Charakterisierung des kardioregenerativen Potenzials adulter humaner endothelialer Progenitorzellen vom Typ ECFC im Infarktmodell der SCID/Beige-Maus

Akute und chronische Myokardischämien ziehen einen irreversiblen Verlust an funktionellem Myokard nach sich und sind mit weitreichenden strukturellen Umbauprozessen am verbleibenden Myokard assoziiert. Häufig entwickelt sich eine progrediente Herzinsuffizienz. Aus Ermangelung an kurativen Behandlungsmöglichkeiten, der schlechten Prognose sowie der immensen sozioökonomischen Bedeutung leitet sich die dringliche Notwendigkeit für die Entwicklung neuer, alternativer Therapiestrategien ab. Vielversprechend erscheint das innovative therapeutische Konzept der zellbasierten myokardialen Regeneration, das zum Ziel hat, über eine Applikation von Stamm- und Progenitorzellen irreversibel verlorengegangene Herzmuskelzellen funktionell zu ersetzen bzw. die Formation neuer Gefäße im geschädigten Herzmuskel zu bewirken. In der vorliegenden Arbeit wurde das kardioregenerative Potenzial von ECFCs, einer neuartigen, bisher im myoregenerativen Kontext wenig charakterisierten endothelialen Progenitorzellpopulation, die aus dem peripheren Blut von KHK-Patienten isoliert wurde, in einem murinen Myokardinfarktmodell untersucht. Unmittelbar nach experimenteller, durch LAD-Ligation realisierter Infarktinduktion wurden 5x105 ECFCs in die Infarktrandzone verabreicht. Versuchstiere der Kontrollgruppe erhielten eine Injektion mit isotoner Kochsalzlösung. Im Vergleich zur Kontrollgruppe führte die intramyokardiale Transplantation von ECFCs zu einer signifikanten Verbesserung der hämodynamischen Funktionsparameter, insbesondere der linksventrikulären Auswurffraktion, sowie einer günstigen Beeinflussung des postischämischen kardialen Remodelings, die sich in geringeren Infarktgrößen bei den behandelten Tieren widerspiegelte. Eine direkte Beteiligung der Zellen an der Neoangiogenese oder eine myokardiale Differenzierung konnte in der vorliegenden Arbeit nicht nachgewiesen werden. Dennoch zeigte sich im Vergleich zur unbehandelten Kontrollgruppe eine deutliche Verbesserung der Vaskularisationsdichte nach Zelltransplantation. In der durchflusszytometrischen Analyse der myoztendepletierten Zellfraktionen der Herzen der Versuchstiere 2 Tage nach Infarktinduktion und Zelltransplantation konnte eine im Vergleich zur unbehandelten Kontrollgruppe deutliche Steigerung der Zahl von CD45-/CD34-/Sca1+ kardial-residenten Stammzellen im Herzen nachgewiesen werden. Dieser indirekte und bislang in der Literatur nicht beschriebene Mechanismus könnte, neben anderen parakrinen Effekten, hauptverantwortlich für die beobachteten positiven Effekte auf Hämodynamik und Remodeling sein. In der Zusammenschau können die beobachteten Effekte am ehesten als Folge sekundär eingewanderter (pro-) angiogener Zellen sowie über positive parakrine Einflüsse auf kardial-residente Stammzellen und ortsständige Endothelzellen mit dem Resultat einer verbesserten postischämischen Neovaskularisation interpretiert werden. In der Subgruppenanalyse scheint die diabetische Stoffwechsellage der Spenderpatienten die zelltherapeutischen Effekte im Tiermodell nicht zu limitieren. Die gemachten Beobachtungen lassen den Schluss zu, dass von der Transplantation von ECFCs nach Myokardischämie ein relevantes therapeutisches Potenzial ausgeht. Die in der vorliegenden Arbeit gemachten Beobachtungen bezüglich des Engraftments der transplantierten Zellen bestätigen die Befunde in der Literatur und verdeutlichen, dass zukünftig weitere Maßnahmen zur Steigerung des permanenten Engraftments ergriffen werden müssen, um die Effekte der Zelltherapie zu maximieren. Weitere Studien müssen detailliertere Einblicke in die genauen Mechanismen der Wirkungsweise von ECFCs bei der Therapie des akuten MIs und den Einfluss von kardiovaskulären Risikofaktoren auf die Funktionalität der zu transplantierenden Zellen erbringen.Objective: The potential therapeutic role of EPCs in ischemic heart disease is subject to intense investigation. It has been shown that the transplantation of unfractioned bone marrow stem cells after myocardial infarction (MI) is moderately effective. Selected stem cell populations may further optimize treatment. The aim of the study was to investigate the pro-regenerative potential of human endothelial colony-forming cells (ECFCs), a very homogenous and highly proliferative endothelial progenitor cell subpopulation, in a murine MI model. Methods: CD34+ peripheral blood mononuclear cells were isolated from patient blood samples using immunomagnetic beads. For generating ECFCs, CD34+ cells were plated on fibronectin-coated dishes and were expanded by culture in endothelial-specific cell medium. Either human ECFCs (5×10^5) or control medium were injected into the peri-infarct region after surgical MI induction in SCID/beige mice. Hemodynamic function was assessed invasively 30 days post-MI. Hearts were analyzed immunohistochemically to assess cell fate, infarct size and neovascularization (ECFCs n=15 vs. control n=10). Flow-cytometric analysis of enzymatically digested whole heart tissue was used to analyze different subsets of migrated CD34+/CD45+ peripheral mononuclear cells as well as CD34-/CD45- cardiac resident stem cells 2 days post-MI (ECFCs n= 10 vs. control n=6). Results: Transplantation of human ECFCs after MI improved hemodynamic function at day 30 post-MI (EF: 30.43±1.20% vs. 22.61±1.73%, p<0.001; ΔP/ΔTmax 5202.28±316.68mmHg/sec vs. 3896.24±534.95mmHg/sec, p<0.05) when compared to controls. In addition, a significantly reduced infarct size (50.3±4.5% vs. 66.1±4.3%, p<0.05) was seen in ECFC treated animals compared to controls. Immunohistochemistry failed to show integration and survival of transplanted cells. However, anti-CD31 immunohistochemistry demonstrated an increased vascular density within the infarct border zone (8.6±0.4 per HPF vs. 6.2±0.5 per HPF, p<0.001). Flow cytometry at day 2 post-MI showed a trend towards increased myocardial homing of CD45+/CD34+ mononuclear cells (1.1±0.3% vs. 0.7±0.1%, p=0.2). Interestingly, we detected a significant increase in CD34-/CD45-/Sca1+ cardiac resident stem cells (11.7±1.7% vs. 4.7±1.7%, p<0.01). In a subgroup analysis no significant differences were seen in the effects of ECFCs derived from diabetic- or non-diabetic patients. Conclusions: In a murine MI model, transplantation of human ECFCs ameliorates myocardial function by attenuation of adverse post-MI remodeling, presumably mainly through paracrine effects. Cardiac repair is enhanced by increasing myocardial neovascularization and the pool of Sca1+ cardiac resident stem cells. The use of human ECFCs for treating ischemic heart disease warrants further investigation

