Deep-Learning based segmentation and quantification in experimental kidney histopathology

BACKGROUND: Nephropathologic analyses provide important outcomes-related data in experiments with the animal models that are essential for understanding kidney disease pathophysiology. Precision medicine increases the demand for quantitative, unbiased, reproducible, and efficient histopathologic analyses, which will require novel high-throughput tools. A deep learning technique, the convolutional neural network, is increasingly applied in pathology because of its high performance in tasks like histology segmentation. METHODS: We investigated use of a convolutional neural network architecture for accurate segmentation of periodic acid-Schiff-stained kidney tissue from healthy mice and five murine disease models and from other species used in preclinical research. We trained the convolutional neural network to segment six major renal structures: glomerular tuft, glomerulus including Bowman\u27s capsule, tubules, arteries, arterial lumina, and veins. To achieve high accuracy, we performed a large number of expert-based annotations, 72,722 in total. RESULTS: Multiclass segmentation performance was very high in all disease models. The convolutional neural network allowed high-throughput and large-scale, quantitative and comparative analyses of various models. In disease models, computational feature extraction revealed interstitial expansion, tubular dilation and atrophy, and glomerular size variability. Validation showed a high correlation of findings with current standard morphometric analysis. The convolutional neural network also showed high performance in other species used in research-including rats, pigs, bears, and marmosets-as well as in humans, providing a translational bridge between preclinical and clinical studies. CONCLUSIONS: We developed a deep learning algorithm for accurate multiclass segmentation of digital whole-slide images of periodic acid-Schiff-stained kidneys from various species and renal disease models. This enables reproducible quantitative histopathologic analyses in preclinical models that also might be applicable to clinical studies

The endothelial-enriched lncRNA LINC00607 mediates angiogenic function

Long non-coding RNAs (lncRNAs) can act as regulatory RNAs which, by altering the expression of target genes, impact on the cellular phenotype and cardiovascular disease development. Endothelial lncRNAs and their vascular functions are largely undefined. Deep RNA-Seq and FANTOM5 CAGE analysis revealed the lncRNA LINC00607 to be highly enriched in human endothelial cells. LINC00607 was induced in response to hypoxia, arteriosclerosis regression in non-human primates, post-atherosclerotic cultured endothelial cells from patients and also in response to propranolol used to induce regression of human arteriovenous malformations. siRNA knockdown or CRISPR/Cas9 knockout of LINC00607 attenuated VEGF-A-induced angiogenic sprouting. LINC00607 knockout in endothelial cells also integrated less into newly formed vascular networks in an in vivo assay in SCID mice. Overexpression of LINC00607 in CRISPR knockout cells restored normal endothelial function. RNA- and ATAC-Seq after LINC00607 knockout revealed changes in the transcription of endothelial gene sets linked to the endothelial phenotype and in chromatin accessibility around ERG-binding sites. Mechanistically, LINC00607 interacted with the SWI/SNF chromatin remodeling protein BRG1. CRISPR/Cas9-mediated knockout of BRG1 in HUVEC followed by CUT&RUN revealed that BRG1 is required to secure a stable chromatin state, mainly on ERG-binding sites. In conclusion, LINC00607 is an endothelial-enriched lncRNA that maintains ERG target gene transcription by interacting with the chromatin remodeler BRG1 to ultimately mediate angiogenesis

Blutdruckmessungen als Gesundheitsmonitoring beim Weißbüschelaffen (Callithrix jacchus)

