Impact of CD362+ -selected mesenchymal stromal cells on left ventricular function in type 2 diabetic db/db mice

Mesenchymal stromal cells (MSCs) are an attractive cell-type for cell therapy, given their immunomodulatory, anti-fibrotic, and endothelial-protective features. The EU Committee for Advanced Therapies highlighted the need for strategies of selecting and purifying MSCs, whereupon the cell-surface sulfated proteoglycan CD362 has been identified as a new candidate. The aim of the present study was to compare the impact of intravenous (i.v.) application of wildtype (WT), CD362-- and CD362+-selected MSCs on left ventricular (LV) function in type 2 diabetic mice and to investigate the underlying mechanisms. For this purpose, 106 WT-MSC, CD362-, or CD362+ cells or PBS were i.v. injected into 20-week-old diabetic BKS. Cg-m+/+Leprdb/BomTac db/db mice. Non-diabetic db+/db mice injected with PBS served as controls. Mice were hemodynamically characterized and sacrificed four weeks after cell or PBS application. Spleens were collected for flow cytometry and LVs for immunohistochemistry, molecular biology, and imaging mass spectrometry. I.v. injection of WT-MSC and CD362- cells, but not of CD362+ cells improved LV function in db/db mice as indicated by an improvement of the contractility parameters dP/dtmax, dP/dtmin and/or Tau (p<0.05). With respect to immune modulation, only CD362+ cells raised the percentage of splenic regulatory T cells (Tregs) by 1.3-fold (p<0.001), whereas WT-MSC, CD362-, and CD362+ cells all decreased the percentage of splenic apoptotic Tregs in db/db mice (p<0.001 each versus db/db mice). Additionally, the number of cardiac CD3+ cells was 1.5-fold (p<0.05), 1.8-fold (p<0.01), and 1.7-fold (p<0.01) reduced in db/db mice injected with WT-MSC, CD362-, and CD362+, respectively, compared to db/db mice. Interestingly, application of WT-MSC, CD362-, and CD362+ cells led to a 1.6-fold (p<0.05), 1.8-fold (p<0.01), and 1.6-fold (p<0.05) decreased number of cardiac CD68+ cells. A hypothesis-free proteome analysis via imaging mass spectrometry further indicated a lower expression of the titin isoform N2B in db/db versus db+/db mice, which could be overcome after i.v. application of all investigated cells. In agreement with the LV function, total titin phosphorylation was 1.8-fold (p<0.01) downregulated in db/db versus db+/db mice, which was only induced after application of WT-MSC (1.6-fold; p<0.05) and CD362- (1.5-fold; p<0.05). In parallel, protein kinase G activity dropped by 1.5-fold (p<0.05) in db/db versus non-diabetic db+/db mice and was normalized to control levels in WT-MSC db/db and CD362- db/db mice. In conclusion, WT-MSC and CD362- cells, but not CD362+ cells improve LV function in diabetic db/db mice via titin regulation.Angesichts ihrer immunmodulatorischen, anti-fibrotischen und endothel-protektiven Eigenschaften, stellen mesenchymale stromale Zellen (MSZ) einen vielversprechenden Zelltyp für zell-therapeutische Interventionen dar. Das EU Komitee für zukunftsweisende Therapien unterstrich die Notwendigkeit von Strategien zur Selektion und Aufreinigung von MSZ, woraufhin das sulfatierte Proteoglykan CD362, welches auf der Zelloberfläche der MSZ zu finden ist, als neuer Kandidat identifiziert wurde. Ziel der vorliegenden Studie war es, den Einfluss von intravenös (i.v.) applizierten Wildtyp (WT), CD362-- und CD362+-selektieren MSZ auf die linksventrikuläre (LV) Funktion in Typ-2 diabetischen Mäusen zu vergleichen und die zugrunde liegenden Mechanismen zu untersuchen. Hierfür wurden 106 WT-MSZ, CD362-- bzw. CD362+-Zellen oder PBS i.v. in 20 Wochen alte diabetische BKS.Cg-m+/+Leprdb/BomTac db/db-Mäuse injiziert. Nicht diabetische db+/db-Mäuse, welchen PBS appliziert wurde, dienten als Kontrolle. Vier Wochen nach Applikation erfolgte die hämodynamische Charakterisierung und anschließende Euthanasie der Tiere. Für durchflusszytometrische Analysen wurde die Milz entnommen, sowie der LV für Immunhistologie, Molekularbiologie und bildgebende Massenspektrometrie. Die i.v. Injektion von WT-MSZ und CD362--Zellen, aber nicht von CD362+-Zellen, verbesserte die LV Funktion in den db/db-Mäuse, was sich in einer Verbesserung der Kontraktilitätsparameter dP/dtmax, dP/dtmin und/oder Tau zeigte (p <0.05). Bezüglich der Immunmodulation, erhöhte nur die Gabe der CD362+-Zellen den prozentualen Anteil an regulatorischen T Zellen (Tregs) in der Milz um das 1.3-fache (p<0.001), wohingegen WT-MSZ, CD362-- und CD362+-Zellen den Anteil an apoptotischen Tregs in der Milz verringerten (p<0.001 jeder Zelltyp gegenüber db/db-Mäuse). Verglichen mit den PBS-behandelten db/db-Mäusen war zusätzlich die Zahl an kardialen CD3+-Zellen um das 1.5-fache (p<0.05), 1.8-fache (p<0.01) und 1.7-fache (p<0.01) nach Injektion von WT-MSZ, CD362-- bzw. CD362+-Zellen reduziert. Interessanterweise führte die Applikation von WT-MSZ, CD362-- bzw. CD362+-Zellen auch zu einer 1.6-fach (p<0.05), 1.8-fach (p<0.01) bzw. 1.6-fach (p<0.05) verringerten Zahl an kardialen CD68+-Zellen. Die hypothesenfreie Proteomanalyse mittels bildgebender Massenspektrometrie zeigte darüber hinaus eine verringerte Expression der Titin-Isoform N2B in den db/db-Mäusen gegenüber den db+/db-Mäusen, welche durch i.v. Anwendung aller untersuchten Zellen verbessert werden konnte. In Übereinstimmung mit der LV Funktion war die totale Titin-Phosphylierung in den db/db-Mäusen, im Vergleich zu den db+/db-Mäusen, um das 1.8-fache (p<0.01) herunterreguliert, welche nur durch die Applikation von WT-MSZ (1.6-fach; p<0.05) und CD362--Zellen (1.5-fach; p<0.05) induziert wurde. Parallel dazu, fiel die Proteinkinase G-Aktivität um das 1.5-fache (p<0.05) in den db/db-Mäusen gegenüber den nicht-diabetische db+/db-Mäusen und war in WT-MSZ und CD362- db/db-Mäusen wieder normalisiert. Abschließend kann gesagt werden, dass WT-MSZ und CD362--Zellen, aber nicht die CD362+-Zellen, die LV Funktion durch eine Regulation von Titin verbessern

