Regeneration of Cryoinjury Induced Necrotic Heart Lesions in Zebrafish Is Associated with Epicardial Activation and Cardiomyocyte Proliferation

In mammals, myocardial cell death due to infarction results in scar formation and little regenerative response. In contrast, zebrafish have a high capacity to regenerate the heart after surgical resection of myocardial tissue. However, whether zebrafish can also regenerate lesions caused by cell death has not been tested. Here, we present a simple method for induction of necrotic lesions in the adult zebrafish heart based on cryoinjury. Despite widespread tissue death and loss of cardiomyocytes caused by these lesions, zebrafish display a robust regenerative response, which results in substantial clearing of the necrotic tissue and little scar formation. The cellular mechanisms underlying regeneration appear to be similar to those activated in response to ventricular resection. In particular, the epicardium activates a developmental gene program, proliferates and covers the lesion. Concomitantly, mature uninjured cardiomyocytes become proliferative and invade the lesion. Our injury model will be a useful tool to study the molecular mechanisms of natural heart regeneration in response to necrotic cell death

Revealing New Mouse Epicardial Cell Markers through Transcriptomics

The epicardium has key functions during myocardial development, by contributing to the formation of coronary endothelial and smooth muscle cells, cardiac fibroblasts, and potentially cardiomyocytes. The epicardium plays a morphogenetic role by emitting signals to promote and maintain cardiomyocyte proliferation. In a regenerative context, the adult epicardium might comprise a progenitor cell population that can be induced to contribute to cardiac repair. Although some genes involved in epicardial function have been identified, a detailed molecular profile of epicardial gene expression has not been available.Using laser capture microscopy, we isolated the epicardial layer from the adult murine heart before or after cardiac infarction in wildtype mice and mice expressing a transgenic IGF-1 propeptide (mIGF-1) that enhances cardiac repair, and analyzed the transcription profile using DNA microarrays.Expression of epithelial genes such as basonuclin, dermokine, and glycoprotein M6A are highly enriched in the epicardial layer, which maintains expression of selected embryonic genes involved in epicardial development in mIGF-1 transgenic hearts. After myocardial infarct, a subset of differentially expressed genes are down-regulated in the epicardium representing an epicardium-specific signature that responds to injury.This study presents the description of the murine epicardial transcriptome obtained from snap frozen tissues, providing essential information for further analysis of this important cardiac cell layer

Сочетанная лучевая терапия у больных раком предстательной железы с поражением семенных пузырьков

