Impact of body mass index on worsening of diastolic function and impairment of left atrial strain in the general female urban population: a subanalysis of the Berlin female risk evaluation echocardiography follow-up study

Background: The association of body mass index (BMI) with diastolic dysfunction (DD) is well described in the literature. However, there is conflicting evidence and long-term follow-up data regarding effects of BMI on preclinical DD and left atrial (LA) function are scarce, highlighting the importance of early detection tools, such as myocardial strain. Purpose: The aim of our study was to prospectively analyze the impact of clinical and demographic parameters, especially of BMI, on worsening of diastolic function and left atrial strain (LAS) in an urban population of women with a low prevalence of cardiovascular risk factors. Methods and Results: An extensive clinical and echocardiographic assessment comprising the analysis of phasic LAS using two-dimensional speckle-tracking echocardiography (2D STE) was performed in 258 participants of the Berlin Female Risk Evaluation (BEFRI) trial between October 2019 and December 2020 after a mean follow-up period of 6.8 years. We compared clinical and echocardiographic parameters stratifying women by BMI < or ≥25 kg/m2, and we analyzed the impact of demographic characteristics on the worsening of DD and LA mechanics in the longer-term follow-up using univariate and multivariate regression analyses. 248 women were suitable for echocardiographic analysis of LAS using 2D STE. After a mean follow-up time of 6.8 years, LA reservoir strain (LASr) and LA conduit strain (LAScd) were significantly reduced in participants with a BMI ≥25 kg/m2 compared with women with a BMI <25 kg/m2 at baseline (30 ± 8% vs. 38 ± 9%, p < 0.0001; −14 ± 7% vs. −22 ± 8%, p < 0.0001). 28% of the overweighted women presented a deterioration of diastolic function at the time of follow-up in contrast with only 7% of the group with a BMI <25 kg/m2 (p < 0.0001). BMI remained significantly associated with LAS reductions after adjustment for other risk factors in multivariate regression analyses. Conclusion: Overweight and obesity are related to impaired LAS and to a worsening of diastolic function after a long-term follow-up in a cohort of randomly selected women

Efficient Killing of Murine Pluripotent Stem Cells by Natural Killer (NK) Cells Requires Activation by Cytokines and Partly Depends on the Activating NK Receptor NKG2D

Natural killer (NK) cells play an important role as cytotoxic effector cells, which scan the organism for infected or tumorigenic cells. Conflicting data have been published whether NK cells can also kill allogeneic or even autologous pluripotent stem cells (PSCs) and which receptors are involved. A clarification of this question is relevant since an activity of NK cells against PSCs could reduce the risk of teratoma growth after transplantation of PSC-derived grafts. Therefore, the hypothesis has been tested that the activity of NK cells against PSCs depends on cytokine activation and specifically on the activating NK receptor NKG2D. It is shown that a subcutaneous injection of autologous iPSCs failed to activate NK cells against these iPSCs and can give rise to teratomas. In agreement with this result, several PSC lines, including two iPSC, two embryonic stem cell (ESC), and two so-called multipotent adult germline stem cell (maGSC) lines, were largely resistant against resting NK cells although differences in killing were found at low level. All PSC lines were killed by interleukin (IL)-2-activated NK cells, and maGSCs were better killed than the other PSC types. The PSCs expressed ligands of the activating NK receptor NKG2D and NKG2D-deficient NK cells from Klrk1−/− mice were impaired in their cytotoxic activity against PSCs. The low-cytotoxic activity of resting NK cells was almost completely dependent on NKG2D. The cytotoxic activity of IL-2-activated NKG2D-deficient NK cells against PSCs was reduced, indicating that also other activating receptors on cytokine-activated NK cells must be engaged by ligands on PSCs. Thus, NKG2D is an important activating receptor involved in killing of murine PSCs. However, NK cells need to be activated by cytokines before they efficiently target PSCs and then also other NK receptors become relevant. These features of NK cells might be relevant for transplantation of PSC-derived grafts since NK cells have the capability to kill undifferentiated cells, which might be present in grafts in trace amount

Impact of body mass index on worsening of diastolic function and impairment of left atrial strain in the general female urban population: a subanalysis of the Berlin female risk evaluation echocardiography follow-up study