Einleitung: Weißbüschelaffen (Callithrix jacchus) sind im Laufe der letzten Jahre vermehrt in den Fokus der Verhaltens-, Alters- und Stoffwechselforschung gerückt. Blutdruckmessungen könnten für die Gesundheitsüberwachung dieser Tiere einen wertvollen Beitrag leisten. Bisher erhobene Daten zeigen jedoch unterschiedliche oder unvollständige Messmethoden und vernachlässigen wichtige physiologische Faktoren wie Alter, Gewicht und Geschlecht der Tiere. Dies erschwert die Reproduzierbarkeit der Werte sowie deren Vergleich untereinander. Ziele der Untersuchungen: Ziel dieser Arbeit war es daher, ein praxistaugliches Protokoll für Blutdruckmessungen beim Weißbüschelaffen zu erstellen. Darauf aufbauend sollten die Tiere der Primatenkolonie des Veterinär-Physiologisch-Chemischen Institutes Leipzig über mehrere Monate untersucht werden, um physiologische Blutdruckwerte unter standardisierten Bedingungen zu erhalten. Tiere, Material und Methoden: Für ein Messprotokoll wurde in einem Vorversuch bei 10 Tieren der Einfluss der Messlokalisation (Gliedmaße oder Schwanz) und bei 6 Tieren der Einfluss der Tageszeit per High-Definition Oszillometrie (HDO)- Blutdruckmessungen (über drei Tage) untersucht. Mit diesen Erkenntnissen wurden dann alle Tiere der Kolonie (n= 56, 25 männlich, 31 weiblich; Altersspanne: 14 - 209 Monate, Gewichte 313 – 499 g) überprüft (Gesamtdauer 30 Monate). Alters- und gewichtsabhängige Blutdruckveränderungen sowie der Unterschied zwischen den Geschlechtern wurde zusätzlich untersucht (Korrelations- und Regressionsanalysen, t-Tests). Bei vier Tieren wurden Blutdruckabweichungen festgestellt, deren weiterführende Analyse in Form von Blut-, Urin- oder Ultraschalluntersuchungen erfolgte. Ergebnisse: Das etablierte Messprotokoll unterschied sich zu denen bei anderen Tierarten, v.a. im Hinblick auf die Messlokalisation (Messungen an den Hintergliedmaßen lieferten präzisere Ergebnisse als am Schwanz). Während 3 - 7 Messungen bei Weißbüschelaffen möglich sind, hatte die Tageszeit keinen Einfluss auf die Werte. Darauf aufbauend konnten Grenzen für physiologische und pathologische Blutdruckwerte beim Weißbüschelaffen festgelegt werden. Sowohl Alter als auch Gewicht beeinflussten die Blutdruckwerte. Das Blutdruckmuster zeigte dabei einen Anstieg der Werte sowohl mit steigendem Alter als auch mit höherem Gewicht an. Das Geschlecht hatte keinen Einfluss auf die Blutdruckwerte, beeinflusste aber ebenso wie das Alter die Messdauer. Messungen an weiblichen und/ oder älteren Tieren konnten schneller durchgeführt werden als bei männlichen und/ oder jungen Individuen. Durch die Früherkennung von Blutdruckabweichungen konnten bei den beschriebenen Patienten die zugrundeliegenden Krankheiten näher untersucht und behandelt werden. Dabei zeigten zwei der vier Tiere progressiv verlaufende Nierenerkrankungen bei gleichzeitigem Vorliegen von Begleiterscheinungen wie Anämie oder Demineralisierung der Knochen. Eine Patientin wies Stoffwechselentgleisungen in Form von erhöhten Triglycerid- und Insulinwerten sowie Übergewicht auf. Bei der vierten Patientin wurden Herzveränderungen in Form einer Endokardiose und Hypertrophie festgestellt. Schlussfolgerungen: Im Rahmen dieser Arbeit wurden sowohl ein Messprotokoll für die nicht-invasive Blutdruckmessung beim Weißbüschelaffen als auch physiologische Blutdruckwerte unter Berücksichtigung von Alter, Gewicht und Geschlecht etabliert. Die Tatsache, dass weibliche und alte Tiere besser zu messen waren, könnte zukünftige Studien in puncto Blutdruckauswertung und -beurteilung erleichtern. Der Nachweis der den veränderten Blutdruckwerten zugrundeliegenden Krankheiten bestätigt die klinische Relevanz von Blutdruckmessungen. Die regelmäßige Akquirierung dieser Daten dient so nicht nur der generellen Gesundheitsüberwachung, sondern bietet sich besonders auch über lange Zeiträume zur Verlaufskontrolle an.Introduction: The common marmoset (Callithrix jacchus) recently moved into focus for behavioral, age and metabolic studies. Blood pressure measurements could be a positive contribution to the health maintenance of these animals. However, the data that has been gathered so far show differing measurement techniques and give little or no information about the protocols applied. In addition, important factors such as age, weight and sex, have not yet been taken into account. This complicates reproducibility and comparison of values. Aim: Aim of this study was therefore to establish a standard protocol for blood pressure measurements in the common marmoset. Based on this, animals from the primate colony of the Institute of Veterinary Physiological Chemistry Leipzig were to be monitored over several months and their data analyzed to gather physiological measurement values under standardized conditions. Animals, materials and methods: For a measurement protocol the influence of measurement localization (thigh or tail) was reviewed in ten animals using blood pressure measurements via High-Definition Oszillometry (HDO). Following this, the influence of daytime was evaluated in six animals over the course of three days. With this knowledge, all animals of the colony (n= 56, 25 males, 31 females; age range: 14 - 209 months, body weight 313 – 499 g) were assessed. Age- and weight-dependent blood pressure changes as well as differences between the sexes were examined (correlation and regression analyses, t-tests). In four animals with conspicuous blood pressure values analyses of blood and urine as well as ultrasonographic examinations were further performed. Results: The established measurement protocol differed from those used in other animal species in terms of measurement localization (thigh measurements resulted in more precise values than measurements at the tail). While 3-7 single measurements were possible in the common marmoset, day time did not influence values. Based on this, threshold values for physiological and pathological blood pressure data could be determined. Age and weight both influenced blood pressure in common marmosets. The blood pressure pattern showed rising values with both increasing age and weight. Sex had no influence on blood pressure values, but affected together with age measurement time. Measurements in old and/ or female individuals could be performed faster than in male and/ or young individuals. Due to the early detection of blood pressure abnormalities, the causative diseases could be analyzed and treated in the described patients. Two of the four patients showed progressing renal diseases simultaneously with co-morbidities like anemia or bone demineralization. One patient displayed metabolic disease in terms of increased triglyceride and insulin values as well as excess weight. The fourth patient was identified as having heart changes in the form of endocardiosis and cardiac hypertrophy. Conclusion: This work outlines the establishment of a measurement protocol for non-invasive blood pressure measurement in the common marmoset as well as physiological values considering age, weight and sex. The fact that female and old animals were easier to measure could facilitate blood pressure evaluation and interpretation in future studies. The identification of the diseases, responsible for the altered blood pressure values confirmed the clinical relevance of blood pressure assessment. The regular acquisition of such data is therefore not only useful in the health monitoring of all individuals, but especially supports follow-up examinations over longer periods of time

