Auswirkungen von hyperglykämisch modifizierter extrazellulärer Matrix von Endothelzellen auf die Charakteristika von Fett-Stroma Zellen

Die diabetische Retinopathie (DRP) ist die häufigste mit Diabetes assoziierte Kompli-kation und Nummer eins der Erblindungsursachen in der westlichen Welt. Als Folge des deregulierten Glukosemetabolismus kommt es bereits in frühen Stadien der DRP zu einer Verdickung der extrazellulären Matrix (EZM) und damit einhergehend einem Absterben von Perizyten. Ursächlich für diesen, auch als „Pericyte drop out“, be-zeichneten Prozess scheint eine qualitative und quantitative Veränderung der EZM zu sein, die zu einer gestörten Kommunikation zwischen Endothel und Perizyten führt. Es kommt zum Verlust der regulierenden und modulierenden Einflüsse von Pe- rizyten auf das vaskuläre Endothel in deren Folge die Permeabilität der Gefäße zu-nimmt und gleichzeitig eine reaktive, deregulierte Neovaskularisation einsetzt. Diese Effekte addieren sich und führen zum charakteristischen Bild der DRP mit insuffizien-ten Gefäßen die auf Dauer zur Ausbildung eines Makulaödems führen. Dem Untergang beziehungsweise Funktionsverlust der Perizyten spricht man eine Schlüsselfunktion bei der Entstehung und Progredienz der DRP zu. Ein Wiederher-stellen der Perizytenfunktion, so hofft man, könnte die im Verlauf der DRP auftreten-den Komplikationen lindern und ihre Progression vermindern. Mesenchymal-stromale Zellen (MSC) haben zahlreiche morphologische und funktio- nelle Gemeinsamkeiten mit Perizyten. Hinzu kommt, dass ihre Differenzierungskapa- zitäten und ihr immunmodulatorisches Potential bereits erfolgreich im Zuge zellthera-peutischer Interventionen gegen andere Erkrankungen eingesetzt wurden. Dies macht sie zu möglichen Substituenten gegen den Perizytenuntergang im Verlauf der DRP. Vorrangehende Untersuchungen konnten nachweisen, dass human retinal pericytes (HRP) die auf hyperglykämisch modifizierter extrazellulärer Matrix kultiviert wurden, in ihrer Adhäsion gehemmt werden und schneller in Apoptose eintreten. Wir stellten die Hypothese auf, dass MSC in ihrem Adhäsions-, Wachstums- und Differenzie- rungsverhalten durch chronisch erhöhte Glukosewerte weniger stark beeinflusst wer-den. Als Vertreter der MSC wählten wir, aus Fettgewebe isolierte adipose derived stem/stromal cells (ASC). Deren Einsatz hat in aktuellen Studien mit Anwendungs- schwerpunkt im perivaskulären Bereich deutliche Vorteile im Vergleich zu MSC, die aus anderen Geweben isoliert wurden, gezeigt. Das Ziel dieser Arbeit war es, die Zusamme nfassung8 Einflüsse von hyperglykämisch modifizierter EZM von Endothelzellen auf das Wachs-tumsverhalten von ASCs und HRPs zu untersuchen und zu vergleichen. Dafür haben wir Endothelzellen in Medien mit verschiedenen Glukosekonzentrationen kultiviert und durch Dezellularisierung die, von ihnen gebildete EZM isoliert. Anschließend ha- ben wir auf den verschieden modifizierten Matrizes ASC und HRP ausgesät und mit- tels „Live cell imaging“ das Proliferationsverhalten der Zellen in Echtzeit dokumentiert und analysiert. Dabei konnten wir nachweisen, dass HRP ein vermindertes Wachs- tum auf hyperglykämisch modifizierter EZM aufweisen. ASC proliferierten im Ver- gleich dazu weitgehend unbeeinflusst von den erhöhten Glukosekonzentrationen. Um die morphologischen und funktionellen Gemeinsamkeiten von ASC und HRP besser zu verstehen, führten wir vergleichende FACS-Analysen, Differenzierungs- assays und Immunfluoreszenzverfahren durch. Dabei konnten wir zeigen, dass ASC und HRP sowohl in ihrer Oberflächenexpression als auch in ihrer Differenzierungs-kapazität zahlreiche Gemeinsamkeiten aufweisen. Als Ursache für das unterschiedliche Verhalten von HRP und ASC auf hyperglykä- misch-modifizierter EZM postulierten wir zunächst Glukose-bedingte Veränderungen der Zusammensetzung der EZM. Deshalb wurden Laminin, Kollagen IV und Fib- ronektin mittels Immunfluoreszenz und ELISA nachgewiesen, zeigten jedoch keine quantitativen Unterschiede. Mit Aussicht auf weiterführende Projekte untersuchten wir im letzten Schritt dieser Arbeit die Veränderungen der Expressionsprofile von ASC, nachdem sie auf EZM kultiviert wurden. Interessanterweise konnten wir dabei feststellen, dass sich die Oberflächenexpression der ASC, durch ihre Kultur auf EZM, in Richtung eines Peri-zyten-assoziierten Profils verändert. Zusammenfassend können wir mit dieser Arbeit zeigen, dass ASC eine höhere Re- sistenz gegenüber hyperglykämisch modifizierter EZM aufweisen als HRP. Des Wei- teren scheint es eine Übereinstimmung wichtiger morphologischer Charakteristika zu geben, sowie Anzeichen dafür, dass ASC, milieubedingt in Perizyten-ähnliche Zellen ausreifen können. Aufgrund dessen möchten wir den Einsatz von ASC gegen Er- krankungen die mit einem Perizytenverlust einhergehen, speziell mit Hinsicht auf die DRP, als vielversprechende Therapieoption einstufen

