Teratogenicity and Reactive Oxygen Species after transient embryonic hypoxia: Experimental and clinical evidence with focus on drugs causing failed abortion in humans.

Teratogenicity and Reactive Oxygen Species after transient embryonic hypoxia: Experimental and clinical evidence with focus on drugs with human abortive potential. Reactive Oxygen Species (ROS) can be harmful to embryonic tissues. The adverse embryonic effects are dependent on the severity and duration of the hypoxic event and when during organongenesis hypoxia occurs. The vascular endothelium of recently formed arteries in the embryo is highly susceptible to ROS damage. Endothelial damage results in vascular disruption, hemorrhage and maldevelopment of organs, which normally should have been supplied by the artery. ROS can also induce irregular heart rhythm in the embryo resulting in alterations in blood flow and pressure from when the tubular heart starts beating. Such alterations in blood flow and pressure during cardiogenesis can result in a variety of cardiovascular defects, for example transpositions and ventricular septal defects. One aim of this article is to review and compare the pattern of malformations produced by transient embryonic hypoxia of various origins in animal studies with malformations associated with transient embryonic hypoxia in human pregnancy due to a failed abortion process. The results show that transient hypoxia and compounds with potential to cause failed abortion in humans, such as misoprostol and hormone pregnancy tests (HPTs) like Primodos, have been associated with a similar spectrum of teratogenicity. The spectrum includes limb reduction-, cardiovascular- and central nervous system defects. The hypoxia-ROS related teratogenicity of misoprostol and HPTs, is likely to be secondary to uterine contractions and compression of uterinoplacental/embryonic vessels during organogenesis.

Outcome prediction for patients assessed by the medical emergency team: a retrospective cohort study.

BACKGROUND: Medical emergency teams (METs) have been implemented to reduce hospital mortality by the early recognition and treatment of potentially life-threatening conditions. The objective of this study was to establish a clinically useful association between clinical variables and mortality risk, among patients assessed by the MET, and further to design an easy-to-use risk score for the prediction of death within 30 days. METHODS: Observational retrospective register study in a tertiary university hospital in Sweden, comprising 2,601 patients, assessed by the MET from 2010 to 2015. Patient registry data at the time of MET assessment was analysed from an epidemiological perspective, using univariable and multivariable analyses with death within 30 days as the outcome variable. Predictors of outcome were defined from age, gender, type of ward for admittance, previous medical history, acute medical condition, vital parameters and laboratory biomarkers. Identified factors independently associated with mortality were then used to develop a prognostic risk score for mortality. RESULTS: The overall 30-day mortality was high (29.0%). We identified thirteen factors independently associated with 30-day mortality concerning; age, type of ward for admittance, vital parameters, laboratory biomarkers, previous medical history and acute medical condition. A MET risk score for mortality based on the impact of these individual thirteen factors in the model yielded a median (range) AUC of 0.780 (0.774-0.785) with good calibration. When corrected for optimism by internal validation, the score yielded a median (range) AUC of 0.768 (0.762-0.773). CONCLUSIONS: Among clinical variables available at the time of MET assessment, thirteen factors were found to be independently associated with 30-day mortality. By applying a simple risk scoring system based on these individual factors, patients at higher risk of dying within 30 days after the MET assessment may be identified and treated earlier in the process.

A Retrospective Study of Posttransplant Amiodarone Exposition on Clad Development and Survival After Lung Transplantation.