Background. It seems advisable to investigate the feasibility of radiation therapy combined with hormone therapy to treat locally advanced prostate cancer. Combination of two ways to deliver ionizing radiation doses enables us to elevate a total tumor dose and to reduce radiation exposure to critical organs. However, the feasibility of combinatorial radiation therapy (CRT) in prostate cancer patients with seminal vesicle invasion remains insufficiently investigated. The number of studies focusing on this problem is still extremely small.Objective of this study is to evaluate the efficacy and toxicity of radiation therapy combined with hormone therapy for prostate cancer with seminal vesicle invasion.Materials and methods. From April 2016 to April 2020, 52 patients with prostate cancer (cT3bN0) received CRT at the clinic of the A.F. Tsyb Medical Radiological Research Center. The median patient follow-up was 29.7 months (from 11.9 to 58.4 months). The mean age of patients was 65.7 years. The initial mean level of PSA was 28.7 ng/ml. Fifty (96.2 %) patients were given radiation therapy together with hormone therapy.Results. The tolerability of CRT appeared satisfactory. Grade I acute radiation-induced reactions of the urinary tract occurred in 13 (25 %) patients; grade II ones - in 2 (3.8 %) patients. Grade I acute radiation-induced reactions of the gastrointestinal tract occurred in 11 (21.5 %) patients; grade II ones - in 1 (1.9 %) patient. Grade I late complications of the urinary tract were noted in 4 (7.7 %) patients; grade II ones - in 2 (3.8 %) patients. Grade I late complications of the gastrointestinal tract were noted in 2 (3.8 %) patients; grade II ones - in 3 (5.8 %) patients.The three-year cancer-specific survival rate was 97 %; the overall survival rate was 83 %. Eight (15.4 %) patients showed prostate cancer progression. Five (9.6 %) patients experienced cancer recurrence in the form of distant bone metastases. In 1 (1.9 %) patient, disease recurrence was associated with involvement of regional lymphatic collectors and distant inguinal lymph node metastases. Local recurrence was noted in 1 (1.9 %) patient. One (1.9 %) patient developed loco-regional recurrence with distant metastasis to bones. The three-year recurrence-free survival rate was 75.6 %.Conclusion. Our study demonstrates that CRT is highly effective in prostate cancer (cT3bN0) treatment while having an acceptable level of complications.Введение. Представляется целесообразным изучение возможности проведения сочетанной лучевой терапии (СЛТ) в объеме гормонолучевого лечения рака предстательной железы у больных с местно-распространенными формами заболевания. Сочетание 2 способов подведения дозы ионизирующего излучения позволяет выполнять эскалацию суммарной очаговой дозы в опухоли при уменьшении лучевой нагрузки на критические органы. Количество исследований, посвященных возможности проведения СЛТ у больных раком предстательной железы с инвазией в семенные пузырьки, крайне незначительно.Цель исследования - анализ эффективности и токсичности СЛТ в объеме гормонолучевого лечения рака предстательной железы с инвазией в семенные пузырьки.Материалы и методы. С апреля 2016 г. по апрель 2020 г. в клинике МРНЦ им. А.Ф. Цыба - филиала НМИЦ радиологии 52 больным раком предстательной железы стадии сT3bN0 проведена СЛТ. Медиана периода наблюдения составила 29,7 (11,9-58,4) мес. Средний возраст пациентов - 65,7 года. Средний уровень инициального простатического специфического антигена - 28,7 нг/мл. У 50 (96,2 %) пациентов СЛТ выполнена в объеме гормонолучевого лечения.Результаты. Отмечена удовлетворительная переносимость СЛТ. Острые лучевые реакции I степени тяжести со стороны мочевыводящих путей зарегистрированы у 13 (25 %) больных, II степени - у 2 (3,8 %). Острые лучевые реакции I степени со стороны желудочно-кишечного тракта отмечены у 11 (21,5 %) пациентов, II степени - у 1 (1,9 %). Поздние осложнения I степени со стороны мочевыводящих путей зафиксированы у 4 (7,7 %) больных, II степени -у 2 (3,8 %). Поздние осложнения I степени со стороны желудочно-кишечного тракта отмечены у 2 (3,8 %) больных, II степени - у 3 (5,8 %).Трехлетняя опухолевоспецифическая выживаемость составила 97 %, общая выживаемость - 83 %. Прогрессирование заболевания наблюдали у 8 (15,4 %) пациентов. У 5 (9,6 %) больных рецидив заболевания был представлен отдаленными метастазами (кости), у 1 (1,9 %) больного - поражением регионарных лимфоколлекторов с отдаленными метастазами в паховые лимфатические узлы. У 1 (1,9 %) пациента отмечен локальный рецидив, у 1 (1,9 %) - локорегионарный рецидив с отдаленным метастазированием (кости). Трехлетняя безрецидивная выживаемость составила 75,6 %.Заключение. Результаты проведенного исследования демонстрируют хорошую эффективность СЛТ у больных раком предстательной железы стадии сT3bN0 при приемлемом уровне осложнений

Long-Distance Signals Are Required for Morphogenesis of the Regenerating Xenopus Tadpole Tail, as Shown by Femtosecond-Laser Ablation

tadpoles has recently emerged as an important model for these studies; we explored the role of the spinal cord during tadpole tail regeneration.Using ultrafast lasers to ablate cells, and Geometric Morphometrics to quantitatively analyze regenerate morphology, we explored the influence of different cell populations. For at least twenty-four hours after amputation (hpa), laser-induced damage to the dorsal midline affected the morphology of the regenerated tail; damage induced 48 hpa or later did not. Targeting different positions along the anterior-posterior (AP) axis caused different shape changes in the regenerate. Interestingly, damaging two positions affected regenerate morphology in a qualitatively different way than did damaging either position alone. Quantitative comparison of regenerate shapes provided strong evidence against a gradient and for the existence of position-specific morphogenetic information along the entire AP axis.We infer that there is a conduit of morphology-influencing information that requires a continuous dorsal midline, particularly an undamaged spinal cord. Contrary to expectation, this information is not in a gradient and it is not localized to the regeneration bud. We present a model of morphogenetic information flow from tissue undamaged by amputation and conclude that studies of information coming from far outside the amputation plane and regeneration bud will be critical for understanding regeneration and for translating fundamental understanding into biomedical approaches

In vitro epithelial-to-mesenchymal transformation in human adult epicardial cells is regulated by TGFβ-signaling and WT1