Quantifying the Effects of Contact Tracing, Testing, and Containment

Contact tracing has the potential to help identify, characterize, and predict disease-spreading human interactions at an unprecedented resolution. However, to realize this potential, we need to utilize data-driven epidemic models that can operate at a high spatiotemporal resolution and make use of and benefit from contact tracing data of individuals. Such data-driven models are currently missing, and in this work we initiate their development using the framework of temporal point processes. Using an efficient sampling algorithm, we can use our model to quantify the effects that different testing and tracing strategies, social distancing measures, and business restrictions may have on the course of the disease. Building on this algorithm, we use Bayesian optimization to estimate the transmission rate due to infectious individuals at the sites they visit and at their households as well as the mobility reduction due to social distancing from longitudinal case data. Simulations using real COVID-19 case data and mobility patterns from several cities and regions in Germany and Switzerland with a wide range of infection levels until today demonstrate that our model may allow individuals and policy makers to make more effective decisions.Comment: Extensive results and additional analysis; refined parameter estimation

Mesenchymal stromal/stem cells as potential therapy in diabetic retinopathy

Diabetic retinopathy (DR) is a multifactorial microvascular disease induced by hyperglycemia and subsequent metabolic abnormalities. The resulting cell stress causes a sequela of events that ultimately can lead to severe vision impairment and blindness. The early stages are characterized by activation of glia and loss of pericytes, endothelial cells (EC) and neuronal cells. The integrity of the retinal microvasculature becomes affected, and, as a possible late response, macular edema may develop as a common reason for vision loss in patients with non proliferative DR. Moreover, the local ischemia can trigger vasoproliferation leading to vision-threating proliferative DR (PDR) in humans. Available treatment options include control of metabolic and hemodynamic factors. Timely intervention of advanced DR stages with laser photocoagulation, intraocular anti-vascular endothelial growth factor (VEGF) or glucocorticoid drugs can reduce vision loss. As the pathology involves cell loss of both the vascular and neuroglial compartments, cell replacement strategies by stem and progenitor cells have gained considerable interest in the past years. Compared to other disease entities, so far little is known about the efficacy and potential mode of action of cell therapy in treatment of DR. In preclinical models of DR different cell types have been applied ranging from embryonic or induced pluripotent stem cells, hematopoietic stem cells, and endothelial progenitor cells to mesenchymal stromal cells (MSC). The latter cell population can combine various modes of action (MoA), thus they are among the most intensely tested cell types in cell therapy. The aim of this review is to discuss the rationale for using MSC as potential cell therapy to treat DR. Accordingly, we will revise identified MoA of MSCs and speculate how these may support the repair of the damaged retina

Pro-angiogenic Activity Discriminates Human Adipose-Derived Stromal Cells From Retinal Pericytes: Considerations for Cell-Based Therapy of Diabetic Retinopathy

