, animal and laboratory studies, ongoing studies, and literature reviews, (ⅱ) patients treated for dysfunctional hemodialysis AVFs with percutaneous transluminal angioplasty using balloons or stents, and (ⅲ) studies in which patients had undergone sAVF creation other than in the arms. Intervention was defined as any percutaneous endovascular approach to create an AVF, including the use of the Ellipsys Vascular Access System  , the eveOutcomes of InterestPrimary outcomes were fistula maturation, procedure-related complications, and patient satisfaction. Secondary outcomes included procedural technical success, procedure time, all adverse events, and medical expenditure. Maturation was defined as AVF blood flow ≥500 ml/min and an outflow vein diameter ≥5 mm at the longest follow-up period after AVF creation, confirmed using duplex ultrasound or similar devices. The definition of procedure-related complications set by the original authors included any unintended complications directly arising from the procedure or device that occurred from the time of procedure initiation to completion. Procedural technical success was defined as the presence of blood flow in the outflow vein(s), confirmed using duplex ultrasound or similar devices. The definition of all adverse events set by the original authors included any postoperative events that required interventions on the sAVF or the eAVF. Medical expenditure was defined as the total medical financial cost including hospitalization, procedures, and materials. Definitions of patient satisfaction and procedure time established by the original authors were used.Search Strategy and Selection of StudiesTrials.gov, and the WHO International Clinical Trials Registry Platform (WHO ICTRP) via their dedicated search portals until May 2021. We manually searched study reference lists, including relevant international guidelines  [We used a set of suitable terms (details provided in Appendix Table updates)  as well Data Abstraction and Quality AssessmentThree reviewers  independently extracted data from the included studies using a standardized data collection form. Disagreements regarding data extraction were resolved through discussion. When necessary, we contacted the authors of studies that did not provide sufficient information. Three reviewers  independently evaluated the risk of bias using the ROBINS-I tool . DiffereData AnalysisAll analyses were conducted using Cochrane Review Manager software . For the dichotomous outcomes of maturation, procedure-related complications, procedural technical success, and all adverse events, pooled odds ratios (ORs) with 95% confidence intervals (CIs) are provided. If adjusted ORs and 95% CIs of the outcomes were not available and if the risk ratios (RRs) had been reported in the primary studies, we used a method reported by Zhang et al.  to transClinicalTrials.gov and the WHO ICTRP to identify trials that had been completed but not published at the time of the review.We evaluated statistical heterogeneity through a visual inspection of forest plots and through calculating the I2 statistic, which interpreted the values as follows: 0% to 40%, negligible heterogeneity; 30 to 60%, mild-to-moderate heterogeneity; 50% to 90%, moderate-to-substantial heterogeneity; and 75% to 100%, considerable heterogeneity. We investigated the underlying reasons for heterogeneity and conducted a chi-squared test, and a p-value <0.10 was considered to indicate statistical significance. We investigated reporting bias by checking Concerning the primary outcomes, the following pre-specified sensitivity analyses were conducted: (ⅰ) exclusion of studies using imputed statistics, and (ii) inclusion of studies with complete case data. We also performed pre-specified subgroup analyses according to the etiology of CKD (diabetes-related nephropathy versus non-diabetes-related nephropathy), type of endovascular device (Ellipsys Vascular Access System versus everlinQ endovascular AVF system), type of control (the use of native arteries and veins versus the use of arteriovenous grafts), type of study , and the number of AVF procedures (the first AVF creation versus the revascularization of AVFs). We created a summary of findings table that included an overall grading of the certainty of the evidence for the following outcomes: maturation, procedural technical success, procedure time, all adverse events, and medical expenditure. Two reviewers (YS and YT) evaluated the certainty of evidence based on the Grading of Recommendation, Assessment, Development, and Evaluation (GRADE) approach . DisagrePatient and Public InvolvementNo patients or members of the public were involved in this meta-analysis.ResultsStudy CharacteristicsAfter removing duplicates, we identified 2838 studies during the search conducted until May 2021. We identified eight studies that fulfilled all eligibility criteria and we included them in the qualitative synthesis -22. AfteTable Primary OutcomesTable Secondary OutcomesFigure DiscussionOur review included seven studies that involved 860 patients with CKD. Our findings indicated that evidence is very uncertain about the effects of eAVF creation on the maturation rate compared with sAVF creation. The certainty of the evidence was downgraded to very low levels because of the inclusion of retrospective cohort studies, a serious risk of bias, and imprecise estimates. We were not able to confirm the effect of eAVF on procedure-related complications due to limited data and a critical risk of bias. Moreover, no study reported on patient satisfaction. Additionally, it remains uncertain whether eAVF increases procedural technical success or reduces all adverse events because the results were based on very low certainty of evidence. However, our findings indicated that eAVF reduced medical expenditures related to patient care, which may suggest a cost-effective alternative to sAVF.The findings of this systematic review and meta-analysis suggest that, based on current evidence, eAVF cannot be confirmed as more efficacious or safer than sAVF. This conclusion is in contrast with conclusions drawn in a recent systematic review  that claOur systematic review comprehensively summarized the available evidence on the efficacy and safety of eAVF compared with sAVF. Another meta-analysis  evaluatiOur finding that medical costs were less with eAVF deserves attention because previous meta-analyses ,9 have nThe results of this meta-analysis may be applicable to patients with excess weight because the populations in our review -21 incluOur results have several clinical and research implications. First, the eAVF was not found to be an alternative to sAVF because it remains unclear whether creating an eAVF is more beneficial or harmful than an sAVF. To determine if eAVFs can be used routinely, high-quality clinical trials aimed at incorporating eAVFs into routine clinical practice for patients with CKD are warranted. Second, patient satisfaction should be measured as a patient-oriented outcome because patients’ opinions offer valuable insight into this topic and contribute to high-quality patient care. For instance, the ease of cannulation between eAVF and sAVF could be used as a measurement item to assess patient satisfaction.Limitations and strengthsThis systematic review had several limitations. First, we excluded studies ,20 with The strengths of our review were that it involved a comprehensive search for evidence in accordance with the PRISMA statement  and the In conclusion, this review showed that evidence for supporting eAVF creation over conventional sAVF was very limited among patients with CKD requiring hemodialysis. We strongly recommend further multicenter, prospective, randomized controlled trials to determine the efficacy and safety of eAVFs in this population.