Blutdruckmessungen als Gesundheitsmonitoring beim Weißbüschelaffen (Callithrix jacchus)

Einleitung: Weißbüschelaffen (Callithrix jacchus) sind im Laufe der letzten Jahre vermehrt in den Fokus der Verhaltens-, Alters- und Stoffwechselforschung gerückt. Blutdruckmessungen könnten für die Gesundheitsüberwachung dieser Tiere einen wertvollen Beitrag leisten. Bisher erhobene Daten zeigen jedoch unterschiedliche oder unvollständige Messmethoden und vernachlässigen wichtige physiologische Faktoren wie Alter, Gewicht und Geschlecht der Tiere. Dies erschwert die Reproduzierbarkeit der Werte sowie deren Vergleich untereinander. Ziele der Untersuchungen: Ziel dieser Arbeit war es daher, ein praxistaugliches Protokoll für Blutdruckmessungen beim Weißbüschelaffen zu erstellen. Darauf aufbauend sollten die Tiere der Primatenkolonie des Veterinär-Physiologisch-Chemischen Institutes Leipzig über mehrere Monate untersucht werden, um physiologische Blutdruckwerte unter standardisierten Bedingungen zu erhalten. Tiere, Material und Methoden: Für ein Messprotokoll wurde in einem Vorversuch bei 10 Tieren der Einfluss der Messlokalisation (Gliedmaße oder Schwanz) und bei 6 Tieren der Einfluss der Tageszeit per High-Definition Oszillometrie (HDO)- Blutdruckmessungen (über drei Tage) untersucht. Mit diesen Erkenntnissen wurden dann alle Tiere der Kolonie (n= 56, 25 männlich, 31 weiblich; Altersspanne: 14 - 209 Monate, Gewichte 313 – 499 g) überprüft (Gesamtdauer 30 Monate). Alters- und gewichtsabhängige Blutdruckveränderungen sowie der Unterschied zwischen den Geschlechtern wurde zusätzlich untersucht (Korrelations- und Regressionsanalysen, t-Tests). Bei vier Tieren wurden Blutdruckabweichungen festgestellt, deren weiterführende Analyse in Form von Blut-, Urin- oder Ultraschalluntersuchungen erfolgte. Ergebnisse: Das etablierte Messprotokoll unterschied sich zu denen bei anderen Tierarten, v.a. im Hinblick auf die Messlokalisation (Messungen an den Hintergliedmaßen lieferten präzisere Ergebnisse als am Schwanz). Während 3 - 7 Messungen bei Weißbüschelaffen möglich sind, hatte die Tageszeit keinen Einfluss auf die Werte. Darauf aufbauend konnten Grenzen für physiologische und pathologische Blutdruckwerte beim Weißbüschelaffen festgelegt werden. Sowohl Alter als auch Gewicht beeinflussten die Blutdruckwerte. Das Blutdruckmuster zeigte dabei einen Anstieg der Werte sowohl mit steigendem Alter als auch mit höherem Gewicht an. Das Geschlecht hatte keinen Einfluss auf die Blutdruckwerte, beeinflusste aber ebenso wie das Alter die Messdauer. Messungen an weiblichen und/ oder älteren Tieren konnten schneller durchgeführt werden als bei männlichen und/ oder jungen Individuen. Durch die Früherkennung von Blutdruckabweichungen konnten bei den beschriebenen Patienten die zugrundeliegenden Krankheiten näher untersucht und behandelt werden. Dabei zeigten zwei der vier Tiere progressiv verlaufende Nierenerkrankungen bei gleichzeitigem Vorliegen von Begleiterscheinungen wie Anämie oder Demineralisierung der Knochen. Eine Patientin wies Stoffwechselentgleisungen in Form von erhöhten Triglycerid- und Insulinwerten sowie Übergewicht auf. Bei der vierten Patientin wurden Herzveränderungen in Form einer Endokardiose und Hypertrophie festgestellt. Schlussfolgerungen: Im Rahmen dieser Arbeit wurden sowohl ein Messprotokoll für die nicht-invasive Blutdruckmessung beim Weißbüschelaffen als auch physiologische Blutdruckwerte unter Berücksichtigung von Alter, Gewicht und Geschlecht etabliert. Die Tatsache, dass weibliche und alte Tiere besser zu messen waren, könnte zukünftige Studien in puncto Blutdruckauswertung und -beurteilung erleichtern. Der Nachweis der den veränderten Blutdruckwerten zugrundeliegenden Krankheiten bestätigt die klinische Relevanz von Blutdruckmessungen. Die regelmäßige