Adult epicardial cells are required for endogenous cardiac repair. After myocardial injury, they are reactivated, undergo epithelial-to-mesenchymal transformation (EMT) and migrate into the injured myocardium where they generate various cell types, including coronary smooth muscle cells and cardiac interstitial fibroblasts, which contribute to cardiac repair. To understand what drives epicardial EMT, we used an in vitro model for human adult epicardial cells. These cells have an epithelium-like morphology and markedly express the cell surface marker vascular cell adhesion marker (VCAM-1). In culture, epicardial cells spontaneously undergo EMT after which the spindle-shaped cells now express endoglin. Both epicardial cells before and after EMT express the epicardial marker, Wilms tumor 1 (WT1). Adding transforming growth factor beta (TGFβ) induces loss of epithelial character and initiates the onset of mesenchymal differentiation in human adult epicardial cells. In this study, we show that TGFβ-induced EMT is dependent on type-1 TGFβ receptor activity and can be inhibited by soluble VCAM-1. We also show that epicardial-specific knockdown of Wilms tumor-1 (WT1) induces the process of EMT in human adult epicardial cells, through transcriptional regulation of platelet-derived growth factor receptor alpha (Pdgfrα), Snai1 and VCAM-1. These data provide new insights into the process of EMT in human adult epicardial cells, which might provide opportunities to develop new strategies for endogenous cell-based cardiac repair

ПОВТОРНОЕ ОБЛУЧЕНИЕ ИНТРАКРАНИАЛЬНЫХ ОПУХОЛЕЙ АКТИВНЫМ СКАНИРУЮЩИМ ПУЧКОМ ПРОТОНОВ

Recently, there has been a growing interest in the use of active beam scanning proton therapy for re-irradiation of CNS tumors. Although this treatment option for recurrent intracranial tumors is considered effective, however, it is rarely used because of severe radiation-induced neurologic complications. We present 3 case reports of using active beam scanning proton therapy for re-irradiation of intracranial tumors. In the first case, the patient with high-grade glioma was treated with proton beam therapy 1.5 years after the previous conventional external beam radiation therapy. Tumor regression lasting 18 months was achieved. In the second case, the patient received repeated proton beam therapy for recurrent esthesioneuroblastoma. After 1.5 years of follow-up, no evidence of recurrence and late toxicity was observed. The third case was unique, since the patient with recurrent hemangiopericitoma of the orbit received 4 courses of radiotherapy to the same area. A significant regression with no evidence of radiation-induced damage was also seen 12 months after the completion of radiation treatment. Conclusion. Recent advances in proton therapy allow for re-irradiation of CNS tumors in effective total doses, while reducing exposure to surrounding normal tissues. The use of PET-CT for contouring the target allows the localization and volume of the metabolically active tumor tissue to be precisely determined.Современные технические достижения лучевой терапии в частности, протонная терапия активным сканирующим пучком и визуальным сопровождением лечения, способствуют возрождению интереса к повторным курсам облучения опухолей центральной нервной системы. Традиционно данная опция лечения при рецидивах интракраниальных новообразований хотя и является эффективной, но спе- циалисты достаточно редко ее используют ввиду опасения тяжелых постлучевых неврологических осложнений. Есть лишь отдельные статьи с небольшим числом наблюдений, посвященные данной теме. Описание клинического случая. Приводим собственные наблюдения – 3 клинических случая повторного облучения интракраниальных опухолей с использованием активного сканирующего пучка протонов. В первом случае представлен опыт лечения больной глиомой высокой степени злокачествен- ности, облученной повторно пучком протонов через 1,5 года после предшествующего конвенционального облучения. Была достигнута значительная регрессия опухоли, подтвержденная данными ПЭТ-КТ, в течение 18 мес. Во втором случае описано повторное протонное облучение пациентки с рецидиви- рующей эстезионейробластомой основания черепа, наблюдаемой нами в течение полутора лет без признаков рецидива и поздней токсичности. Третий случай является уникальным, так как пациентке с рецидивирующей гемангиоперицитомой орбиты был проведен четвертый курс облучения на одну зону. У больной также достигнута значительная регрессия без признаков осложнений облучения в течение 12 мес. Заключение. Использование современных технических достижений протонной терапии по- зволяет проводить повторное облучение опухолей центральной нервной системы в эффективных суммарных очаговых дозах, с возможностью значительно снизить лучевую нагрузку на окружающие нормальные структуры. Выполнение ПЭТ-КТ для оконтуривания мишени позволяет уточнить лока- лизацию и объем метаболически активной ткани опухоли. Следование принципу ALARA позволяет минимизировать токсичность лечения