Pulsatile ex vivo perfusion of human saphenous vein grafts under controlled pressure conditions increases MMP-2 expression

<p>Abstract</p> <p>Background</p> <p>The use of human saphenous vein grafts (HSVGs) as a bypass conduit is a standard procedure in the treatment of coronary artery disease while their early occlusion remains a major problem.</p> <p>Methods</p> <p>We have developed an <it>ex vivo </it>perfusion system, which uses standardized and strictly controlled hemodynamic parameters for the pulsatile and non-static perfusion of HSVGs to guarantee a reliable analysis of molecular parameters under different pressure conditions. Cell viability of HSVGs (n = 12) was determined by the metabolic conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) into a purple formazan dye.</p> <p>Results</p> <p>Under physiological flow rates (10 mmHg) HSVGs remained viable for two weeks. Their exposure to arterial conditions (100 mmHg) was possible for one week without important reduction in viability. Baseline expression of matrix metalloproteinase-2 (MMP-2) after venous perfusion (2.2 ± 0.5, n = 5) was strongly up-regulated after exposure to arterial conditions for three days (19.8 ± 4.3) or five days (23.9 ± 6.1, p < 0.05). Zymographic analyses confirmed this increase on the protein level. Our results suggest that expression and activity of MMP-2 are strongly increased after exposure of HSVGs to arterial hemodynamic conditions compared to physiological conditions.</p> <p>Conclusion</p> <p>Therefore, our system might be helpful to more precisely understand the molecular mechanisms leading to an early failure of HSVGs.</p

Myeloid Zinc Finger 1 (Mzf1) Differentially Modulates Murine Cardiogenesis by Interacting with an Nkx2.5 Cardiac Enhancer