Akquirierung dieser Daten dient so nicht nur der generellen Gesundheitsüberwachung, sondern bietet sich besonders auch über lange Zeiträume zur Verlaufskontrolle an.Introduction: The common marmoset (Callithrix jacchus) recently moved into focus for behavioral, age and metabolic studies. Blood pressure measurements could be a positive contribution to the health maintenance of these animals. However, the data that has been gathered so far show differing measurement techniques and give little or no information about the protocols applied. In addition, important factors such as age, weight and sex, have not yet been taken into account. This complicates reproducibility and comparison of values. Aim: Aim of this study was therefore to establish a standard protocol for blood pressure measurements in the common marmoset. Based on this, animals from the primate colony of the Institute of Veterinary Physiological Chemistry Leipzig were to be monitored over several months and their data analyzed to gather physiological measurement values under standardized conditions. Animals, materials and methods: For a measurement protocol the influence of measurement localization (thigh or tail) was reviewed in ten animals using blood pressure measurements via High-Definition Oszillometry (HDO). Following this, the influence of daytime was evaluated in six animals over the course of three days. With this knowledge, all animals of the colony (n= 56, 25 males, 31 females; age range: 14 - 209 months, body weight 313 – 499 g) were assessed. Age- and weight-dependent blood pressure changes as well as differences between the sexes were examined (correlation and regression analyses, t-tests). In four animals with conspicuous blood pressure values analyses of blood and urine as well as ultrasonographic examinations were further performed. Results: The established measurement protocol differed from those used in other animal species in terms of measurement localization (thigh measurements resulted in more precise values than measurements at the tail). While 3-7 single measurements were possible in the common marmoset, day time did not influence values. Based on this, threshold values for physiological and pathological blood pressure data could be determined. Age and weight both influenced blood pressure in common marmosets. The blood pressure pattern showed rising values with both increasing age and weight. Sex had no influence on blood pressure values, but affected together with age measurement time. Measurements in old and/ or female individuals could be performed faster than in male and/ or young individuals. Due to the early detection of blood pressure abnormalities, the causative diseases could be analyzed and treated in the described patients. Two of the four patients showed progressing renal diseases simultaneously with co-morbidities like anemia or bone demineralization. One patient displayed metabolic disease in terms of increased triglyceride and insulin values as well as excess weight. The fourth patient was identified as having heart changes in the form of endocardiosis and cardiac hypertrophy. Conclusion: This work outlines the establishment of a measurement protocol for non-invasive blood pressure measurement in the common marmoset as well as physiological values considering age, weight and sex. The fact that female and old animals were easier to measure could facilitate blood pressure evaluation and interpretation in future studies. The identification of the diseases, responsible for the altered blood pressure values confirmed the clinical relevance of blood pressure assessment. The regular acquisition of such data is therefore not only useful in the health monitoring of all individuals, but especially supports follow-up examinations over longer periods of time