Cardiac Hypertrophy Involves Both Myocyte Hypertrophy and Hyperplasia in Anemic Zebrafish

Background: An adult zebrafish heart possesses a high capacity of regeneration. However, it has been unclear whether and how myocyte hyperplasia contributes to cardiac remodeling in response to biomechanical stress and whether myocyte hypertrophy exists in the zebrafish. To address these questions, we characterized the zebrafish mutant tr265/tr265, whose Band 3 mutation disrupts erythrocyte formation and results in anemia. Although Band 3 does not express and function in the heart, the chronic anemia imposes a sequential biomechanical stress towards the heart. Methodology/principal findings: Hearts of the tr265/tr265 Danio rerio mutant become larger than those of the sibling by week 4 post fertilization and gradually exhibit characteristics of human cardiomyopathy, such as muscular disarray, re-activated fetal gene expression, and severe arrhythmia. At the cellular level, we found both increased individual cardiomyocyte size and increased myocyte proliferation can be detected in week 4 to week 12 tr265/tr265 fish. Interestingly, all tr265/tr265 fish that survive after week-12 have many more cardiomyocytes of smaller size than those in the sibling, suggesting that myocyte hyperplasia allows the long-term survival of these fish. We also show the cardiac hypertrophy process can be recapitulated in wild-type fish using the anemia-inducing drug phenylhydrazine (PHZ). Conclusions/significance: The anemia-induced cardiac hypertrophy models reported here are the first adult zebrafish cardiac hypertrophy models characterized. Unlike mammalian models, both cardiomyocyte hypertrophy and hyperplasia contribute to the cardiac remodeling process in these models, thus allowing the effects of cardiomyocyte hyperplasia on cardiac remodeling to be studied. However, since anemia can induce effects on the heart other than biomechanical, non-anemic zebrafish cardiac hypertrophy models shall be generated and characterized

Dedifferentiation and Proliferation of Mammalian Cardiomyocytes

It has long been thought that mammalian cardiomyocytes are terminally-differentiated and unable to proliferate. However, myocytes in more primitive animals such as zebrafish are able to dedifferentiate and proliferate to regenerate amputated cardiac muscle.Here we test the hypothesis that mature mammalian cardiomyocytes retain substantial cellular plasticity, including the ability to dedifferentiate, proliferate, and acquire progenitor cell phenotypes. Two complementary methods were used: 1) cardiomyocyte purification from rat hearts, and 2) genetic fate mapping in cardiac explants from bi-transgenic mice. Cardiomyocytes isolated from rodent hearts were purified by multiple centrifugation and Percoll gradient separation steps, and the purity verified by immunostaining and RT-PCR. Within days in culture, purified cardiomyocytes lost their characteristic electrophysiological properties and striations, flattened and began to divide, as confirmed by proliferation markers and BrdU incorporation. Many dedifferentiated cardiomyocytes went on to express the stem cell antigen c-kit, and the early cardiac transcription factors GATA4 and Nkx2.5. Underlying these changes, inhibitory cell cycle molecules were suppressed in myocyte-derived cells (MDCs), while microRNAs known to orchestrate proliferation and pluripotency increased dramatically. Some, but not all, MDCs self-organized into spheres and re-differentiated into myocytes and endothelial cells in vitro. Cell fate tracking of cardiomyocytes from 4-OH-Tamoxifen-treated double-transgenic MerCreMer/ZEG mouse hearts revealed that green fluorescent protein (GFP) continues to be expressed in dedifferentiated cardiomyocytes, two-thirds of which were also c-kit(+).Contradicting the prevailing view that they are terminally-differentiated, postnatal mammalian cardiomyocytes are instead capable of substantial plasticity. Dedifferentiation of myocytes facilitates proliferation and confers a degree of stemness, including the expression of c-kit and the capacity for multipotency

Dedifferentiation of Foetal CNS Stem Cells to Mesendoderm-Like Cells through an EMT Process