BackgroundThe association of body mass index (BMI) with diastolic dysfunction (DD) is well described in the literature. However, there is conflicting evidence and long-term follow-up data regarding effects of BMI on preclinical DD and left atrial (LA) function are scarce, highlighting the importance of early detection tools, such as myocardial strain.PurposeThe aim of our study was to prospectively analyze the impact of clinical and demographic parameters, especially of BMI, on worsening of diastolic function and left atrial strain (LAS) in an urban population of women with a low prevalence of cardiovascular risk factors.Methods and ResultsAn extensive clinical and echocardiographic assessment comprising the analysis of phasic LAS using two-dimensional speckle-tracking echocardiography (2D STE) was performed in 258 participants of the Berlin Female Risk Evaluation (BEFRI) trial between October 2019 and December 2020 after a mean follow-up period of 6.8 years. We compared clinical and echocardiographic parameters stratifying women by BMI &lt; or ≥25 kg/m2, and we analyzed the impact of demographic characteristics on the worsening of DD and LA mechanics in the longer-term follow-up using univariate and multivariate regression analyses. 248 women were suitable for echocardiographic analysis of LAS using 2D STE. After a mean follow-up time of 6.8 years, LA reservoir strain (LASr) and LA conduit strain (LAScd) were significantly reduced in participants with a BMI ≥25 kg/m2 compared with women with a BMI &lt;25 kg/m2 at baseline (30 ± 8% vs. 38 ± 9%, p &lt; 0.0001; −14 ± 7% vs. −22 ± 8%, p &lt; 0.0001). 28% of the overweighted women presented a deterioration of diastolic function at the time of follow-up in contrast with only 7% of the group with a BMI &lt;25 kg/m2 (p &lt; 0.0001). BMI remained significantly associated with LAS reductions after adjustment for other risk factors in multivariate regression analyses.ConclusionOverweight and obesity are related to impaired LAS and to a worsening of diastolic function after a long-term follow-up in a cohort of randomly selected women

Regeneration of myocardial infarction via cardiovascular progenitors from murine and human pluripotent stem cells