“Empowering” Cardiac Cells via Stem Cell Derived Mitochondrial Transplantation- Does Age Matter?

With cardiovascular diseases affecting millions of patients, new treatment strategies are urgently needed. The use of stem cell based approaches has been investigated during the last decades and promising effects have been achieved. However, the beneficial effect of stem cells has been found to being partly due to paracrine functions by alterations of their microenvironment and so an interesting field of research, the “stem- less” approaches has emerged over the last years using or altering the microenvironment, for example, via deletion of senescent cells, application of micro RNAs or by modifying the cellular energy metabolism via targeting mitochondria. Using autologous muscle-derived mitochondria for transplantations into the affected tissues has resulted in promising reports of improvements of cardiac functions in vitro and in vivo. However, since the targeted treatment group represents mainly elderly or otherwise sick patients, it is unclear whether and to what extent autologous mitochondria would exert their beneficial effects in these cases. Stem cells might represent better sources for mitochondria and could enhance the effect of mitochondrial transplantations. Therefore in this review we aim to provide an overview on aging effects of stem cells and mitochondria which might be important for mitochondrial transplantation and to give an overview on the current state in this field together with considerations worthwhile for further investigations

Dysregulation of Krüppel-like Factor 2 and Myocyte Enhancer Factor 2D Drive Cardiac Microvascular Inflammation and Dysfunction in Diabetes

Cardiovascular complications are the main cause of morbidity and mortality from diabetes. Herein, vascular inflammation is a major pathological manifestation. We previously characterized the cardiac microvascular inflammatory phenotype in diabetic patients and highlighted micro-RNA 92a (miR-92a) as a driver of endothelial dysfunction. In this article, we further dissect the molecular underlying of these findings by addressing anti-inflammatory Krüppel-like factors 2 and 4 (KLF2 and KLF4). We show that KLF2 dysregulation in diabetes correlates with greater monocyte adhesion as well as migratory defects in cardiac microvascular endothelial cells. We also describe, for the first time, a role for myocyte enhancer factor 2D (MEF2D) in cardiac microvascular dysfunction in diabetes. We show that both KLFs 2 and 4, as well as MEF2D, are dysregulated in human and porcine models of diabetes. Furthermore, we prove a direct interaction between miR-92a and all three targets. Altogether, our data strongly qualify miR-92a as a potential therapeutic target for diabetes-associated cardiovascular disease

Dysregulation of Krüppel-like Factor 2 and Myocyte Enhancer Factor 2D Drive Cardiac Microvascular Inflammation and Dysfunction in Diabetes

Cardiovascular complications are the main cause of morbidity and mortality from diabetes. Herein, vascular inflammation is a major pathological manifestation. We previously characterized the cardiac microvascular inflammatory phenotype in diabetic patients and highlighted micro-RNA 92a (miR-92a) as a driver of endothelial dysfunction. In this article, we further dissect the molecular underlying of these findings by addressing anti-inflammatory Krüppel-like factors 2 and 4 (KLF2 and KLF4). We show that KLF2 dysregulation in diabetes correlates with greater monocyte adhesion as well as migratory defects in cardiac microvascular endothelial cells. We also describe, for the first time, a role for myocyte enhancer factor 2D (MEF2D) in cardiac microvascular dysfunction in diabetes. We show that both KLFs 2 and 4, as well as MEF2D, are dysregulated in human and porcine models of diabetes. Furthermore, we prove a direct interaction between miR-92a and all three targets. Altogether, our data strongly qualify miR-92a as a potential therapeutic target for diabetes-associated cardiovascular disease