Cerebrovascular diseases are a leading cause of death and disability globally. The development of new therapeutic targets for cerebrovascular diseases  is limited by a lack of knowledge of the cellular and molecular biology of health and disease conditions and the factors that cause injury to cerebrovascular structures. Here, we describe the role of advances in omics technology, particularly RNA sequencing, in studying high-dimensional, multifaceted profiles of thousands of individual blood and vessel cells at single-cell resolution. This analysis enables the dissection of the heterogeneity of diseased cerebral vessels and their atherosclerotic plaques, including the microenvironment, cell evolutionary trajectory, and immune response pathway. In animal models, RNA sequencing permits the tracking of individual cells  that compose atherosclerotic plaques and their alteration under experimental settings such as phenotypic transition. We describe how single-cell RNA transcriptomics in humans allows mapping to the molecular and cellular levels of atherosclerotic plaques in cerebral arteries, tracking individual lymphocytes and macrophages, and how these data can aid in identifying novel immune mechanisms that could be exploited as therapeutic targets for cerebrovascular diseases. Single-cell multi-omics approaches will likely provide the unprecedented resolution and depth of data needed to generate clinically relevant cellular and molecular signatures for the precise treatment of cerebrovascular diseases. Neurovascular diseases are a top cause of disability, morbidity, and death globally , with anTherapeutic approaches in acute stroke are targeted at rapid recanalizing of the blocked artery, either with intravenous thrombolysis or with thrombus lysis by endovascular thrombectomy ,4. OftenAtherosclerosis is important in the onset and progression of cerebral vascular disease. Cells and fibrous and lipid-rich material can accumulate and form arterial plaques in mid-size and large arteries . After tGenomic studies have highlighted potential genetic risk variants in cerebrovascular diseases, underscoring the relevance of identifying genetic factors associated with specific cell types. Dissecting the dynamic spectrum of a plaque’s cellular components is a challenge, but the resulting information may break new ground for therapeutic development.Bulk RNA sequencing (RNA-seq) of rodent and human atherosclerotic plaques has yielded data on the cellular composition and main molecular pathways implicated in the plaque formation. Nevertheless, this technique does not offer cell-specific resolution and does not allow for identification of individual cells responsible for a signal, and the contributions of uncommon but significant cell subtypes can be lost in the noise of more common cellular populations.Methods that could resolve this information at the single-cell level include single-cell RNA-seq and others under the single-cell omics umbrella, all of which can deepen and broaden results of pathological, imaging, and clinical investigations. Single-cell omics offers fundamental new tools for uncovering the mechanisms underlying atherosclerosis and cerebrovascular diseases. These techniques allow for the depiction of cellular phenotypes and their trajectory, including cellular plasticity and clonal expansion. Single-cell omics also can distinguish gene/protein expression signatures between healthy and disease conditions and various clinical phenotypes.Single-cell methods likewise can aid understanding of the vascular modification process during stroke and other brain diseases. Here, as an example of the potential of these approaches, we review in detail the cellular landscape of atherosclerotic plaques, thrombi, and immune signaling as uncovered by single-cell omics investigations focusing on the potential this information holds to significantly influence treatment of human cerebral vessel disorders.A search of the literature was performed in PubMed using key words searching and citation single cell sequencing and carotid atherosclerosis or single cell sequencing and cerebral vessel to identify articles published in English between 1 January 2000 and 25 June 2022.With recent technological advancements, biological processes can be studied at the resolution of a single cell . Single-The most widely used technology is scRNA-seq , which cThe general workflow of scRNA-seq experiments usually includes: (1) isolation of single cells, i.e., by microfluidic platforms to separate cells; (2) cell lysis, mRNA capture, and reverse transcription into cDNA and its amplification; (3) preparation of barcoded cDNA libraries for next-generation sequencing (NGS); and (4) bioinformatic analysis. New types of cells and more clearly defined cellular subsets have been identified using cluster algorithms and unbiased dimension reduction. Single-cell RNA bioinformatic methods have been described in detail previously ,14,15,16scRNA-seq makes it possible to divide cell populations into groups with different transcriptional features that confer on them characteristic metabolic states and biological functions ,18,19,20Other single-cell sequencing omics technologies can complement scRNA-seq, which does not capture cell–surface protein signatures. Cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq)  can partAnother approach that operates in tandem with scRNA-seq is the single-cell assay for transposase-accessible chromatin with sequencing (scATAC-seq) technique . scATAC-Methylation sequencing (methyl-seq) also can generate an epigenetic profile, depicting methylation in single cells . Other tAmong the single-cell proteomic technologies, cytometry by time of flight (CyTOF) relies on mass spectroscopy to quantify metal isotope-labeled antibodies and other tags on individual cells. The basic methods for effectively analyzing complex CyTOF experiments have been described previously ,33.scRNA-seq techniques that isolate cells from a sample cannot capture local intercellular communication networks, and some spatial transcriptomic and proteomic methods have been developed to address this issue. These strategies integrate fluorescence in situ hybridization with transcription data or proteomics, permitting assessment of gene or protein expression in a single cell while showing cell location. Recent advances in spatial transcriptomics have enabled profiling of single cells . Other eAnimal models are used to elucidate the link between blood flow and atherosclerosis lesion.Indeed, disturbed flow alters gene expression in the vascular wall, leading to infiltration of leukocytes that can promote atherosclerosis. The process of transcriptome modification in different cell populations  after alteration of blood flow is a main process in the pathogenesis. The most widely used in vivo platform to experimentally study flow perturbation in preclinical animal models is partial carotid ligation (PCL), which Kumar et al. have detailed in the mouse with a video explanation of endothelial cell collection for single-cell omics .Andueza et al.  comparedBy reanalyzing these scRNA-seq and scATACseq datasets, the same group demonstrated a key role of kallikrein-related peptidase 10  as a flow-regulated endothelial protein with positive anti-inflammatory, anti-atherogenic activities . KLK10 eIn an independent study, Li et al.  used a s+ and CXCR6+ T cells, and granulocytes also have been detected under disturbed flow. Li et al. [Birc5 has been described as critical to macrophage function in atherosclerosis, namely, as a key element of macrophage apoptosis and recruitment. Disturbed flow promotes the epithelial-to-mesenchymal cell transition, and vascular smooth muscle cells exposed to disturbed flow express markers related to osteoblast differentiation that can contribute to arterial stiffness. Of other inflammation-related cell subpopulations, NK cells, CD4i et al.  generateAnimal models are also useful for studying post-stenting carotid restenosis. Neointimal hyperplasia from vascular smooth vessel proliferation is the most important process in restenosis. Gao et al.  used sinAtherosclerosis and related cerebrovascular diseases develop with age and are linked to the accumulation of a spectrum of senescent cells and dysregulated inflammatory events. These conditions involve multifaceted players, including different vascular and metabolic components, age-associated chronic inflammatory processes within the blood circulation and at focal vascular walls, and the interplay between systemic and local sites. The highly diverse plaque cellular composition determines the clinical manifestations such as stroke. However, specific cell subpopulation phenotypes and status changes during atherogenesis (from initiation to a complex plaque) are still largely unknown. Single cell omics studies of human atherosclerotic lesions have demonstrated that T cells and macrophages are the most represented populations in atherosclerotic lesions ,41,42. SMost published scRNA-seq studies were conducted on tissues obtained during carotid endarterectomy . In this+ cells from six atherosclerotic plaques using scRNA-seq and CITE-seq, and >90,000 CD45+ cells using CyTOF mass cytometry [+ and CD8+ T cells, and the most common in blood were CD8+ T cells, presenting considerable phenotypic heterogeneity. The phenotypes of the T cells and macrophages differed fundamentally between plaques from symptomatic patients  and those from asymptomatic patients (no recent stroke). Distinct transcriptional changes in migration, activation, and