Vertebrate heart development is strictly regulated by temporal and spatial expression of growth and transcription factors (TFs). We analyzed nine TFs, selected by in silico analysis of an Nkx2.5 enhancer, for their ability to transactivate the respective enhancer element that drives, specifically, expression of genes in cardiac progenitor cells (CPCs). Mzf1 showed significant activity in reporter assays and bound directly to the Nkx2.5 cardiac enhancer (Nkx2.5 CE) during murine ES cell differentiation. While Mzf1 is established as a hematopoietic TF, its ability to regulate cardiogenesis is completely unknown. Mzf1 expression was significantly enriched in CPCs from in vitro differentiated ES cells and in mouse embryonic hearts. To examine the effect of Mzf1 overexpression on CPC formation, we generated a double transgenic, inducible, tetOMzf1-Nkx2.5 CE eGFP ES line. During in vitro differentiation an early and continuous Mzf1 overexpression inhibited CPC formation and cardiac gene expression. A late Mzf1 overexpression, coincident with a second physiological peak of Mzf1 expression, resulted in enhanced cardiogenesis. These findings implicate a novel, temporal-specific role of Mzf1 in embryonic heart development. Thereby we add another piece of puzzle in understanding the complex mechanisms of vertebrate cardiac development and progenitor cell differentiation. Consequently, this knowledge will be of critical importance to guide efficient cardiac regenerative strategies and to gain further insights into the molecular basis of congenital heart malformations

A novel de novo TBX5 mutation in a patient with Holt-Oram syndrome leading to a dramatically reduced biological function

BACKGROUND: The Holt-Oram syndrome (HOS) is an autosomal dominant disorder affecting 1/100.000 live births. It is defined by upper limb anomalies and congenital heart defects with variable severity. We describe a dramatic phenotype of a male, 15-month-old patient being investigated for strict diagnostic criteria of HOS. ----- METHODS AND RESULTS: Genetic analysis revealed a so far unpublished TBX5 mutation, which occurs de novo in the patient with healthy parents. TBX5 belongs to the large family of T-box transcription factors playing major roles in morphogenesis and cell-type specification. The mutation located in the DNA-binding domain at position 920 (C→A) leads to an amino acid change at position 85 (proline → threonine). Three-dimensional analysis of the protein structure predicted a cis to trans change in the respective peptide bond, thereby probably provoking major conformational and functional alterations of the protein. The p.Pro85Thr mutation showed a dramatically reduced activation (97%) of the NPPA promoter in luciferase assays and failed to induce NPPA expression in HEK 293 cells compared to wild-type TBX5 protein. The mutation did not interfere with the nuclear localization of the protein. ----- CONCLUSION: These results suggest that the dramatic functional alteration of the p.Pro85Thr mutation leads to the distinctive phenotype of the patient

Early and late outcomes after minimally invasive direct coronary artery bypass vs. full sternotomy off-pump coronary artery bypass grafting

ObjectivesMinimally-invasive direct coronary artery bypass (MIDCAB) is a less-invasive alternative to full sternotomy off-pump coronary artery bypass (FS-OPCAB) revascularization of the left anterior descending artery (LAD). Some studies suggested that MIDCAB is associated with a greater risk of graft occlusion and repeat revascularization than FS-OPCAB LIMA-to-LAD grafting. Data comparing MIDCAB to FS-OPCAB with regard to long-term follow-up is scarce. We compared short- and long-term results of MIDCAB vs. FS-OPCAB revascularization over a maximum follow-up period of 10 years.Patients and methodsFrom December 2009 to June 2020, 388 elective patients were included in our retrospective study. 229 underwent MIDCAB, and 159 underwent FS-OPCAB LIMA-to-LAD grafting. Inverse probability of treatment weighting (IPTW) was used to adjust for selection bias and to estimate treatment effects on short- and long-term outcomes. IPTW-adjusted Kaplan–Meier estimates by study group were calculated for all-cause mortality, stroke, the risk of repeat revascularization and myocardial infarction up to a maximum follow-up of 10 years.ResultsMIDCAB patients had less rethoracotomies (n = 13/3.6% vs. n = 30/8.0%, p = 0.012), fewer transfusions (0.93 units ± 1.83 vs. 1.61 units ± 2.52, p &lt; 0.001), shorter mechanical ventilation time (7.6 ± 4.7 h vs. 12.1 ± 26.4 h, p = 0.005), and needed less hemofiltration (n = 0/0% vs. n = 8/2.4%, p = 0.004). Thirty-day mortality did not differ significantly between the two groups (n = 0/0% vs. n = 3/0.8%, p = 0.25). Long-term outcomes did not differ significantly between study groups. In the FS-OPCAB group, the probability of survival at 1, 5, and 10 years was 98.4%, 87.8%, and 71.7%, respectively. In the MIDCAB group, the corresponding values were 98.4%, 87.7%, and 68.7%, respectively (RR1.24, CI0.87–1.86, p = 0.7). In the FS group, the freedom from stroke at 1, 5, and 10 years was 97.0%, 93.0%, and 93.0%, respectively. In the MIDCAB group, the corresponding values were 98.5%, 96.9%, and 94.3%, respectively (RR0.52, CI0.25–1.09, p = 0.06). Freedom from repeat revascularization at 1, 5, and 10 years in the FS-OPCAB group was 92.2%, 84.7%, and 79.5%, respectively. In the MIDCAB group, the corresponding values were 94.8%, 90.2%, and 81.7%, respectively (RR0.73, CI0.47–1.16, p = 0.22).ConclusionMIDCAB is a safe and efficacious technique and offers comparable long-term results regarding mortality, stroke, repeat revascularization, and freedom from myocardial infarction when compared to FS-OPCAB