Evaluation of MR-safe bioptomes for MR-guided endomyocardial biopsy in minipigs: a potential radiation-free clinical approach

Abstract Background Diagnostic accuracy of endomyocardial biopsy could improve if clinically safe magnetic resonance (MR)-compatible bioptomes were available. We explored two novel MR-compatible cardiac bioptomes for performance, safety, and clinical viability, employing in vivo minipig trials and phase-contrast synchrotron radiation computed microtomography (SRµCT). Methods Analysis of ex vivo obtained pig endomyocardial biopsies was performed using phase-contrast SRµCT and conventional two-dimensional histology. The technical performance was evaluated by measuring volume, inner and outer integrities, compression, and histological diagnostic value in 3 sets (6 per set) of biopsies for each experimental bioptome. The bioptomes were tested in vivo in 3 healthy minipigs per bioptome. The clinical feasibility was evaluated by procedural and cutting success as well as histological diagnostic value. Results The bioptome with the ‘grind-grind’ design achieved similar values to control in compression (p = 0.822), inner (p = 0.628), and outer (p = 0.507), integrities ex vivo. It showed a better performance in the in vivo real-time MRI setting demonstrating a higher cutting success (91.7%) than the ‘grind-anvil’ (86.2%) design. In both ex vivo and in vivo evaluations, the ‘grind-grind’ design displayed sufficient diagnostic value (83% and 95%). The ‘grind-anvil’ design showed adequate diagnostic value both ex vivo and in vivo (78% and 87.5%) but was not comparable to control according to the three-dimensional (3D) analysis. Conclusion A novel MR-compatible bioptome was identified as plausible in a clinical setting. Additionally, SRµCT and subsequent 3D structural analysis could be valuable in the label-free investigation of myocardial tissue at a micrometer level. Relevance statement Implementation of MR-guided biopsy can improve animal studies on structural myocardial changes at any point in an experimental setup. With further improvements in guiding catheters, MR-guided biopsy, using the new bioptome, has a potential to increase quality and diagnostic accuracy in patients both with structural and inflammatory cardiomyopathies. Key points • Novel MR-compatible bioptomes show promise for a clinical application. • SRµCT enabled detailed analysis of endomyocardial biopsies. • The bioptomes showed adequate in vivo performance without major complications. Graphical Abstrac

The endothelial-enriched lncRNA LINC00607 mediates angiogenic function

Long non-coding RNAs (lncRNAs) can act as regulatory RNAs which, by altering the expression of target genes, impact on the cellular phenotype and cardiovascular disease development. Endothelial lncRNAs and their vascular functions are largely undefined. Deep RNA-Seq and FANTOM5 CAGE analysis revealed the lncRNA LINC00607 to be highly enriched in human endothelial cells. LINC00607 was induced in response to hypoxia, arteriosclerosis regression in non-human primates, post-atherosclerotic cultured endothelial cells from patients and also in response to propranolol used to induce regression of human arteriovenous malformations. siRNA knockdown or CRISPR/Cas9 knockout of LINC00607 attenuated VEGF-A-induced angiogenic sprouting. LINC00607 knockout in endothelial cells also integrated less into newly formed vascular networks in an in vivo assay in SCID mice. Overexpression of LINC00607 in CRISPR knockout cells restored normal endothelial function. RNA- and ATAC-Seq after LINC00607 knockout revealed changes in the transcription of endothelial gene sets linked to the endothelial phenotype and in chromatin accessibility around ERG-binding sites. Mechanistically, LINC00607 interacted with the SWI/SNF chromatin remodeling protein BRG1. CRISPR/Cas9-mediated knockout of BRG1 in HUVEC followed by CUT&RUN revealed that BRG1 is required to secure a stable chromatin state, mainly on ERG-binding sites. In conclusion, LINC00607 is an endothelial-enriched lncRNA that maintains ERG target gene transcription by interacting with the chromatin remodeler BRG1 to ultimately mediate angiogenesis