differentiation of the cells accompanied the increased effector memory CD4+ T cell counts in plaques from symptomatic stroke patients. Furthermore, some T cell subgroups in these plaques showed RNA signatures compatible with T cell exhaustion  A (GMZA), GZMK, and perforin 1 (PRF1) levels and decreased GMZB levels, indicating the presence of cytotoxic CD4+ T cells in vasculature associated with disease. Five unique subsets of CD4+ and CD8+ T cell clusters. Several genes encoding cytokines present an open chromatin pattern, supporting their active transcription. In particular, IFNG has open chromatin in two subtypes of T cells, the cytotoxic and effector T cells. IFNG can activate pro-inflammatory macrophages, and CD4/CD8 cells can be activated by dendritic cells that express IL12 (characterized by open chromatin in the enhancer). The coordinated IL12-IFNG signals may play a key role in activation of T cells in the plaque, with subsequent myeloid cell recruitment at the atherosclerotic sites. This pattern is consistent with other data showing the atherogenic action of IL12- IFNG in cerebrovascular disorders [Open chromatin at the GZMB and GZMH loci validated the cytotoxic phenotype of CD4isorders .Depuydt et al.  also ideAmong the smooth muscle cells, the authors identified two main subgroups of cells: one with a contractile phenotype and the other with a low level of typical muscle markers and a higher proportion of extracellular matrix proteins.A study by Pan et al.  challengLi et al.  reanalyz− and Lgals3+ population , showing a gene expression pattern that was chondrocyte-like and diminished by Klf4 silencing. The activation of Lgals3 is pivotal to generating a population of cells that express genes characterized as stem cell markers and those associated with extracellular matrix remodeling. This subset gives rise to three other smooth muscle cell clusters, including Klf4-dependent osteogenic phenotypes that are coincident with plaque calcification and disruption. Overall, Klf4 pivoted transition of smooth muscle cells to a spectrum of phenotypes including Lgals3+ osteogenic cells that significantly contributed to late-stage atherosclerotic plaque pathogenesis. Whether calcification proves to be detrimental or protective in atherosclerotic plaques remains to be elucidated, as does the druggability of these transitions.The utility of scRNA-seq for understanding how the smooth muscle cell phenotypic transition is generated and affects plaque stability also was confirmed in other work . That stAlsaigh et al.  performeAn essential point in the cerebrovascular diseases field is identifying relevant genetic risk factors that might represent possible therapeutic targets. Hundreds of candidate genetic variants for atherosclerotic disease and cardio/cerebrovascular risk factors have emerged in genome-wide association studies (GWAS), besides monogenetic risk factors . SlenderAnother example of a correlation of genomic and single-cell RNA-seq data has been published . Polymor+), pericytes (KCNJ8+), endothelial cells (CLDN5+), and perivascular fibroblasts (DCN+). Using marker genes defined from single-cell transcriptomes, they performed a spatial multiplexed transcriptomic analysis of different cerebrovascular cell states by combining multiplexed fluorescent in situ hybridization and immunostaining. Then, they applied multiplexed spatial transcriptomics to the tissues. This dataset highlighted the signatures responsible for arteriovenous phenotypic changes known as zonation. Besides endothelial cells, they also characterized the principal perivascular cellular subclasses in the brain, which are pericytes, smooth muscle cells, and perivascular fibroblasts. These findings confirm the presence of perivascular fibroblasts and of smooth muscle-like cells known as fibromyocytes in the human cerebrovasculature.Recently, Winkler et al.  generate+), smooth muscle cells (TAGLN+), fibromyocytes (CCL19+), and perivascular fibroblasts (COL1A2+) in AVMs. A comparison of control and AVM data showed that endothelial cells in the AVMs presented mainly an arterial/venous phenotype without a transcriptional phenotype related to venules or capillaries, suggesting loss of normal zonation in the endothelial cells.To apply this strategy in a practical context, these authors also applied scRNA-seq to cells isolated from arteriovenous malformation (AVM) samples. AVM is a relatively frequent cerebrovascular disease of arteriovenous patterning in which patients are prone to bleeding and stroke and represents a major cause of stroke in young people. Working with AVMs from the brains of five patients who underwent surgery for these lesions, the authors performed scRNA-seq on 106,853 cells from the dissected samples and identified 11 significant clusters. Spatial transcriptomic analysis characterized endothelial cells  and were labeled as GPNMB+ Mo3, were highly present in ruptured AVMs and presented an activated phenotype.Hemorrhagic stroke is an extremely important consequence of AVMs, so characterizing the pathological cell states associated with AVM rupture is crucial. In this study, a spectrum of cellular and spatially heterogeneous immune reactions was observed in the AVMs. RNA analysis connected 871 differentially expressed genes to AVM rupture, many associated with vascular developmental pathways and inflammatory processes. The authors identified the cellular ontology of the cerebrovascular immune cells present in the AVM and detected the infiltration of distinct immune cell states. In particular, P2RY12+ monocytes contribute to vascular smooth muscle cell depletion, leading to AVM rupture and hemorrhage. These findings are expected to lead to new investigations in other cerebrovascular disorders to contribute to the mechanistic understanding and therapeutic development targeting the human cerebrovascular system.The authors hypothesize that GPNMBAn atlas of human and experimental cerebrovascular diseases at the single-cell level would support precision neurology. Precise patient selection and stratification based on molecular markers are required to maximize therapeutic success. Data from single-cell omics reveal that vascular cells during atherosclerosis have increased tissue specialization, heterogeneity, and plasticity. By allowing analysis of cells from human samples that usually carry the limitation of size and quantity, single-cell omics enables the longitudinal characterization of the delicate cellular architecture of diseased human arteries.Models of atherosclerosis in humans and mice differ fundamentally. The effects of high cholesterol, metabolic dysregulation, and environmental risk factors on different genetic backgrounds and aging are difficult to replicate in animal models. Animal models do not mimic human immune activity at plaques or spontaneous plaque instability . Animal scRNA-seq technology is clearly a robust and rapidly developing tool for generating expression profiles at the individual cell level and supporting the dissection of mechanisms of atherosclerosis, thrombosis, and tissue reaction in stroke. Indeed, single-cell experiments have revealed a more complex landscape of individual cell phenotypes, known and novel, than previously anticipated. In fact, the peculiarity of single-cell omics is its ability to capture heterogeneity and dynamic transformations in cell subpopulations. Cluster analysis has been used in the vast majority of the published data set, permitting the description of previously unknown cell types/states. Pseudo-time analysis has further contributed to the identification of relevant processes in atherosclerosis development. These analyses emphasize that there is some degree of transdifferentiation of a subset of cells into other phenotypes (such as smooth muscle cells into fibromyocytes), suggesting the presence of cell plasticity. The implementation of spatial gene expression information will expand the spectrum of possibilities for analysis, providing crucial new information on cell plasticity, spatio-temporal transcriptome profile, and intercellular communications in atherosclerotic lesions. In fact, while some degree of paracrine communication, i.e., through cytokines, is captured by conventional single-cell analysis, local ligand–receptor cell communications are largely lost.A crucial aspect of single-cell omics data is the integration and comparative analysis of a dataset originating from different research groups, patients, batches, platforms, or species. The final objective is to describe in a reproducible way common cell phenotypes detected across various scRNA-seq datasets by ensuring that clustering is robust and consistent and dimensionality reduced among studies. The generation of a consensus nomenclature of cell types will allow comparison of the results obtained from different groups and facilitate the evaluation of common and divergent properties of cell types across species and thus, translational studies from animals to humans.http://plaqview.uvadcos.io/, accessed 25 May 2022). Overall, the volume and complexity of data that will be generated will provide a challenge in the framework of big data that might be solved with artificial intelligence applications coupled with user friendly interfaces. Single-cell transcriptomics offers new opportunities for unraveling the intricacy of cerebrovascular illness, and analysis of cerebral artery disease, atherosclerotic tissue, and thrombi can be facilitated by single-cell technology. Recent studies have produced a reference map of immunological abnormalities associated