Charakterisierung des kardioregenerativen Potenzials adulter humaner endothelialer Progenitorzellen vom Typ ECFC im Infarktmodell der SCID/Beige-Maus

Akute und chronische Myokardischämien ziehen einen irreversiblen Verlust an funktionellem Myokard nach sich und sind mit weitreichenden strukturellen Umbauprozessen am verbleibenden Myokard assoziiert. Häufig entwickelt sich eine progrediente Herzinsuffizienz. Aus Ermangelung an kurativen Behandlungsmöglichkeiten, der schlechten Prognose sowie der immensen sozioökonomischen Bedeutung leitet sich die dringliche Notwendigkeit für die Entwicklung neuer, alternativer Therapiestrategien ab. Vielversprechend erscheint das innovative therapeutische Konzept der zellbasierten myokardialen Regeneration, das zum Ziel hat, über eine Applikation von Stamm- und Progenitorzellen irreversibel verlorengegangene Herzmuskelzellen funktionell zu ersetzen bzw. die Formation neuer Gefäße im geschädigten Herzmuskel zu bewirken. In der vorliegenden Arbeit wurde das kardioregenerative Potenzial von ECFCs, einer neuartigen, bisher im myoregenerativen Kontext wenig charakterisierten endothelialen Progenitorzellpopulation, die aus dem peripheren Blut von KHK-Patienten isoliert wurde, in einem murinen Myokardinfarktmodell untersucht. Unmittelbar nach experimenteller, durch LAD-Ligation realisierter Infarktinduktion wurden 5x105 ECFCs in die Infarktrandzone verabreicht. Versuchstiere der Kontrollgruppe erhielten eine Injektion mit isotoner Kochsalzlösung. Im Vergleich zur Kontrollgruppe führte die intramyokardiale Transplantation von ECFCs zu einer signifikanten Verbesserung der hämodynamischen Funktionsparameter, insbesondere der linksventrikulären Auswurffraktion, sowie einer günstigen Beeinflussung des postischämischen kardialen Remodelings, die sich in geringeren Infarktgrößen bei den behandelten Tieren widerspiegelte. Eine direkte Beteiligung der Zellen an der Neoangiogenese oder eine myokardiale Differenzierung konnte in der vorliegenden Arbeit nicht nachgewiesen werden. Dennoch zeigte sich im Vergleich zur unbehandelten Kontrollgruppe eine deutliche Verbesserung der Vaskularisationsdichte nach Zelltransplantation. In der durchflusszytometrischen Analyse der myoztendepletierten Zellfraktionen der Herzen der Versuchstiere 2 Tage nach Infarktinduktion und Zelltransplantation konnte eine im Vergleich zur unbehandelten Kontrollgruppe deutliche Steigerung der Zahl von CD45-/CD34-/Sca1+ kardial-residenten Stammzellen im Herzen nachgewiesen werden. Dieser indirekte und bislang in der Literatur nicht beschriebene Mechanismus könnte, neben anderen parakrinen Effekten, hauptverantwortlich für die beobachteten positiven Effekte auf Hämodynamik und Remodeling sein. In der Zusammenschau können die beobachteten Effekte am ehesten als Folge sekundär eingewanderter (pro-) angiogener Zellen sowie über positive parakrine Einflüsse auf kardial-residente Stammzellen und ortsständige Endothelzellen mit dem Resultat einer verbesserten postischämischen Neovaskularisation interpretiert werden. In der Subgruppenanalyse scheint die diabetische Stoffwechsellage der Spenderpatienten die zelltherapeutischen Effekte im Tiermodell nicht zu limitieren. Die gemachten Beobachtungen lassen den Schluss zu, dass von der Transplantation von ECFCs nach Myokardischämie ein relevantes therapeutisches Potenzial ausgeht. Die in der vorliegenden Arbeit gemachten Beobachtungen bezüglich des Engraftments der transplantierten Zellen bestätigen die Befunde in der Literatur und verdeutlichen, dass zukünftig weitere Maßnahmen zur Steigerung des permanenten Engraftments ergriffen werden müssen, um die Effekte der Zelltherapie zu maximieren. Weitere Studien müssen detailliertere Einblicke in die genauen Mechanismen der Wirkungsweise von ECFCs bei der Therapie des akuten MIs und den Einfluss von kardiovaskulären Risikofaktoren auf die Funktionalität der zu transplantierenden Zellen erbringen.Objective: The potential therapeutic role of EPCs