Diabetic retinopathy (DR) is a frequent diabetes-associated complication. Pericyte dropout can cause increased vascular permeability and contribute to vascular occlusion. Adipose-derived stromal cells (ASC) have been suggested to replace pericytes and restore microvascular support as potential therapy of DR. In models of DR, ASC not only generated a cytoprotective and reparative environment by the secretion of trophic factors but also engrafted and integrated into the retina in a pericyte-like fashion. The aim of this study was to compare the pro-angiogenic features of human ASC and human retinal microvascular pericytes (HRMVPC) in vitro. The proliferation and the expression of ASC and HRMVPC markers were compared. Adhesion to high glucose-conditioned endothelial extracellular matrix, mimicking the diabetic microenvironment, was measured. The angiogenesis-promoting features of both cell types and their conditioned media on human retinal endothelial cells (EC) were assessed. To identify a molecular basis for the observed differences, gene expression profiling was performed using whole-genome microarrays, and data were validated using PCR arrays and flow cytometry. Based on multiplex cytokine results, functional studies on selected growth factors were performed to assess their role in angiogenic support. Despite a distinct heterogeneity in ASC and HRMVPC cultures with an overlap of expressed markers, ASC differed functionally from HRMVPC. Most importantly, the pro-angiogenic activity was solely featured by ASC, whereas HRMVPC actively suppressed vascular network formation. HRMVPC, in contrast to ASC, showed impaired adhesion and proliferation on the high glucose-conditioned endothelial extracellular matrix. These data were supported by gene expression profiles with differentially expressed genes. The vessel-stabilizing factors were more highly expressed in HRMVPC, and the angiogenesis-promoting factors were more highly expressed in ASC. The vascular endothelial growth factor receptor-2 inhibition efficiently abolished the ASC angiogenic supportive capacities, whereas the addition of angiopoietin-1 and angiopoietin-2 did not alter these effects. Our results clearly show that ASC are pro-angiogenic, whereas HRMVPC are marked by anti-angiogenic/EC-stabilizing features. These data support ASC as pericyte replacement in DR but also suggest a careful risk-to-benefit analysis to take full advantage of the ASC therapeutic features

Pro-angiogenic Activity Discriminates Human Adipose-Derived Stromal Cells From Retinal Pericytes: Considerations for Cell-Based Therapy of Diabetic Retinopathy

Diabetic retinopathy (DR) is a frequent diabetes-associated complication. Pericyte dropout can cause increased vascular permeability and contribute to vascular occlusion. Adipose-derived stromal cells (ASC) have been suggested to replace pericytes and restore microvascular support as potential therapy of DR. In models of DR, ASC not only generated a cytoprotective and reparative environment by the secretion of trophic factors but also engrafted and integrated into the retina in a pericyte-like fashion. The aim of this study was to compare the pro-angiogenic features of human ASC and human retinal microvascular pericytes (HRMVPC) in vitro. The proliferation and the expression of ASC and HRMVPC markers were compared. Adhesion to high glucose-conditioned endothelial extracellular matrix, mimicking the diabetic microenvironment, was measured. The angiogenesis-promoting features of both cell types and their conditioned media on human retinal endothelial cells (EC) were assessed. To identify a molecular basis for the observed differences, gene expression profiling was performed using whole-genome microarrays, and data were validated using PCR arrays and flow cytometry. Based on multiplex cytokine results, functional studies on selected growth factors were performed to assess their role in angiogenic support. Despite a distinct heterogeneity in ASC and HRMVPC cultures with an overlap of expressed markers, ASC differed functionally from HRMVPC. Most importantly, the pro-angiogenic activity was solely featured by ASC, whereas HRMVPC actively suppressed vascular network formation. HRMVPC, in contrast to ASC, showed impaired adhesion and proliferation on the high glucose-conditioned endothelial extracellular matrix. These data were supported by gene expression profiles with differentially expressed genes. The vessel-stabilizing factors were more highly expressed in HRMVPC, and the angiogenesis-promoting factors were more highly expressed in ASC. The vascular endothelial growth factor receptor-2 inhibition efficiently abolished the ASC angiogenic supportive capacities, whereas the addition of angiopoietin-1 and angiopoietin-2 did not alter these effects. Our results clearly show that ASC are pro-angiogenic, whereas HRMVPC are marked by anti-angiogenic/EC-stabilizing features. These data support ASC as pericyte replacement in DR but also suggest a careful risk-to-benefit analysis to take full advantage of the ASC therapeutic features