with atherosclerotic plaques in human carotid arteries, which may help in development of immunomodulation therapies, such as immune checkpoint regulation. Targeting inflammation to halt or reverse cerebrovascular disease [Cost reductions for single-cell sequencing experiments will be helpful in opening the way to larger sample sizes and increased power in studies as well as in facilitating the association of other technologies with single-cell sequencing. A large majority of scRNA-seq datasets are available in public databases for independent analysis or analysis using online tools such as PlaqView  (http:// disease  has beenIndividualized treatment must be linked to underlying plaque heterogeneity and specific mechanisms of disease development. scRNA-seq could generate precise diagnostic tools and biomarkers for risk assessment and individualized therapy. Single-cell omics may facilitate understanding of the implications of cerebrovascular risk variables such as smoking, age, gender, or sex, and diabetes for disease onset and progression, as well as regression of plaques with lipid-lowering drugs, and the molecular and cellular events that influence cerebrovascular phenotypes and outcomes. Combining plaque single-cell omics data with CT/MRI/scan or doppler imaging data will maximize clinical value.Standardized cross-study methods can be used to assess atherosclerosis and thrombosis in humans to identify relevant preclinical platforms that can validate recognized targets and related pathways. These methods also can be used to develop new candidate therapies to propose for clinical studies of cerebrovascular diseases.


Stroke represents a major cause of mortality and long-term disability among adult populations, leaving a devastating socioeconomic impact globally. Clinical manifestation of stroke is characterized by great diversity, ranging from minor disability to considerable neurological impairment interfering with activities of daily living and even death. Prognostic ambiguity has stimulated the interest for implementing stroke recovery biomarkers, including those provided by structural neuroimaging techniques, i.e., diffusion tensor imaging (DTI) and tractography for the study of white matter (WM) integrity. Considering the necessity of prompt and accurate prognosis in stroke survivors along with the potential capacity of DTI as a relevant imaging biomarker, the purpose of our study was to review the pertinent literature published within the last decade regarding DTI as a prognostic tool for recovery in acute and hyperacute stroke. We conducted a thorough literature search in two databases (MEDLINE and Science Direct) in order to trace all relevant studies published between 1 January 2012 and 16 March 2022 using predefined terms as key words. Only full-text human studies published in the English language were included. Forty-four studies were identified and are included in this review. We present main findings and by describing several methodological issues, we highlight shortcomings and gaps in the current literature so that research priorities for future research can be outlined. Our review suggests that DTI can track longitudinal changes and identify prognostic correlates in acute and hyperacute stroke patients. Stroke represents a major cause of mortality and long-term disability among the adult population, leaving a devastating socioeconomic impact globally. More specifically, in Western countries, up to 4% of healthcare resources are expended on stroke. In the United States, the mean stroke-related cost per individual, including rehabilitation, is nearly USD 140,000 ,3. In thClinical manifestation of stroke is characterized by great diversity, ranging from minor disability to considerable neurological impairment interfering with activities of daily living (ADL) and even causing death. Prognostic ambiguity has stimulated an interest in implementing stroke recovery biomarkers. Ideally, prognostic markers hold high sensitivity and specificity, enabling appropriate management of healthcare resources and individualization of rehabilitation treatments. Efficiency of the selected biomarker is also based on its capacity to accurately depict underlying mechanisms of disease. Moreover, it should be non-invasive, readily accessible to patients and clinicians, easily interpreted by physicians, reproducible, and cost-effective ,5.Stroke diagnosis has been facilitated by the incorporation of advanced neuroimaging modalities in clinical practice, especially diffusion weighted imaging (DWI), a technique based on the motion of water molecules. Nevertheless, conventional magnetic resonance imaging (MRI) is incapable of accurately illustrating microstructural impairment on white matter (WM) tracts, rendering its role in predicting stroke outcome limited. Conversely, diffusion tensor imaging (DTI) is an extension of this modality for in vivo mapping of white matter (WM) directionality and organization, allowing the qualitative and quantitative evaluation of major WM tracts and their microstructural integrity . DTI is The majority of studies assessing DTI value as a stroke recovery biomarker have focused primarily on corticospinal tract (CST) integrity . CST is Research on DTI metrics as stroke outcome biomarker is not limited to the acute and subacute phases, as it is also implemented on chronic stroke patients ,21. DireConsidering the necessity of prompt and accurate prognosis in stroke survivors along with the potential capacity of DTI as a relevant imaging biomarker, the purpose of our study was to review the pertinent literature published within the last decade regarding DTI as a prognostic tool in acute and hyperacute stroke.The Preferred Reporting Items for Systematic Reviews (PRISMA checklist) was used to guide this study. Our study’s methods were designed a priori.A literature research of two databases (MEDLINE and Science Direct) was conducted by one investigator in order to trace all relevant studies published between 1 January 2012 and 16 March 2022, using either “diffusion tensor imaging” as a keyword or related term “DTI” as a search criterion. Moreover, the terms “stroke prognosis” or “stroke outcome” or “stroke recovery” were used as a second search criterion. The retrieved articles were also hand-searched for any further potential eligible articles. Any disagreement regarding screening or selection process was solved by a second investigator until consensus was reached. Only full-text original articles published in the English language were included. Secondary analyses, reviews, case reports, guidelines, meeting summaries, comments, unpublished abstracts or studies conducted in animals were excluded. There was no restriction on study design or sample characteristics.Data extraction was performed using a predefined data form created in Excel. We recorded author, year of publication, number and age of participants, study design with regards to DTI acquisition , type of stroke, time of DTI acquisition, main DTI parameters , anatomical regions examined, scales utilized, and main findings.No statistical analysis or meta-analysis was performed due to the high heterogeneity among studies. Thus, the data were only descriptively analyzed.Overall, 767 records were retrieved from the database search. Duplicates and irrelevant studies were excluded; hence, a total of 84 articles were selected. After screening the full text of the articles, 44 studies were eligible for inclusion.Forty-four publications fulfilled our inclusion criteria. They were classified into three groups, according to the type of stroke. The first group comprised 29 studies focusing on ischemic stroke , the secTwenty-three studies were cross-sectional, and 21 studies were longitudinal with regards to the DTI acquisition. Of note, the majority of ischemic stroke studies were longitudinal (18/29 studies), the majority of hemorrhagic stroke studies were cross-sectional (8/11 studies) while all of the studies including both cohorts were cross-sectional (4/4 studies).n = 3 . The practical demands of clinical imaging require relatively short acquisition times, transparent and reliable data interpretation processes, and ease of harmonizing protocols across clinical sites to conduct multicenter studies. There is a relative urgency to shift the emphasis from descriptive, qualitative studies to the development of protocols with practical clinical utility that can be applied in large sample sizes and provide accurate biomarkers for the prediction of stroke recovery. Finally, its potential as a putative monitoring biomarker for pharmacological and non-pharmacological interventions in stroke patients should be carefully evaluated to further identify both reorganization and degeneration processes.In conclusion, the results of the present review are indicative of the utility of DTI and specific parameters  for tracking longitudinal changes and identifying prognostic correlates in acute and hyperacute stroke patients. However, it is worth mentioning that additional efforts are needed to translate the insights gained from DTI studies in stroke patients into practical applications in clinical settings and routine clinical practice. Of note, ROC curves and various machine-learning frameworks in structural imaging in other neurological and psychiatric groups suggest that DTI may have a role in predicting patients’ outcomes with adequate accuracy at a single-patient level, thus providing valuable information for patients’ therapeutic management and both short- and long-term outcomes.