in ischemic heart disease is subject to intense investigation. It has been shown that the transplantation of unfractioned bone marrow stem cells after myocardial infarction (MI) is moderately effective. Selected stem cell populations may further optimize treatment. The aim of the study was to investigate the pro-regenerative potential of human endothelial colony-forming cells (ECFCs), a very homogenous and highly proliferative endothelial progenitor cell subpopulation, in a murine MI model. Methods: CD34+ peripheral blood mononuclear cells were isolated from patient blood samples using immunomagnetic beads. For generating ECFCs, CD34+ cells were plated on fibronectin-coated dishes and were expanded by culture in endothelial-specific cell medium. Either human ECFCs (5×10^5) or control medium were injected into the peri-infarct region after surgical MI induction in SCID/beige mice. Hemodynamic function was assessed invasively 30 days post-MI. Hearts were analyzed immunohistochemically to assess cell fate, infarct size and neovascularization (ECFCs n=15 vs. control n=10). Flow-cytometric analysis of enzymatically digested whole heart tissue was used to analyze different subsets of migrated CD34+/CD45+ peripheral mononuclear cells as well as CD34-/CD45- cardiac resident stem cells 2 days post-MI (ECFCs n= 10 vs. control n=6). Results: Transplantation of human ECFCs after MI improved hemodynamic function at day 30 post-MI (EF: 30.43±1.20% vs. 22.61±1.73%, p<0.001; ΔP/ΔTmax 5202.28±316.68mmHg/sec vs. 3896.24±534.95mmHg/sec, p<0.05) when compared to controls. In addition, a significantly reduced infarct size (50.3±4.5% vs. 66.1±4.3%, p<0.05) was seen in ECFC treated animals compared to controls. Immunohistochemistry failed to show integration and survival of transplanted cells. However, anti-CD31 immunohistochemistry demonstrated an increased vascular density within the infarct border zone (8.6±0.4 per HPF vs. 6.2±0.5 per HPF, p<0.001). Flow cytometry at day 2 post-MI showed a trend towards increased myocardial homing of CD45+/CD34+ mononuclear cells (1.1±0.3% vs. 0.7±0.1%, p=0.2). Interestingly, we detected a significant increase in CD34-/CD45-/Sca1+ cardiac resident stem cells (11.7±1.7% vs. 4.7±1.7%, p<0.01). In a subgroup analysis no significant differences were seen in the effects of ECFCs derived from diabetic- or non-diabetic patients. Conclusions: In a murine MI model, transplantation of human ECFCs ameliorates myocardial function by attenuation of adverse post-MI remodeling, presumably mainly through paracrine effects. Cardiac repair is enhanced by increasing myocardial neovascularization and the pool of Sca1+ cardiac resident stem cells. The use of human ECFCs for treating ischemic heart disease warrants further investigation

Direct Reprogramming—The Future of Cardiac Regeneration?

Today, the only available curative therapy for end stage congestive heart failure (CHF) is heart transplantation. This therapeutic option is strongly limited by declining numbers of available donor hearts and by restricted long-term performance of the transplanted graft. The disastrous prognosis for CHF with its restricted therapeutic options has led scientists to develop different concepts of alternative regenerative treatment strategies including stem cell transplantation or stimulating cell proliferation of different cardiac cell types in situ. However, first clinical trials with overall inconsistent results were not encouraging, particularly in terms of functional outcome. Among other approaches, very promising ongoing pre-clinical research focuses on direct lineage conversion of scar fibroblasts into functional myocardium, termed “direct reprogramming” or “transdifferentiation.” This review seeks to summarize strategies for direct cardiac reprogramming including the application of different sets of transcription factors, microRNAs, and small molecules for an efficient generation of cardiomyogenic cells for regenerative purposes