Amino Acid Homeostasis and Fatigue in Chronic Hemodialysis Patients

Patients dependent on chronic hemodialysis treatment are prone to malnutrition, at least in part due to insufficient nutrient intake, metabolic derangements, and chronic inflammation. Losses of amino acids during hemodialysis may be an important additional contributor. In this study, we assessed changes in plasma amino acid concentrations during hemodialysis, quantified intradialytic amino acid losses, and investigated whether plasma amino acid concentrations and amino acid losses by hemodialysis and urinary excretion are associated with fatigue. The study included a total of 59 hemodialysis patients (65 +/- 15 years, 63% male) and 33 healthy kidney donors as controls (54 +/- 10 years, 45% male). Total plasma essential amino acid concentration before hemodialysis was lower in hemodialysis patients compared with controls (p = 0.006), while total non-essential amino acid concentration did not differ. Daily amino acid losses were 4.0 +/- 1.3 g/24 h for hemodialysis patients and 0.6 +/- 0.3 g/24 h for controls. Expressed as proportion of protein intake, daily amino acid losses of hemodialysis patients were 6.7 +/- 2.4% of the total protein intake, compared to 0.7 +/- 0.3% for controls (p < 0.001). Multivariable regression analyses demonstrated that hemodialysis efficacy (Kt/V) was the primary determinant of amino acid losses (Std. beta = 0.51; p < 0.001). In logistic regression analyses, higher plasma proline concentrations were associated with higher odds of severe fatigue (OR (95% CI) per SD increment: 3.0 (1.3; 9.3); p = 0.03), while higher taurine concentrations were associated with lower odds of severe fatigue (OR (95% CI) per log2 increment: 0.3 (0.1; 0.7); p = 0.01). Similarly, higher daily taurine losses were also associated with lower odds of severe fatigue (OR (95% CI) per log2 increment: 0.64 (0.42; 0.93); p = 0.03). Lastly, a higher protein intake was associated with lower odds of severe fatigue (OR (95% CI) per SD increment: 0.2 (0.04; 0.5); p = 0.007). Future studies are warranted to investigate the mechanisms underlying these associations and investigate the potential of taurine supplementation

Creatine homeostasis and protein energy wasting in hemodialysis patients

Muscle wasting, low protein intake, hypoalbuminemia, low body mass, and chronic fatigue are prevalent in hemodialysis patients. Impaired creatine status may be an often overlooked, potential contributor to these symptoms. However, little is known about creatine homeostasis in hemodialysis patients. We aimed to elucidate creatine homeostasis in hemodialysis patients by assessing intradialytic plasma changes as well as intra- and interdialytic losses of arginine, guanidinoacetate, creatine and creatinine. Additionally, we investigated associations of plasma creatine concentrations with low muscle mass, low protein intake, hypoalbuminemia, low body mass index, and chronic fatigue. Arginine, guanidinoacetate, creatine and creatinine were measured in plasma, dialysate, and urinary samples of 59 hemodialysis patients. Mean age was 65 ± 15 years and 63% were male. During hemodialysis, plasma concentrations of arginine (77 ± 22 to 60 ± 19 μmol/L), guanidinoacetate (1.8 ± 0.6 to 1.0 ± 0.3 μmol/L), creatine (26 [16–41] to 21 [15–30] μmol/L) and creatinine (689 ± 207 to 257 ± 92 μmol/L) decreased (all P < 0.001). During a hemodialysis session, patients lost 1939 ± 871 μmol arginine, 37 ± 20 μmol guanidinoacetate, 719 [399–1070] μmol creatine and 15.5 ± 8.4 mmol creatinine. In sex-adjusted models, lower plasma creatine was associated with a higher odds of low muscle mass (OR per halving: 2.00 [1.05–4.14]; P = 0.04), low protein intake (OR: 2.13 [1.17–4.27]; P = 0.02), hypoalbuminemia (OR: 3.13 [1.46–8.02]; P = 0.008) and severe fatigue (OR: 3.20 [1.52–8.05]; P = 0.006). After adjustment for potential confounders, these associations remained materially unchanged. Creatine is iatrogenically removed during hemodialysis and lower plasma creatine concentrations were associated with higher odds of low muscle mass, low protein intake, hypoalbuminemia, and severe fatigue, indicating a potential role for creatine supplementation.ISSN:1479-587