BACKGROUND: Lung transplantation is a lifesaving procedure, still marred by worse results than other solid organ transplants. The 1-year mortality is 10%, and within 5 years after the procedure, half of patients develop chronic lung allograft dysfunction (CLAD), which also is the main limiting factor for long-term survival. Heart arrhythmias are also common directly after a lung transplant, and 1 treatment for this is the drug amiodarone. Recent research suggests that amiodarone exposure leads to activation of fibroblasts, a cell type that synthesizes stroma in the lung, associated with acute respiratory distress syndrome and CLAD. This study aims to retrospectively investigate the effect of posttransplant amiodarone treatment on survival and CLAD. MATERIAL AND METHODS: All patients transplanted at Sahlgrenska University Hospital between 2007 and 2018 were reviewed, and adult patients with a follow-up within Sweden were included. Of the 394 patients who met this inclusion criteria, retrospective data concerning postoperative complications and long-term outcomes were retrieved. A multivariable Cox proportional hazards model was applied to identify a set of independently significant predictors. RESULTS: Posttransplant use of amiodarone was associated with shorter survival (hazard ratio = 1.65; 95% confidence interval, 1.08-2.54; P = .02). Amiodarone exposure was not associated with CLAD (hazard ratio = 0.64; 95% confidence interval, 0.33-1.22; P = .17). CONCLUSIONS: An increased risk of death but not CLAD was observed in patients treated with amiodarone postoperatively after lung transplantation in the current cohort.

Haemophagocytic lymphohistiocytosis after heart transplantation: a case report.

BACKGROUND: Haemophagocytic lymphohistiocytosis (HLH) is an uncommon but serious systemic inflammatory response with high mortality rates. It can be triggered by malignancy or infectious agents, often in the context of immunosuppression. Literature covering HLH in heart transplantation (HTx) is scarce. CASE SUMMARY: A 25-year-old male with a history of celiac disease underwent HTx at Sahlgrenska Hospital in 2011 due to giant cell myocarditis and was treated with tacrolimus, mycophenolate mofetil (MMF), and prednisolone. He developed several episodes of acute cellular rejections (ACR) during the first 3 post-HTx years, which subsided after addition of everolimus. In May 2017, the patient was admitted to the hospital due to fever without focal symptoms. He had an extensive inflammatory reaction, but screening for infectious agents was negative. Haemophagocytic lymphohistiocytosis was discussed early, but first dismissed since two bone marrow biopsies revealed no signs of haemophagocytosis. Increasing levels of soluble IL-2 were considered confirmative of the diagnosis. Even with intense immunosuppressant treatment, the patient deteriorated and died in progressive multiorgan failure within 2 weeks of the symptom onset. DISCUSSION: A 25-year-old HTx recipient with an extensive inflammatory response, fulfilled criteria for HLH, but the diagnosis was delayed due to normal bone marrow biopsies. A background with autoimmune reactivity and immunosuppressive therapy may have contributed to HLH, but the actual trigger was not identified. Haemophagocytic lymphohistiocytosis can occur in HTx recipients in the absence of malignancy, identifiable infectious triggers and signs of haemophagocytosis. Early diagnosis and intervention are likely to be of importance for a favourable outcome.

Outcomes and Long-term Survival After Pulmonary Retransplantation: A Single-Center Experience.

BACKGROUND: The median survival after lung retransplantation (ReLTx) reported to the International Society of Heart and Lung Transplantation is restricted to 2.5 years. We report the results after ReLTx from our center. METHODS: A retrospective data collection was performed for the 635 patients who underwent lung transplantation between 1991 and 2017 at our center. Recipient variables were compared between patients undergoing only primary lung transplantation (PLTx) and those undergoing PLTx and later ReLTx. Time to death was compared using the Kaplan-Meier method. The risk of ReLTx was analyzed in Cox regression models. Any interaction between type of transplantation, single/double, and PLTx/ReLTx was investigated. RESULTS: ReLTx was performed in 49 patients. Survival after ReLTx at 30 days and 1, 2, and 5 years was 90%, 76%, 71%, and 55%, respectively, and the corresponding survival after PLTx was 94%, 82%, 76%, and 61%, respectively. A hazard ratio of 1.73 for ReLTx was shown (95% confidence interval [CI], 1.14 to 2.63; P = .011). After adjustments for sex, age, diabetes, renal function, preoperative ventilator, and extracorporeal membrane oxygenation, the hazard ratio was 1.43 (95% CI, 0.90 to 2.26; P = .13). ReLTx was performed in 8 patients (16%) within the first year after PLTx. The 1-year survival for this group was 50% compared with 81% (P = .18) for patients who underwent ReLTx later than 1 year after the PLTx. One-year survival after double ReLTx was 60% (95% CI, 25% to 83%) compared with 79% (95% CI, 63% to 89%) for single ReLTx. CONCLUSIONS: ReLTx is a reasonable option for a selected group of patients. Ideally, a number of well-established risk factors are avoided and the ReLTx is performed more than 1 year after the PLTx.