Stammzellen nehmen in der regenerativen Therapie von Herzkreislauferkrankungen einen hohen Stellenwert ein. Aufgrund der ethischen Kontroverse besitzen adulte Stammzellen mit einem Differenzierungspotential von embryonalen Stammzellen einen Vorteil gegenüber den humanen embryonalen Stammzellen. In dieser Arbeit konnten spermatogoniale Stammzellen aus einem transgenen Mausmodell (MHC-NEO/MHC-eGFP) isoliert und mittels lentiviraler Überexpression von OCT4 allein in den pluripotenten Zustand überführt werden. Die Pluripotenz dieser induzierten pluripotenten Stammzellen (iPSCs) wurde durch verschiedene charakteristische Analysen nachgewiesen. Für therapeutische Einsätze zur Myokardinfarktregeneration stellen kardiovaskuläre Vorläuferzellen, die die Fähigkeit aufweisen sowohl zu proliferieren als auch in Kardiomyozyten, glatte Muskelzellen und endotheliale Zellen zu differenzieren, eine geeignete Zellart dar. Diese Vorläuferzellen sind durch die Expression des Oberflächenrezeptors fetal liver kinase 1 (FLK1) gekennzeichnet. In dieser Arbeit wurden iPSCs generiert und in FLK1 positive Zellen durch Cokultivierung differenziert und mit Hilfe der fluorescence activated cell sorting (FACS)-Technik separiert. Es wurden 30% FLK1 positive Zellen gewonnen. Auch hESCs konnten durch Cokultivierung auf OP9 Zellen (30%) und über das mass culture Verfahren (50%) in kinase domain region (KDR) positive Zellen differenziert werden. Die Vitalität und Differenzierungsfähigkeit der FLK1 positiven Zellen nach der FACS-Separierung wurden bestätigt. Zur Analyse des therapeutischen Effekts dieser FLK1 positiven Zellen wurde ein Myokardinfarkt-Mausmodell etabliert. Die FLK1 positiven Zellen wurden allogen in Wildtypmäuse injiziert, die mit dem Immunsuppresivum Ciclosporin A (CsA) behandelt wurden. Nach Injektion dieser Vorläuferzellen wurde eine signifikante Steigerung der Ejektionsfraktion an Tag 56 nach Injektion nachgewiesen. Dieser funktionssteigernde Trend an Tag 56 durch die Zellinjektion wurde auch bei der Verkürzungsfraktion beobachtet. Die injizierten Zellen konnten zu den frühen Untersuchungszeitpunkten im Myokard durch Zellkernansammlungen nachgewiesen werden. Zu späteren Zeitpunkten war dies nicht mehr der Fall. Während der in vivo Studie wurde zu keinem Zeitpunkt eine Teratombildung beobachtet. Immunologische Untersuchungen zeigten, dass die FLK1 positiven Zellen eine Tumorigenität in immundefizienten Mäusen aufwiesen und somit in dieser Form nicht direkt für die regenerative Therapie eingesetzt werden können. Ebenfalls wurde gezeigt, dass das Immunsystem der Versuchstiere und die Behandlung mit dem Immunsuppressivum CsA Auswirkungen auf die Teratombildung in der in vivo Studie besitzen. Die injizierten Zellen führten zu einer Aktivierung der natürlichen Killer (NK)-Zellen der Versuchstiere, die hingegen zu einer Lyse der injizierten Zellen führen konnten. CsA inhibierte die Teratombildung, besaß jedoch keinen Einfluss auf die NK-Zellaktivität. Diese Ergebnisse deuteten darauf hin, dass die injizierten Zellen vom Immunsystem der Mäuse abgestoßen worden sind und somit zu späteren Versuchszeiten nicht mehr nachgewiesen werden konnten. Trotz der eventuellen Abstoßung im späteren Versuchsverlauf führte die Injektion der FLK1 positiven Zellen zu einer signifikanten Steigerung der Herzleistung. Die Tumorigenität von Zellen spielt eine entscheidende Rolle in der stammzellbasierenden Therapie. Die virale Methode der Reprogrammierung in iPSCs hatte keinen Einfluss auf die Tumorigenität dieser Zellen. Bei Injektion von autologen viral iPSCs in entsprechende Akzeptormäuse wurden Teratombildungen beobachtet. Die NK-Zellaktivität wurde durch die Injektion jedoch nicht aktiviert. Es wurde somit gezeigt, dass die viral iPSCs vom Immunsystem der autologen Versuchstiere nicht abgestoßen wurden. Die Tumorigenität schien somit von zelllinienspezifischen Immunogenen beeinflusst. Für klinische Anwendungen könnten die hier untersuchten kardiovaskulären Vorläuferzellen, die ein regeneratives Potential aufwiesen, aufgrund ihrer Tumorigenität jedoch nicht eingesetzt werden. Zukünftig müssten die zu transplantierenden Zellen erneut selektiert werden, um eventuelle Unreinheit von undifferenzierten Zellen zu umgehen. Dies könnte durch Kultivierung der Population mit NK-Zellen erfolgen, da diese Arbeit gezeigt hat, dass eine NK-Zellaktivität gegen tumorigene Zellen vorliegt. Eine andere Möglichkeit wäre diese kardiovaskulären Vorläuferzellpopulation im Folgenden auch zur Herstellung von künstlichem Herzgewebe einzusetzen, um größere Bereiche von geschädigtem Myokard zu regenerieren.Stem cells hold a great promise in regenerative therapies for cardiovascular diseases. Heart regeneration with stem cells requires a delicate balance between the loss of donor cell pluripotency (so that teratoma formation is avoided) and retention of donor cell proliferative capacity prior to terminal differentiation (so that a sufficient number of target cells are generated to effect a therapeutic benefit). In this study, spermatogonial stem cells (SSCs) derived from double transgenic adult mice (MHC-NEO/MHC-EGFP) were isolated and reprogrammed into induced pluripotent stem cells (iPSCs) by over expression of OCT4 alone. The reprogrammed cells were positive for pluripotency markers and showed similar characteristics as pluripotent stem cells. For a therapeutic application, a cardiovascular progenitor cell population that has the ability to differentiate into cardiomyocytes, endothelial cells and smooth muscle cells is interesting. This cardiovascular cell population is characterised by the expression of the cell surface receptor fetal liver kinase 1 (FLK1). A protocol to induce the generated iPSCs to differentiate into FLK1+ cardiovascular progenitors via coculture with OP9 cells was established. After differentiation for 6 days, 30% of the cells were positive for FLK1. In addition, human embryonic stem cells could be differentiated into kinase domain region (KDR) positive cells by both coculture with OP9 cells (30%) and mass culture method (50%). The FLK1+ cells were then sorted using the fluorescence-activated cell sorting technique. For investigating the regenerative potential of these cells, we used a myocardial infarction mouse model and transplanted FLK1+ cells in the hearts of allogenic mice. The mice were treated daily with the immunosuppressive drug cyclosporin A (CsA). Echocardiographic investigations showed a functional improvement of the heart parameters like ejection fraction and fractional shortening after injection of FLK1+ cells at day 56. At early time points the injected cells could be found again in the myocardium. Later time points showed no more distinct cell aggregations. Moreover, no teratoma formation could be observed in the in vivo study. Immunological investigations showed that FLK1 positive cells were tumorigenic in immunodeficient mice, hence the cells could not be transferred directly for a therapeutic application. In addition, experiments showed that both the immune system of the acceptor mice and the CsA injection interfered with the teratoma formation in the in vivo study. The injected cells activated the natural killer (NK) cells of the acceptor mice. CsA inhibited the teratoma formation but had no effect of the NK cell activity. These results pointed out that the injected cells could be rejected by the immune system of the acceptor mice and could not be detected by histological analysis. In spite of the feasible rejection of the FLK1 positive cells at later time points, the injection of these cells resulted in a significant improvement of the heart function. Tumorigenicity of cells plays a certain role in the stem cell based therapy. The reprogramming by a viral method had no influence on the teratoma formation. By injection of viral iPSCs in autologous acceptor mice, teratoma formation could be observed. The NK cell activity was not activated and the experiments showed that injected viral iPSCs were not rejected by the immune system of the autologous mice. The tumorigenicity seems to be influenced by cell line specific immunogens. The generated cardiovascular progenitor cells showed therapeutic benefits but were also tumorigenic. Therefore the cells could not be used for direct clinical applications. Prospectively, the transplanted cells have to be selected for a second time to purify the cell population. Undifferentiated cells could be selected by cocultivating with NK cells because our data indicated that the NK cells had an activity against tumorigenic cells. Another possibility is to generate engineered heart tissue with cardiovascular progenitor cells to repopulate the damaged heart