Deep Attention-based Representation Learning for Heart Sound Classification

Cardiovascular diseases are the leading cause of deaths and severely threaten human health in daily life. On the one hand, there have been dramatically increasing demands from both the clinical practice and the smart home application for monitoring the heart status of subjects suffering from chronic cardiovascular diseases. On the other hand, experienced physicians who can perform an efficient auscultation are still lacking in terms of number. Automatic heart sound classification leveraging the power of advanced signal processing and machine learning technologies has shown encouraging results. Nevertheless, human hand-crafted features are expensive and time-consuming. To this end, we propose a novel deep representation learning method with an attention mechanism for heart sound classification. In this paradigm, high-level representations are learnt automatically from the recorded heart sound data. Particularly, a global attention pooling layer improves the performance of the learnt representations by estimating the contribution of each unit in feature maps. The Heart Sounds Shenzhen (HSS) corpus (170 subjects involved) is used to validate the proposed method. Experimental results validate that, our approach can achieve an unweighted average recall of 51.2% for classifying three categories of heart sounds, i. e., normal, mild, and moderate/severe annotated by cardiologists with the help of Echocardiography

Deep wavelets for heart sound classification

A paper in ISPACS 201

Speckle-tracking echocardiography combined with imaging mass spectrometry assesses region-dependent alterations