Exploration of human, rat, and rabbit embryonic cardiomyocytes suggests K-channel block as a common teratogenic mechanism.

AIMS: Several drugs blocking the rapidly activating potassium (K(r)) channel cause malformations (including cardiac defects) and embryonic death in animal teratology studies. In humans, these drugs have an established risk for acquired long-QT syndrome and arrhythmia. Recently, associations between cardiac defects and spontaneous abortions have been reported for drugs widely used in pregnancy (e.g. antidepressants), with long-QT syndrome risk. To investigate whether a common embryonic adverse-effect mechanism exists in the human, rat, and rabbit embryos, we made a comparative study of embryonic cardiomyocytes from all three species. METHODS AND RESULTS: Patch-clamp and quantitative-mRNA measurements of K(r) and slowly activating K (K(s)) channels were performed on human, rat, and rabbit primary cardiomyocytes and cardiac samples from different embryo-foetal stages. The K(r) channel was present when the heart started to beat in all species, but was, in contrast to human and rabbit, lost in rats in late organogenesis. The specific K(r)-channel blocker E-4031 prolonged the action potential in a species- and development-dependent fashion, consistent with the observed K(r)-channel expression pattern and reported sensitive periods of developmental toxicity. E-4031 also increased the QT interval and induced 2:1 atrio-ventricular block in multi-electrode array electrographic recordings of rat embryos. The K(s) channel was expressed in human and rat throughout the embryo-foetal period but not in rabbit. CONCLUSION: This first comparison of mRNA expression, potassium currents, and action-potential characteristics, with and without a specific K(r)-channel blocker in human, rat, and rabbit embryos provides evidence of K(r)-channel inhibition as a common mechanism for embryonic malformations and death.

Islet-1 cells are cardiac progenitors present during the entire lifespan: from the embryonic stage to adulthood.

The aim of this study was to longitudinally characterize the distribution of cells actively expressing the progenitor transcription factor islet-1 (Isl1+) during the embryonic life, the postnatal period, and adulthood. In this study, we have used direct immunohistochemical staining toward the protein Isl1 in a longitudinal rat model. Cells actively expressing Isl1 were traced in embryos from gestational day (GD) 11 until adulthood. In early cardiac development (GD 11), the Isl1+ progenitors were located in a greater abundance in the paracardiac regions, areas suggested to be the second heart field. To a lesser extent, Isl1+ cells were present within the bulbotruncal region and the truncus arteriosus. During the following days until GD 15, the Isl1+ cells were mainly observed at the proximal outflow tract (OFT) and at the inflow area of the right atrium. No Isl1+ cells were detected in the left ventricle. Compared with GD 11, more Isl1+ cells seemed to co-express cardiomyocyte markers and a minority of the Isl1+ cells was undifferentiated. Unexpectedly, only few undifferentiated Isl1+ cells were Ki67+ while a lot of TnT+ cardiomyocytes were proliferating in the ventricles. After birth, immature Isl1+ cells were still present in the OFT where they resided until adulthood. Our data suggest that during embryogenesis, Isl1+ cells migrate from extracardiac regions into the proximal part of the heart, proliferating and giving rise to cardioblasts. Unexpectedly, only a minority of the Isl1+ cells while a majority of ventricular cardiomyocytes were proliferating. The Isl1+ cell pool persists into adulthood, which might open up new strategies to repair damaged myocardium.

Early first trimester human embryonic cardiac Islet-1 progenitor cells and cardiomyocytes: Immunohistochemical and electrophysiological characterization.