Generation of iPSC-lines from two independent Takotsubo syndrome patients with recurrent Takotsubo events

The Takotsubo syndrome (TTS) is characterized by acute transient left ventricular dysfunction in the absence of obstructive coronary lesions. An enhanced β-adrenergic signaling and higher sensitivity to catecholamine-induced-toxicity were identified as mechanisms associated with TTS. It is still elusive, whether TTS patients with recurrent events show similar underlying signaling pathomechanism. Induced pluripotent stem cell (iPSC)-lines were generated from skin fibroblasts of two independent female Takotsubo syndrome patients with a severe phenotype characterized by recurrent TTS events. For reprogramming, a non-integrative plasmid technique was used. All generated iPSCs maintained full pluripotency, genomic integrity, and spontaneous in vitro and in vivo differentiation capacity

Efficient Killing of Murine Pluripotent Stem Cells by Natural Killer (NK) Cells Requires Activation by Cytokines and Partly Depends on the Activating NK Receptor NKG2D

Natural killer (NK) cells play an important role as cytotoxic effector cells, which scan the organism for infected or tumorigenic cells. Conflicting data have been published whether NK cells can also kill allogeneic or even autologous pluripotent stem cells (PSCs) and which receptors are involved. A clarification of this question is relevant since an activity of NK cells against PSCs could reduce the risk of teratoma growth after transplantation of PSC-derived grafts. Therefore, the hypothesis has been tested that the activity of NK cells against PSCs depends on cytokine activation and specifically on the activating NK receptor NKG2D. It is shown that a subcutaneous injection of autologous iPSCs failed to activate NK cells against these iPSCs and can give rise to teratomas. In agreement with this result, several PSC lines, including two iPSC, two embryonic stem cell (ESC), and two so-called multipotent adult germline stem cell (maGSC) lines, were largely resistant against resting NK cells although differences in killing were found at low level. All PSC lines were killed by interleukin (IL)-2-activated NK cells, and maGSCs were better killed than the other PSC types. The PSCs expressed ligands of the activating NK receptor NKG2D and NKG2D-deficient NK cells from Klrk1−/− mice were impaired in their cytotoxic activity against PSCs. The low-cytotoxic activity of resting NK cells was almost completely dependent on NKG2D. The cytotoxic activity of IL-2-activated NKG2D-deficient NK cells against PSCs was reduced, indicating that also other activating receptors on cytokine-activated NK cells must be engaged by ligands on PSCs. Thus, NKG2D is an important activating receptor involved in killing of murine PSCs. However, NK cells need to be activated by cytokines before they efficiently target PSCs and then also other NK receptors become relevant. These features of NK cells might be relevant for transplantation of PSC-derived grafts since NK cells have the capability to kill undifferentiated cells, which might be present in grafts in trace amounts