Left ventricular (LV) contraction is characterized by shortening and thickening of longitudinal and circumferential fibres. To date, it is poorly understood how LV deformation is altered in the pathogenesis of streptozotocin (STZ)-induced type 1 diabetes mellitus-associated diabetic cardiomyopathy and how this is associated with changes in cardiac structural composition. To gain further insights in these LV alterations, eight-week-old C57BL6/j mice were intraperitoneally injected with 50 mg/kg body weight STZ during 5 consecutive days. Six, 9, and 12 weeks (w) post injections, echocardiographic analysis was performed using a Vevo 3100 device coupled to a 30-MHz linear-frequency transducer. Speckle-tracking echocardiography (STE) demonstrated impaired global longitudinal peak strain (GLS) in STZ versus control mice at all time points. 9w STZ animals displayed an impaired global circumferential peak strain (GCS) versus 6w and 12w STZ mice. They further exhibited decreased myocardial deformation behaviour of the anterior and posterior base versus controls, which was paralleled with an elevated collagen I/III protein ratio. Additionally, hypothesis-free proteome analysis by imaging mass spectrometry (IMS) identified regional- and time-dependent changes of proteins affecting sarcomere mechanics between STZ and control mice. In conclusion, STZ-induced diabetic cardiomyopathy changes global cardiac deformation associated with alterations in cardiac sarcomere proteins

Machine listening for heart status monitoring: introducing and benchmarking HSS — the Heart Sounds Shenzhen corpus

An article in IEEE Journal of Biomedical and Health Informatic

MALDI‐IMS as a Tool to Determine the Myocardial Response to Syndecan‐2‐Selected Mesenchymal Stromal Cell Application in an Experimental Model of Diabetic Cardiomyopathy

Purpose: Mesenchymal stromal cells (MSC) are an attractive tool for treatment of diabetic cardiomyopathy. Syndecan-2/CD362 has been identified as a functional marker for MSC isolation. Imaging mass spectrometry (IMS) allows for the characterization of therapeutic responses in the left ventricle. This study aims to investigate whether IMS can assess the therapeutic effect of CD362+-selected MSC on early onset experimental diabetic cardiomyopathy. Experimental Design: 1 × 106 wild type (WT), CD362−, or CD362+ MSC are intravenously injected into db/db mice. Four weeks later, mice are hemodynamically characterized and subsequently sacrificed for IMS combined with bottom-up mass spectrometry, and isoform and phosphorylation analyses of cardiac titin. Results: Overall alterations of the cardiac proteome signatures, especially titin, are observed in db/db compared to control mice. Interestingly, only CD362+ MSC can overcome the reduced titin intensity distribution and shifts the isoform ratio toward the more compliant N2BA form. In contrast, WT and CD362− MSCs improve all-titin phosphorylation and protein kinase G activity, which is reflected in an improvement in diastolic performance. Conclusions and Clinical Relevance: IMS enables the characterization of differences in titin intensity distribution following MSC application. However, further analysis of titin phosphorylation is needed to allow for the assessment of the therapeutic efficacy of MSC

A novel and high-efficient method for the preparation of heat-stable antifungal factor from Lysobacter enzymogenes by high-speed counter-current chromatography

Heat-stable antifungal factor (HSAF) produced by the biocontrol bacterium Lysobacter enzymogenes shows considerable antifungal activity and has broad application potential in the agricultural and medical fields. There is a great demand for pure HSAF compounds in academic or industrial studies. However, an efficient preparation method that produces a high yield and high purity of HSAF is lacking, limiting the development of HSAF as a new drug. In the present study, high-speed counter-current chromatography (HSCCC) combined with column chromatography was successfully developed for the separation and preparation of HSAF from the crude extract of L. enzymogenes OH11. The crude extract was obtained by macroporous resin adsorption and desorption, and the main impurities were partly removed by ultraviolet light (254 nm) and gel filtration (Sephadex LH-20). In the HSCCC procedure, the selected suitable two-phase solvent system (n-hexane/ethyl acetate/methanol/water = 3:5:4:5, v/v, the lower phase added with 0.1% TFA) with a flow rate of 2.0 mL/min and a sample loading size of 100 mg was optimized for the separation. As a result, a total of 42 mg HSAF with a purity of 97.6% and recovery of 91.7% was yielded in one separation. The structure elucidation based on HR-TOF-MS, 1H and 13C NMR, and antifungal activities revealed that the isolated compound was unambiguously identified as HSAF. These results are helpful for separating and producing HSAF at an industrial scale, and they further demonstrate that HSCCC is a useful tool for isolating bioactive constituents from beneficial microorganisms