The aims of this study were to systematically characterize the distribution, proliferation, and differentiation of Islet-1(+)(Isl1(+)) progenitor cells in the early first trimester human embryonic heart during which period most of the organogenesis takes place. In hearts of gestational week 5 to 10 Isl1(+)cells were identified and mainly clustered in the outflow tract and to a lesser extent in the atria and in the right ventricle. Some of the clusters were also troponin T(+). Unexpectedly a only few Isl1(+)cells were Ki67(+)while in the ventricles a majority of Isl1(-)troponinT(+)cells were Ki67(+). Cultures derived from the digested embryonic heart developed into spontaneously beating cardiospheres. At harvest cells in these cardiospheres showed frequent expression of troponin T(+)and Nkx2.5(+), while Isl1 was expressed only in scattered cells. Only a minority of the cultured cells expressed Ki67. The cardiospheres could be frozen, thawed, and recultured to beating cardiospheres. In a multielectrode array system, the beating cardiospheres were responsive to adrenergic stimulation and exhibited rate-dependent action potential duration. In conclusion, the early first trimester human embryonic heart expresses clusters of Isl1(+)cells, some of which differentiate into cardiomyocytes. Unexpectedly, only a minority of the Isl1(+)cells, while a majority of ventricular cardiomyocytes, were proliferating. Spontaneously beating cardiospheres could be derived from the human embryonic heart and these cardiospheres showed functional frequency control.

Costimulation blockade induces tolerance to HESC transplanted to the testis and induces regulatory T-cells to HESC transplanted into the heart.

In order to study the ability of costimulation blockade to induce tolerance to human embryonic stem cells (HESC), severe combined immunodeficient (SCID), and immunocompetent C57BL/6 mice treated with costimulation blockade received intratesticular and intramyocardial HESC transplants. All SCID mice with intratesticular HESC transplants developed teratoma. When SCID mice were transplanted intramyocardially, only two of five mice developed teratoma-like tumors. C57BL/6 mice transplanted intratesticularly and treated with costimulation blockade all developed teratoma and were surrounded by CD4(+)CD25(+)Foxp3(+) T-cells, while isotype control treated recipients rejected their grafts. Most C57BL/6 mice transplanted intramyocardially and treated with costimulation blockade demonstrated lymphocytic infiltrates 1 month after transplantation, whereas one maintained its graft. Isolation of regulatory T-cells from intramyocardial transplanted recipients treated with costimulation blockade demonstrated specificity toward undifferentiated HESC and down-regulated naive T-cell activation toward HESC. These results demonstrate that costimulation blockade is sufficiently robust to induce tolerance to HESC in the immune-privileged environment of the testis. HESC specific regulatory T-cells developed to HESC transplanted to the heart and the success of transplantation was similar to that seen in SCID mice.

Erythropoietin has an antiapoptotic effect after myocardial infarction and stimulates in vitro aortic ring sprouting.

Aims were to explore if darbepoietin-alpha in mouse can induce angiogenesis and if moderate doses after myocardial infarction stimulates periinfarct capillary and arteriolar densities, cell proliferation, and apoptosis. Myocardial infarction was induced by ligation of LAD. Mouse aortic rings (0.8mm) were cultured in matrigel and the angiogenic sprouting was studied after addition of darbepoietin-alpha with and without VEGF-165. After 12 days the hemoglobin concentration was 25% higher in the darbepoietin-alpha treated mice than in the control group. No difference in capillary densities in the periinfarct or noninfarcted areas was seen with darbepoietin-alpha. Cell proliferation was about 10 times higher in the periinfarct area than in the noninfarcted wall. Darbepoietin-alpha treatment led to a decrease of cell proliferation (BrdU, (p<0.02)) and apoptosis (TUNEL, p<0.005) with about 30% in the periinfarct area. Darbepoietin-alpha and VEGF-165 both independently induced sprouting from aortic rings. The results suggest that darbepoietin-alpha can induce angiogenesis but that moderate doses after myocardial infarction are not angiogenic but antiapoptotic.