The process by which aging leads to increased risk for Alzheimer’s disease and dementia is not entirely understood, but one hypothesized contributor is the occurrence of low-grade inflammation in older age. In this study, we examined how peripheral levels of C-reactive protein (CRP), a measure of systemic inflammation, relate to brain structure and diffusion in a group of older adult men. After excluding twenty-seven participants for confounding medical conditions, we analyzed a final sample of 426 community-dwelling men from the Vietnam Era Twin Study of Aging, who were assessed at average age 68 for plasma levels of CRP and underwent structural and diffusion brain imaging. Linear mixed models adjusting for family relatedness, age, medical morbidity, and BMI examined associations of CRP with whole brain volume, whole gray and white matter volumes, global fractional anisotropy (FA), and global mean diffusivity (MD). Higher CRP was related to lower whole brain volume , including lower whole white matter volume  but not whole gray matter volume (p = .08). Higher CRP was related to lower global FA  but not global MD (p = .203). Regionally, a relationship of higher CRP to lower FA was found in the anterior thalamic radiation , which is implicated in a variety of higher order cognitive processes. These results suggest a link between peripheral inflammation and lower white matter integrity in older adult men, the implications of which for cognitive aging and dementia should be further explored.


Here, we aim to delineate mechanisms driving lung IRI and review both experimental and clinical data on the effects of aging in augmenting the consequences of IRI and PGD in lung transplantation.Transplant centers around the world have been using extended criteria donors to remedy the ongoing demand for lung transplantation. With a rapidly aging population, older donors are increasingly considered. Donor age, at the same time has been linked to higher rates of lung ischemia reperfusion injury (IRI). This process of acute, sterile inflammation occurring upon reperfusion is a key driver of primary graft dysfunction (PGD) leading to inferior short- and long-term survival. Understanding and improving the condition of older lungs is thus critical to optimize outcomes. Notably,  The world population is aging rapidly: predications suggest that more than 1.5 billion people above the age of 65 will inhabit our planet in 2050, accounting for 30 percent of the population , 5. UnitWhile lung donor selection criteria have historically been strict, thus limiting the donor pool, more recently, several transplant centers have used extended criteria donors including those from older donors with non-inferior outcomes –20. The 2/FiO2 ratio) the classification contains both a time- and severity component, facilitating mechanistic and clinical trials  including superoxide, hydroxyl radicals and hydrogen peroxide play a key role in the development of IRI –57. Alth+H+ exchange  located at the inner mitochondrial membrane . ActivatAs part of the events during reperfusion, calcium overload and the excessive ROS generation trigger both, apoptosis and necrosis causing a further release of ROS, proinflammatory cytokines and damage-associated molecular patterns (DAMPs) consisting of peptides, proteins and nucleotide fragments . DAMPs rSubsequently, the inflammatory cascade is initiated through, ROS- and DAMP-triggered activated resident lung macrophages as a key early source of multiple proinflammatory mediators that orchestrate lung IRI . This inAll these inflammatory pathways and mechanism contribute to an increased pulmonary vascular resistance and microvascular permeability , 98 leadAllogeneic lung transplantation represents the only curative approach for selected patients with end-stage lung disease. Advances in surgical techniques and immunosuppression therapy have improved graft survival rates to a median of 6.7 years compared to 4.7 year two decades ago . Of releWhile conclusive data in lung transplantation are currently lacking, data from other organ transplants support the concept that organ age augments damages subsequent to IRI: in hearts, this process has been driven, at least in part, through alterations in gene expression, signal transduction cascades, and mitochondrial dysfunction, resulting in an impaired intrinsic tolerance against damaging stress , 106. NoAging also impacts graft immunogenicity. In a broad clinical analysis of renal transplant recipients listed in the UNOS data base, we have been able to show that older grafts had higher rates of acute rejection within the first post-transplant year. Conversely, acute rejections were significantly lower in older recipients although they were more likely to receive an older organ . That grClinically, elevated perioperative levels of cell-free circulating plasma mitochondrial DNA (cf-mt-DNA) have been observed in lung transplant recipients with moderate or severe PGD . We haveEx vivo lung perfusion (EVLP) have facilitated the evaluation and reconditioning of marginal donor lungs including those from older lungs, thus augmenting the pool of prospective donor organs , the Organ Care System™ Lung , and the XPS™ XVIVO Perfusion AB system   .ex-vivo by the XVIVO Perfusion AB system. During this two-year period, 116 non-high-risk lungs were transplanted without EVLP and used as controls. Lungs in the EVLP group showed an improved PaO2/FiO2 ratio after EVLP resulting into a reduced PGD incidence  of the German Academic Exchange Service (MJR and AS), the Osaka Medical College Foundation (to TM), NIH grant R01AG064165 (to SGT) and a grant by the Pepper Foundation (to HZ).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