Impact of Syndecan-2-Selected Mesenchymal Stromal Cells on the Early Onset of Diabetic Cardiomyopathy in Diabetic db/db Mice

Background: Mesenchymal stromal cells (MSCs) are an attractive cell type for cell therapy given their immunomodulatory, anti-fibrotic, and endothelial-protective features. The heparin sulfate proteoglycan, syndecan-2/CD362, has been identified as a functional marker for MSC isolation, allowing one to obtain a homogeneous cell product that meets regulatory requirements for clinical use. We previously assessed the impact of wild-type (WT), CD362(-), and CD362(+) MSCs on local changes in protein distribution in left ventricular (LV) tissue and on LV function in an experimental model of early-onset diabetic cardiomyopathy. The present study aimed to further explore their impact on mechanisms underlying diastolic dysfunction in this model. Materials: For this purpose, 1 x 10(6) WT, CD362(-), or CD362(+) MSCs were intravenously (i.v.) injected into 20-week-old diabetic BKS.Cg-m+/+Lepr(db)/BomTac, i.e., db/db mice. Control animals (db+/db) were injected with the equivalent volume of phosphate-buffered saline (PBS) alone. After 4 weeks, mice were sacrificed for further analysis. Results: Treatment with all three MSC populations had no impact on blood glucose levels in db/db mice. WT, CD362(-), and CD362(+) MSC application restored LV nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) levels in db/db mice, which correlated with a reduction in cardiomyocyte stiffness. Furthermore, all stromal cells were able to increase arteriole density in db/db mice. The effect of CD362(+) MSCs on NO and cGMP levels, cardiomyocyte stiffness, and arteriole density was less pronounced than in mice treated with WT or CD362(-) MSCs. Analysis of collagen I and III protein expression revealed that fibrosis had not yet developed at this stage of experimental diabetic cardiomyopathy. All MSCs reduced the number of cardiac CD3(+) and CD68(+) cells in db/db mice, whereas only splenocytes from CD362(-)- and CD362(+)-db/db mice exhibited a lower pro-fibrotic potential compared to splenocytes from db/db mice. Conclusion: CD362(+) MSC application decreased cardiomyocyte stiffness, increased myocardial NO and cGMP levels, and increased arteriole density, although to a lesser extent than WT and CD362(-) MSCs in an experimental model of early-onset diabetic cardiomyopathy without cardiac fibrosis. These findings suggest that the degree in improvement of cardiomyocyte stiffness following CD362(+) MSC application was insufficient to improve diastolic function

CX3CR1 knockout aggravates Coxsackievirus B3-induced myocarditis

Studies on inflammatory disorders elucidated the pivotal role of the CX3CL1/CX3CR1 axis with respect to the pathophysiology and diseases progression. Coxsackievirus B3 (CVB3)-induced myocarditis is associated with severe cardiac inflammation, which may progress to heart failure. We therefore investigated the influence of CX3CR1 ablation in the model of acute myocarditis, which was induced by inoculation with 5x105 plaque forming units of CVB3 (Nancy strain) in either CX3CR1-/- or C57BL6/j (WT) mice. Seven days after infection, myocardial inflammation, remodeling, and titin expression and phosphorylation were examined by immunohistochemistry, real-time PCR and Pro-Q diamond stain. Cardiac function was assessed by tip catheter. Compared to WT CVB3 mice, CX3CR1-/- CVB3 mice exhibited enhanced left ventricular expression of inflammatory cytokines and chemokines, which was associated with an increase of immune cell infiltration/presence. This shift towards a pro- inflammatory immune response further resulted in increased cardiac fibrosis and cardiomyocyte apoptosis, which was reflected by an impaired cardiac function in CX3CR1-/- CVB3 compared to WT CVB3 mice. These findings demonstrate a cardioprotective role of CX3CR1 in CVB3-infected mice and indicate the relevance of the CX3CL1/CX3CR1 system in CVB3-induced myocarditis