New proposals for testing drugs with IKr-blocking activity to determine their teratogenic potential.

Drugs blocking the potassium current IKr, either as an intended pharmacologic effect (eg antiarrhythmics dofetilide and almokalant) or as an unwanted side-effect (eg antihistamine astemizole, propulsive drug cisapride, antidepressive drugs and macrolide antibiotics) are potential human teratogens. It is the contention of this paper that the existing repeat dose regimen used in teratology studies to fulfil regulatory requirements, does not properly identify the teratogenic risk of these drugs. Results from conventional studies for dofetilide and almokalant showed high rates of postimplantation embryonic death with few malformed fetuses. For astemizole and cisapride only embryonic death was seen. These latter results were not considered important because they occurred either in the presence of maternal toxicity and/or at high doses. Subsequent studies have shown that IKr-blockers are highly teratogenic when administered on single gestational days (GD) during a sensitive period of rat pregnancy (GD 10-14) when they induce a high incidence of stage-specific malformations. This teratogenic activity of astemizole and cisapride was missed in the original teratology studies. Mechanistically IKr-blockers cause bradycardia and arrhythmia of the embryonic heart and while an embryo may be able to survive a single day exposure to a teratogenic dose, repeat dosing often leads to death of the embryo. With this review we suggest that new drugs identified at the preclinical stage of development as having IKr-blocking properties, should undergo more comprehensive teratology testing including single GD dosing and studies using embryo culture. This would further help identify and characterise their teratogenic potential.

Polytherapy with hERG-blocking antiepileptic drugs: increased risk for embryonic cardiac arrhythmia and teratogenicity.

BACKGROUND: The antiepileptic drugs (AEDs) phenytoin, phenobarbital, dimethadione, and carbamazepine cause a similar pattern of malformations in humans, with an increased risk after polytherapy. The teratogenicity has been linked to cardiac rhythm disturbances and hypoxic damage as a consequence of their common potential to inhibit a specific potassium ion current (IKr). The IKr is of major importance for embryonic cardiac repolarization and rhythm regulation. This study investigated whether these AEDs cause irregular rhythm and if various combinations of AEDs result in higher arrhythmia risk than exposure to a single AED. METHODS: The effects on heart rhythm of a single AED (monotherapy), and of various combinations (polytherapy) of AEDs, in gestational day 10 C57BL mouse embryos in culture were analyzed and graphically illustrated during a 25 s recording with a digitalization technique. RESULTS: All of the studied AEDs caused increased intervals between heartbeats (resulting in bradycardia) and large variations in the interval between heartbeats (resulting in irregular rhythm) in a concentration-dependent manner in cultured mouse embryos. Dimethadione caused irregular rhythm at concentrations within and phenytoin slightly above the therapeutic ranges. Polytherapy resulted in more substantial prolongation of the mean interval between heartbeats (>60 ms) than monotherapy at clinically relevant concentrations. CONCLUSIONS: The results suggest that polytherapy more than monotherapy causes substantial prolongation of the cardiac repolarization, a marker associated with high risk of developing irregular rhythm during longer exposure periods (days to months). This supports the idea that the increased risk for malformations following polytherapy is linked to an increased risk for cardiac rhythm disturbances.

Embryonic cardiac arrhythmia and generation of reactive oxygen species: common teratogenic mechanism for IKr blocking drugs.