This study describes the clinical workflow for occlusal vertical dimension (OVD) increase in patients showing extensive tooth wear and mild teeth misalignment/crowding. A patient affected by dental erosion and occlusal abnormalities was treated to improve her situation. After ascertaining sound condyle and temporomandibular joint (TMJ) conditions, an OVD increase was sought to provide enough inter-occlusal space for the restorations. The use of TMJ three-dimensional imaging throughout the clinical procedures allowed to objectively track the condyle and disk position and confirm a steady condyle–glenoid cavity relationship before definitive restorations placement. Sectional clear aligner therapy prior to totally additive prosthetic rehabilitation allowed obtaining slight derotation and movements of anterior teeth, thus maximizing sound tissue preservation. Adhesively luted restorations were finally delivered on both anterior and posterior sectors. At the end of the treatment, the pre-operative TMJ balance appeared successfully preserved, and the patient was satisfied with the aesthetic and function achieved. Aesthetic concerns are nowadays part and parcel of every restorative and prosthetic rehabilitation: a satisfactory aesthetic result is as considerable as a proper functional outcome or a minimally invasive approach.Simple cases involving frontal teeth aesthetics in the presence of balanced occlusion and proper inter-occlusal space are part of routine clinical practice , 2. SituAdditive adhesive dentistry allows to get the most out of OVD increase. Without the need to seek mechanical retention with traditional preparation designs, in fact, sound cervical tooth structure can be saved , 2, 21, In the present clinical case, a safe clinical workflow optimized to aesthetically restore the smile of an asymptomatic patient affected by tooth wear and mild teeth misalignment is described. Adhesive and totally additive lithium disilicate indirect restorations were used in combination with SCAs, to preserve all residual sound tissues and to be as much conservative as possible. An MRI-based diagnostic approach was employed to evaluate the condyle–fossa relationship and keep it unchanged throughout the whole treatment.A 65-year-old woman with unremarkable medical history was referred with a chief complaint concerning the unsatisfactory aesthetic of her smile. The intra-oral examination revealed extensive tooth wear involving anterior and posterior teeth on both upper and lower arches, signs of cervical abrasion/erosion, slight misalignment in the upper anterior, and mild crowding in the lower-anterior areas Figures . As showT1-weighted sagittal sequences with a 2 mm slice thickness were obtained for each TMJ [The presence of physiological and healthy pre-operative TMJ conditions was objectified by MRI sequences taken in maximum intercuspidation using a 3 Tesla MRI system  with 6-channel dS Flex M surface coil. After placing a standard cheek/lip retractor, the patient was asked to perform a slight and slow mouth opening, starting from the maximum intercuspidation, up to a degree of tooth separation sufficient to assure enough inter-occlusal clearance for the restorative needs, trying to limit the OVD increase within the boundaries of pure condylar rotation. Any kind of operator-guided manipulation and/or bite registration medium in between the arches were avoided during this step of the procedure. Based on this, an OVD increase of 5 mm interincisally was supposed as appropriate. An indelible marker was used to mark the upper and lower canines and central incisors as references, so as to make the mandible positioning easily repeatable. After pushing two softened three-layer wax wafers (3 mm each) against the occlusal surfaces of left and right mandibular posterior teeth, the patient was asked to close while the operator guided the mandible in the new position, using the previously described marker signs on incisors and canines to prevent unsought excursive movements. The wax wafers were removed, cooled down, and placed back on the teeth to verify accuracy, stability, and fit.The next step was aimed at objectively verifying that the new mandibular position, in the empirically proposed OVD, was truly within the limit of condylar pure rotation. For this purpose, self-curing low shrinkage acrylic pattern resin  in dough stage was placed onto the upper anterior teeth before asking the patient to bite with the posterior wax wafers in place, thus obtaining an anterior jig. Following a facebow transfer, the jig, together with the wax wafers, served as inter-occlusal record to mount pre-operative casts on a semi-adjustable articulator . On these casts, temporary and additive posterior splints extending from the second lower premolar to the second lower molar were manufactured with orthodontic acrylic resin  through salt-and-pepper technique. The splints extended bilaterally from the second lower premolar to the second lower molar and were fixed intra-orally on the unprepared teeth using a glass-ionomer cement . The patient was, then, subjected to a second TMJ MRI evaluation while dressing the posterior fixed splints, using the same parameters and the same corrected cross-sections described for the pre-operative exam. Comparing the two MRIs, the condylar position appeared substantially unchanged in respect to the articular fossa , which cThe posterior splints allowed for an additional diagnostic step, planned to clinically verify in a totally reversible way the real patient adaptation. After having dressed the fixed splints for two months, the patient reported a reassuring subjective adaptation to the new mandibular position, in the absence of TMD and muscle tenderness, which represented the required validation for the new proposed mandible position.The prosthetic finalization was performed adopting the splint-induced occlusal position as inter-maxillary relationship and took place starting from the left posterior sector. Left-side fixed splint was removed, providing access for restoring both upper and lower left premolars and molars. Sound posterior teeth were left unprepared. Old fillings were removed and replaced with direct composite  build-ups, and the residual sound tissues were not prepared. Polyvinylsiloxane impressions of the upper and lower arches were taken, holding the right-side fixed splint in place. Lithium disilicate  indirect overlays were manufactured using standard laboratory procedures, etched with 5% hydrofluoric acid, silanized, and adhesively luted: Enabond (Micerium S.p.A.) and warm Enamel Plus BioFunction composite were, respectively, used as adhesive and cement. Once stable and definite occlusal contacts were established on the left sector, the right-side fixed splint was removed, and both upper and lower right premolars and molars were restored as just described for the left side. Since the patient wanted to keep her pre-existing implant-supported fixed dental prostheses, lithium disilicate overlays were adhesively luted even on the old implant-supported ceramic crowns (teeth 1.5 and 1.6). At this stage, in order to close the residual open bite between upper and lower frontal teeth following the OVD incrementation, SCA therapy in both arches  was performed. A digital impression was acquired by means of an intra-oral scanner  and sent to the manufacturer for the 3D alignment simulation through the ClinCheck software , is always necessary to re-establish good aesthetics in these patients.Paired with adhesively luted restorations, SCA therapy may represent an additional tool to approach these complex clinical cases in the least invasive way possible.