Catecholamine-dependent β-adrenergic signaling in a pluripotent stem cell model of takotsubo cardiomyopathy

BACKGROUND: Takotsubo syndrome (TTS) is characterized by an acute left ventricular dysfunction and is associated with life-threating complications in the acute phase. The underlying disease mechanism in TTS is still unknown. A genetic basis has been suggested to be involved in the pathogenesis. OBJECTIVES: The aims of the study were to establish an in vitro induced pluripotent stem cell (iPSC) model of TTS, to test the hypothesis of altered β-adrenergic signaling in TTS iPSC-cardiomyocytes (CMs), and to explore whether genetic susceptibility underlies the pathophysiology of TTS. METHODS: Somatic cells of patients with TTS and control subjects were reprogrammed to iPSCs and differentiated into CMs. Three-month-old CMs were subjected to catecholamine stimulation to simulate neurohumoral overstimulation. We investigated β-adrenergic signaling and TTS cardiomyocyte function. RESULTS. Enhanced β-adrenergic signaling in TTS-iPSC-CMs under catecholamine-induced stress increased expression of the cardiac stress marker NR4A1; cyclic adenosine monophosphate levels; and cyclic adenosine monophosphate-dependent protein kinase A-mediated hyperphosphorylation of RYR2-S2808, PLN-S16, TNI-S23/24, and Cav1.2-S1928, and leads to a reduced calcium time to transient 50% decay. These cellular catecholamine-dependent responses were mainly mediated by β-adrenoceptor signaling in TTS. Engineered heart muscles from TTS-iPSC-CMs showed an impaired force of contraction and a higher sensitivity to isoprenaline-stimulated inotropy compared with control subjects. In addition, altered electrical activity and increased lipid accumulation were detected in catecholamine-treated TTS-iPSC-CMs, and were confirmed by differentially expressed lipid transporters CD36 and CPT1C. Furthermore, we uncovered genetic variants in different key regulators of cardiac function. CONCLUSIONS. Enhanced β-adrenergic signaling and higher sensitivity to catecholamine-induced toxicity were identified as mechanisms associated with the TTS phenotype. (International Takotsubo Registry [InterTAK Registry] [InterTAK]; NCT01947621)

A High-Throughput Method as a Diagnostic Tool for HIV Detection in Patient-Specific Induced Pluripotent Stem Cells Generated by Different Reprogramming Methods

Induced pluripotent stem cells (iPSCs) provide a unique opportunity for generation of patient-specific cells for use in translational purposes. We aimed to compare iPSCs generated by different reprogramming methods regarding their reprogramming efficiency, pluripotency capacity, and the possibility to use high-throughput PCR-based methods for detection of human pathogenic viruses. iPSCs from skin fibroblasts (FB), peripheral blood mononuclear cells (PBMCs), or mesenchymal stem cells (MSCs) were generated by using three different reprogramming systems including chromosomal integrating and nonintegrating methods. Reprogramming efficiencies were in accordance with the literature, indicating that the parental cell type and the reprogramming method play a major role for the reprogramming efficiencies (FB: STEMCCA: 1.30 +/- 0.18, Sendai virus: 1.37 +/- 0.01, and episomal plasmids: 0.04 +/- 0.02; PBMCs: Sendai virus: 0.002 +/- 0.001, episomal plasmids: 0) but result in the same characteristics of pluripotency. We found the highest reprogramming efficiencies for MSC with 3.32 +/- 1.2 by using episomal plasmids. Since GMP standard working procedures and screening units need virus contamination-free cell lines, we studied HIV-1 contamination in the generated iPSCs. We used the high-throughput cobas (R) 6800/8800 system, which is normally used for detection of HIV-1 in plasma of patients, and found that footprint-free reprogramming methods as episomal plasmids and Sendai virus are useful for the described virus detection method. This fast, cost-effective, robust, and reliable assay demonstrates the feasibility to use high-throughput PCR-based methods for detection of human pathogenic viruses in ps-iPSC lines that were generated with nongenome integrating reprogramming methods