In the adult organism, it is well established that hypoxia followed by reperfusion may be fatal and result in generation of reactive oxygen species (ROS) and subsequent tissue damage. There is also considerable evidence that temporary decrease or interruption in oxygen supply to the embryo and ROS generation during reperfusion result in tissue damage in embryonic tissues. A wide spectrum of different malformations by transient embryonic hypoxia could be produced, depending on the duration, extent, and timing of the hypoxic event. It is the contention of this paper that drugs that block the potassium channel IKr, either as an intended pharmacologic effect or as an unwanted side-effect, are potentially teratogenic by a common ROS related mechanism. Drugs blocking the IKr channel, such as almokalant, dofetilide, phenytoin, cisapride and astemizole, do all produce a similar pattern of hypoxia-related malformations. Mechanistic studies show that the malformations are preceded by embryonic cardiac arrhythmia and periods of hypoxia/reoxygenation in embryonic tissues. Pretreatment or simultaneous treatment with radical scavengers with capacity to capture ROS, markedly decrease the teratogenicity of different IKr blocking drugs. A second aim of this review is to demonstrate that the conventional design of teratology studies is not optimal to detect malformations caused by IKr blocking drugs. Repeated high doses result in high incidences of embryonic death due embryonic cardiac arrhythmia, thus masking their teratogenic potential. Instead, single dosing on specific days is proposed to be a better way to characterize the teratogenic potential of Ikr blocking drugs.

Phenytoin teratogenicity: hypoxia marker and effects on embryonic heart rhythm suggest an hERG-related mechanism.

BACKGROUND: The antiepileptic drug phenytoin (PHT) is a human and animal teratogen. The teratogenicity has been linked to PHT-induced embryonic cardiac arrhythmia and hypoxic damage during a period when regulation of embryonic heart rhythm is highly dependent on a specific K(+) ion current (I(Kr)). PHT has been shown to inhibit I(Kr). The aims of this study were to investigate whether teratogenic doses cause embryonic hypoxia during and after the I(Kr) susceptible period and to further characterize PHT effects on embryonic heart rhythm. METHODS: Pregnant C57BL mice were administered the hypoxia marker pimonidazole followed by PHT or saline (controls) on GD 10 or GD 15. The embryos were fixed and sectioned, and the immunostained sections were analyzed with a computer assisted image analysis. Effects of PHT (0-250 microM) on heart rhythm in GD 10 embryos cultured in vitro were videotaped and then analyzed by using a digitalization technique. RESULTS: PHT dose-dependently increased the hypoxia staining (6- and 11-fold after maternal dosing of 100 and 150 mg/kg, respectively) during the period I(Kr) is expressed and functional (GD 10). In contrast, there were no differences between the PHT doses in hypoxia staining, and much less pronounced hypoxia after this period (GD 15). With increasing PHT concentrations, increased length of the interval (bradycardia) and large variations in length between individual heartbeats (arrhythmia) were recorded. CONCLUSIONS: PHT induced bradycardia/arrhythmia and severe embryonic hypoxia during the I(Kr) susceptible period, supporting the idea of an I(Kr)-arrhythmia-hypoxia-related teratogenic mechanism.

Teratogenicity of the I(Kr)-blocker cisapride: relation to embryonic cardiac arrhythmia.

Cisapride and mosapride are structurally and pharmacologically related prokinetic agents. In contrast to mosapride, cisapride causes embryonic lethality in teratology studies, and has been related to fatal cardiac arrhythmia in the adult. The arrhythmogenic potential of cisapride is linked to its potential to inhibit a specific ion channel (I(Kr)) as a side effect. Mosapride lacks I(Kr)-blocking properties. The aims of this study were (1) to compare the effects of cisapride and mosapride on embryonic heart rhythm in vitro and (2) to investigate if cisapride in vivo, has potential to induce hypoxia-related teratogenic effects as has been shown for selective I(Kr)-blockers. Cisapride induced severe embryonic bradycardia (approximately 60% decrease), and arrhythmia in 94% of the cultured rat embryos at 1000 ng/ml. Mosapride did not induce any bradycardia or arrhythmia up to 2000 ng/ml. In vivo, single dose administration of cisapride to rats on gestational day (GD) 13 caused digital reductions (8/108 fetuses, 4/9 litters) at 75 mg/kg and high incidence of embryonic death (55-82%) at 100-200 mg/kg. Identical developmental toxic effects have been described after temporary interruption of oxygen supply, and after single dose administration of selective I(Kr)-blockers, on the same GD. The results support the idea that all potent I(Kr)-blocking agents have the potential to cause embryolethality and teratogenicity, and that the adverse effects are mediated via hypoxic episodes due to embryonic arrhythmia.