The dense neuropil of the central nervous system leaves only limited space for extracellular substances free. The advent of immunohistochemistry, soon followed by advanced diagnostic tools, enabled us to explore the biochemical heterogeneity and compartmentalization of the brain extracellular matrix in exploratory and clinical research alike. The composition of the extracellular matrix is critical to shape neuronal function; changes in its assembly trigger or reflect brain/spinal cord malfunction. In this study, we focus on extracellular matrix changes in neurodegenerative disorders. We summarize its phenotypic appearance and biochemical characteristics, as well as the major enzymes which regulate and remodel matrix establishment in disease. The specifically built basement membrane of the central nervous system, perineuronal nets and perisynaptic axonal coats can protect neurons from toxic agents, and biochemical analysis revealed how the individual glycosaminoglycan and proteoglycan components interact with these molecules. Depending on the site, type and progress of the disease, select matrix components can either proactively trigger the formation of disease-specific harmful products, or reactively accumulate, likely to reduce tissue breakdown and neuronal loss. We review the diagnostic use and the increasing importance of medical screening of extracellular matrix components, especially enzymes, which informs us about disease status and, better yet, allows us to forecast illness. The expression “extracellular matrix” refers to a network of proteins and complex sugar molecules built up around the cellular components of every tissue. Throughout the past decades, our concept of the role of the extracellular matrix has dramatically changed. It became clear that the extracellular matrix dynamically shapes parenchymal function instead of passively supporting the cells. The extracellular matrix tightly regulates the micromilieu around cells and influences signal transduction, thereby contributing to virtually every aspect of cell life, both in physiological and pathological circumstances, such as development, migration, inflammation, healing processes, fibrosis or tumorigenesis.We soon recognized that—similarly to any other organs—the extracellular matrix is an integral part of the brain nervous tissue as well, which vastly improved our understanding about synaptic structure and function, the locus of inter-neuronal connections. Whilst neurons and surrounding glial cells are mandatory cellular components of the synapse, previously referred to as the tripartite synapse paradigm , it has This review provides insight into the change of the brain extracellular matrix in disease. We first give a short overview about the major components, location/compartmentalization and phenotypic appearance of this specific extracellular substance and subsequently spotlight their role and typical changes in disease. We address the intriguing question of whether reassembly of the extracellular matrix is a cause or consequence of neuronal degeneration and subsequent malfunction in major neurodegenerative diseases, identify their triggers and present the diagnostic and therapeutic use of monitorable extracellular matrix-related changes in patients.When classifying the brain extracellular matrix, two major viewpoints emerge. Phenotypical characterization goes back to Camillo Golgi and Ramón y Cajal, who were first to recognize perineuronal nets over 100 years ago . FurtherIn general, extracellular matrix is built up by fibrous proteins (such as elastins or collagen) embedded in an amorphous gel formed by nonfibrous components ,16. ThesSchematic representations of molecules were redrawn based on previous publications ,43,44,45Most prominent proteoglycan representatives belong to the lectican family, which include aggrecan, versican, neurocan and brevican , the las(i) degraded matrix proteins activate previously bound tissue growth factors [(ii) secreted inactive precursors can be later activated by limited proteolysis  the local limiting of proteinase effect by binding them to membrane receptors (eg. MMPs) [Brain extracellular matrix is not permanent, its structure and composition undergoes major changes during pre- and postnatal development ,46,47, w factors  that stig. MMPs) ,80,81.Compared to MMPs, enzymes showing chondroitinase activity have minor significance in vivo, although they are present in humans and other higher organisms and contribute to extracellular matrix turnover ,83. Of nThe architectural, physical and functional differences of mesenchymal tissues rely on the vast diversity of components which accumulate in the abundant extracellular space. The advent of electronmicroscopical analysis of the nervous tissue suggested that the dense neuropil—i.e., the network of neurites, dendritic and glial cell processes and the microvasculature—leaves hardly any space for extracellular components free . Neverth+ material was described around the soma and dendrites of some neurons [As a first step, perineuronal nets were described by Camillo Golgi, but fell into oblivion for decades after Golgi lost his famous debate with Camón y Rajal over the basic concept of the nervous system and the cellular theory became widely accepted . Redisco neurons . Of note neurons . Our viePerineuronal nets are indeed distinct phenotypic matrix assemblies which enwrap the somatodendritic compartments of neurons, depending on neuronal type and brain area. Thus, perineuronal nets typically surround parvalbumin-containing, fast-firing interneurons ,93,94). In addition to perineuronal nets which surround the bulk neuronal compartment, tiny, isolated ring-like structures were identified later around synapses which were not embedded in perineuronal nets. These structures, termed axonal coats ,106, ens(i) if functional change of the synapse triggers transitional, short-term matrix re-assembly and its significance; (ii) if perineuronal/perisynaptic matrix assemblies go undetected presently due its unknown composition or limited amount/thickness; (iii) left–right brain asymmetries remained largely unaddressed; (iv) whilst major immunohistochemical and morphological characteristics of brain perineuronal matrix are actually identical in human and nonhuman vertebrates, does any specific composition or difference in relative content appear in the human brain?We may have the misleading impression that the exploration of extracellular matrix phenotype in the central nervous system has reached its zenith in the last decade. Albeit, while the major chemical and morphological characteristics of perineuronal matrix assemblies are known, several intriguing questions emerge including, but not limited to: Shortly after the first description of the perineuronal nets by Golgi, their possible contribution to neurodegenerative diseases was suggested by Nissl and Alzheimer, and later by Besta, Belloni and Donaggio. Besta argued that perineuronal nets were distinct anatomical structures, not part of the neuron itself, and likely produced by glia . BelloniHere we attempt to describe extracellular matrix-related brain pathology from two perspectives: based on their phenotype and biochemical assembly.During the past years, a series of observations led us to hypothesize that perineuronal nets protect cells from degeneration ,111. AmoThe role of perineuronal nets in disease development was implicated in other clinical aspects, too. The protective role of perineuronal nets was identified and a similar mechanism and cell-type specificity suggested in Parkinson’s disease, based on rodent models ,118,119.Symptoms of neurodegenerative diseases are typically associated with synaptic loss ,122. WhiThe nervous tissue benefits from a tight vascular barrier to protect its neurons which are most sensitive to agents that are relatively harmless to most other cell/tissue types. This blood–brain barrier consists of endothelium, pericytes, astrocytic endfeet, neuronal processes, microglia and the basement membrane, which is a specialized form of the extracellular matrix ,127. In It is unclear if changes in major extracellular matrix components of the basement membrane-like collagen IV, laminin, nidogen, or the heparin sulfate proteoglycans agrin and perlecan-are cause or consequence of neurodegeneration. Actually, basal lamina thickens also during normal aging, which is caused by altered expression levels of the different matrix components . In neur+ T cells [A further aspect of blood–brain barrier disruption is that it allows inflammatory cells to enter the brain tissue . Typical T cells , and acc T cells . In addition to the challenge posed by perineuronal and especially perisynaptic matrix assemblies that are presently undetectable immunohistochemically—due to their low amount or unknown composition—which could significantly improve our knowledge on disease detection and mechanism, several questions have remained largely unaddressed until now. Neuron-glia and neuron-microglia talk can be a critical trigger in matrix re-assembly, and the identification of specific inter- and intracellular downstream mechanisms can offer druggable targets in the future. It has remained unknown if the pre- or the postsynaptic neuron “cares” for its own safety and survival in neurodegenerative diseases, and whether it reflects or results in extracellular matrix rearrangements. Finally, despite our accumulating knowledge, we still know little about the dynamic composition change of perineuronal nets in disease, especially around the synapse.Hyaluronic acid concentration typically increases in neurodegenerative diseases. In multiple sclerosis, two different types of hyaluronic acid accumulate in demyelinated lesions. Acute lesions are characterized by a smaller molecular weight form, which is thought to further elicit inflammation. On the other hand, chronic lesions are enriched in high molecular weight hyaluronan in both human and mouse tissues. This latter form plays a role in the inhibition of oligodendrocyte precursor cell maturation which ultimately leads to impaired remyelinization . The foc+ astrocytes [(i) it disrupts the blood–brain barrier, decreases blood flow with consequent loss of glucose and oxygen-supply, but (ii) also affects the composition of perineuronal nets, which become less effective in terms of protecting neurons from ischemia. Abnormal production can also be the consequence of altered hyaluronan synthase pattern: hyperphosphorylated tau remodels the activity and compartmentalization of different hyaluronic acid synthase types, which finally leads to defunct perineuronal net formation [In Alzheimer’s disease, hyaluronic acid accumulates at the perimeter, but less so towards the center of plaques. Excess hyaluronan might be produced by reactive CD44trocytes ,155, whitrocytes ,157, theormation . Of noteormation ,160. Enrormation ,162. The expression of proteoglycans typically increases in neurodegenerative diseases. Despite previous progress, why these essential extracellular matrix components undergo remodeling in brain pathology remains ambiguous. Instead of being byproducts during the development or restorative processes of neurodegenerative diseases, they likely accumulate to either protect/maintain function or, conversely, to trigger tissue breakdown. In fact, separating protective and causative actions of proteoglycans brain pathology is challenging: different proteoglycans act at different loci and at different stages of disease development, which can reflect an independent, complementary, synergistic or antagonistic role/effect. Much of the future research in the field must aim to understand the functional importance of these time- and locus-specific changes.In multiple sclerosis, lecticans,  and dermatan sulfate-proteoglycans (DSPGs) are upregulated around the edges of acute lesions alongside astrogliosis. This pathological, likely reactive change is responsible for inhibition of axonal growth and remyelination. Towards the center of the lesion, lecticans and DSPGs were found to be phagocytosed by macrophages together with damaged myelin components. Increased CSPG concentration directly activates inflammatory cells and enhances their ability to migrate into the brain parenchyma in active lesions. In mice, a specific mechanism was unveiled when versican was shown to promote T helper 17 cytotoxic inflammation and to impede oligodendrocyte precursor cell remyelination . Thus, rA series of studies investigated proteoglycans in Alzheimer’s disease. Similarly to spinal cord lesions , CSPG acHigh and low molecular weight heparin promote or inhibit β-amyloid aggregation/structure stabilization, respectively . HSPGs aAgrin appears as critical proteoglycan in Parkinson’s disease as well: it enhances the insolubility of alpha-synuclein (at least in vitro) and co-localizes with it in Lewy bodies in the human tissue . Both LeGlucosaminoglycans (side chains of proteoglycans) are especially critical molecules in alpha-synuclein formation; their high concentration, long polymer isoforms, high protein:glycosaminoglycan ratio, charge density and select orientation of sulfate groups trigger and promote pathology. Different types of glucosaminoglycans lead to changes in fibril structure and cytotoxicity and influence the cellular uptake of alpha-synuclein . HoweverThe glycoprotein composition of the extracellular matrix reflects both phase/status and the type of neurodegeneration. Expression of different glycoproteins may change oppositely, likely reflecting their different roles or fate in the disease mechanism. In multiple sclerosis, tenascin concentrations decrease in the active lesions, while fibronectin and vitronectin are upregulated . Loss ofGlycoprotein shifts are also typical in Alzheimer’s disease. In corresponding animal models, tenascin-C deficiency reduces pro- but enhances anti-inflammatory activation, associated with a reduced cerebral β-amyloid load and higher levels of the postsynaptic density protein 95 (PSD-95) . TenasciAmongst other glycoproteins, thrombospondin-1 attracted attention because it supports synapse maintenance . ThromboIn addition to the reactive and proactive regulatory changes, proteoglycan expression in Alzheimer’s disease is also regulated at the epigenetic level: a large-scale sex-specific meta-analysis of DNA methylation uncovered 14 proteoglycans, including tenascins, associated with the Braak stage in females, but not in males . A plethora of enzymes dynamically shapes the composition of brain extracellular matrix. They either are activated because of excess matrix depletion or emerge as the trigger of pathology in neurodegeneration. Whilst many studies report about the effect of MMPs, the exact source remains unexplored. Thus, neurons, astrocytes, oligodendrocytes, microglias as well as pericytes can equally produce MMPs, but the trigger, timing and select cellular source of release in the different neurodegenerative diseases remain ambiguous. Obviously, human studies can offer only limited insight into molecular mechanisms and report rather about altered MMP levels in disease. Further, most of the in vitro and animal studies focus on the triggers or effects of increased or decreased MMPs expression/release and on its monitorable and measurable pathohistological or pathobiochemical effects. Without going into detailed cell biological discussion about cell-specific molecular regulation of MMPs’ production and release in the healthy brain, we focus on and summarize the relevant information which is related to neurodegenerative processes. In addition to diagnostically useful data about MMPs expression in human clinical studies, we also summarized known molecular mechanisms explored in neurodegenerative disease models in vitro and in vivo.Enzymes belonging to the TIMP family form non-covalent bonds with matrix metalloproteases to modulate their function . TIMP1 aIn multiple sclerosis, transient upregulation of extracellular matrix molecules reflects a physiological response. Whereas MMPs help in the remyelination processes, their dysregulation or dysfunction leads to insufficient extracellular matrix degradation/accumulation, with consequential demyelination and disruption of the blood–brain barrier. In this action, activation of astrocytes, microglia and macrophages are critical; additionally, they help inflammatory cell attraction and invasion. The complex action of the different MMPs are summarized in Changes in the level of MMPs carry diagnostic value: elevated MMP-9 levels were detected in the CSF of patients with relapsing-remitting multiple sclerosis and patients suffering from the primary progressive form of the disease showed similar results . MMP-9 cThe role of tissue enzymatic activity in Alzheimer’s disease emerged from the basic idea that the amyloid precursor protein is processed by α-secretases of the ADAM family in physiological conditions, which show structural and functional similarities with MMPs. In human tissue, MMP-9 expression increased already in moderate-stage Alzheimer’s disease, whereas MMP-2 expression increased only in late-stage Alzheimer’s disease . Other sThe causal role, its reactive expression change and the diagnostic use of MMPs are still ambiguous. In mice, intracerebroventricular injection of several β-amyloid fragments increase MMP-9, but not MMP-2 activity and protein expression in hippocampal neurons and glias . BecauseAlthough MMP-9 seems to play a casual role in disease development, its increased expression emerges as a reactive response. Cerebrovascular β-amyloid induces expression and activation of MMP-9 in cerebral vessels, and topical application of recombinant MMP-9 results in a time- and dose-dependent cerebral hemorrhage . FurtherMMP2 rs243865 and MMP3 rs3025058 promoter polymorphisms were shown to impact on the onset of Alzheimer’s disease [Diagnostically, the investigation of CSF and plasma samples attracted attention. CSF concentrations of MMP-9 decrease, concentrations of MMP-3 increase, whilst neither MMP-2 nor TIMPs show significant changes in Alzheimer’s disease patients . Other s disease . On the Obviously, valid data to understand neurodegenerative diseases shall derive from human studies. Brain areas typically affected in neurodegenerative diseases show different/higher phylogenetic maturation in humans, which poses a difficult challenge to find usable animal or in vitro models to model human pathology. Especially cell biological and regulatory processes call for experimental paradigms which cannot be carried out in humans. Thus, due to species differences, and despite thorough experiment planning and critical data analysis, results gained from animal models can be incomplete or misleading. In The mainstream of neurobiology studies in the field of neurodegenerative diseases concentrate upon intra- and intercellular alterations, which manifest in changes of the physiological and morphological character of the neuron. We believe that understanding the fine balance in brain extracellular matrix composition provides a major contribution to understanding these changes and offers valuable diagnostic and therapeutic tools for scientists and clinicians alike. The enzymatic machinery of brain extracellular matrix emerges as an especially promising druggable target, where enzyme manipulation can shape the destruction or protection of characteristic matrix assemblies. Their in vivo diagnostic imaging as well as their selective therapeutic access are the major challenges which scientists and physicians currently face.