Tissue-specific stem cells are considered to have a limited differentiation potential. Recently, this notion was challenged by reports that showed a broader differentiation potential of neural stem cells, in vitro and in vivo, although the molecular mechanisms that regulate plasticity of neural stem cells are unknown. Here, we report that neural stem cells derived from mouse embryonic cortex respond to Lif and serum in vitro and undergo epithelial to mesenchymal transition (EMT)-mediated dedifferentiation process within 48 h, together with transient upregulation of pluripotency markers and, more notably, upregulation of mesendoderm genes, Brachyury (T) and Sox17. These induced putative mesendoderm cells were injected into early gastrulating chick embryos, which revealed that they integrated more efficiently into mesoderm and endoderm lineages compared to non-induced cells. We also found that TGFβ and Jak/Stat pathways are necessary but not sufficient for the induction of mesendodermal phenotype in neural stem cells. These results provide insights into the regulation of plasticity of neural stem cells through EMT. Dissecting the regulatory pathways involved in these processes may help to gain control over cell fate decisions

Сочетанная лучевая терапия у больных раком предстательной железы высокого риска. Анализ осложнений лечения

Purpose: to analyze toxicity of combined modality radiotherapy in high-risk prostate cancer patients.Material and methods. Short-term outcomes after combined modality radiotherapy were analyzed in 88 prostate cancer patients treated at clinic of the a.F. tsyb medical Radiological Research center between april 2016 and February 2018. the median follow-up time was 13 months (range, 3–23 months). the mean age of the patients was 64.8 years (range, 49–80 years). an initial psawas in the range of 3.5–114 ng/ml (mean 16.7 ng/ml). all patients were considered high-risk according to the d’amico classification. the patients were divided into two groups according to the sequence of combined modality radiotherapy. in group 1, the patients (n=45) received conformal EBRtto a total dose of 44–46 gy, and 2–3 weeks later, they underwent high–dose rate 192ir- brachytherapy (one single fraction of 15 gy) as a boost to EBRt. in group 2, the patients (n=43) were treated with interstitial brachytherapy followed by EBRtdelivering at total doses of 44–46 gy.Results. All patients eventually completed the combined modality radiotherapy course planned for them. acute, grade 1 genitourinary (gu) toxicities (Rtog/EoRtc) occurred in 29 (32.9 %) patients. acute gastrointestinal (gi) toxicity was grade 1 in 17 (19.3 %) and grade 2 in 2 (2.3 %) patients. late gutoxicity was grade 1 in 6 (6.8 %) and grade 2 in 3 (3.4 %) patients. an urethral stricture developed in 1 (1.1 %) patient. late gitoxicity was grade 1 in 8 (9.1 %) patients and grade 2 in 6 (6.8 %) patients.Conclusion. The preliminary results of this study suggest satisfactory tolerability of combined modality radiotherapy by prostate cancer patients. the level of acute toxicity and complications in critical organs is quite acceptable, which maintains high quality of life for patients and does not exceed the published data.Цель исследования – анализ токсичности сочетанной лучевой терапии (СЛТ) у больных раком предстательной железы (РПЖ) высокого риска прогрессирования.Материал и методы. Проведенанализ непосредственных результатов СЛТ у 88 больных РПЖ, получивших лечение в клинике МРНЦ им. А.Ф. Цыба с апреля 2016 г. по февраль 2018 г. Медиана наблюдения составила 13 мес (от 3 до 23 мес). Возраст пациентов варьировал от 49 до 80 лет, в среднем – 64,8 года. Инициальный уровень ПСА составлял от 3,5 до 114 нг/мл (в среднем – 16,7 нг/мл). Все пациенты относились к группе высокого риска прогрессирования заболевания (d’amico et al.). В зависимости от последовательности этапов СЛТ больные были разделены на две группы. У больных первой группы (n=45) проводили курс конформной дистанционной лучевой терапии (ДЛТ) в суммарной очаговой дозе (СОД) 44–46 Гр с последующим (через 2–3 нед) сеансом брахитерапии (192ir в дозе 15 Гр, однократно). Во второй группе (n=43) пациентам проводили внутритканевую терапию с последующим курсом ДЛТ в СОД 44–46 Гр.Результаты. Запланированный курс СЛТ выполнен в полном объёме у всех больных РПЖ. Острые лучевые реакции I степени тяжести (Rtog/EoRtc) со стороны нижних отделов мочевыводящих путей (МВП) наблюдали у 29 (32,9 %) пациентов. Острые лучевые реакции со стороны прямой кишки I степени отмечены у 17 (19,3 %), II степени – у 2 (2,3 %) пациентов. Поздние лучевые осложнения I степени со стороны нижних отделов МВП отмечены у 6 (6,8 %), II степени – у 3 (3,4 %) пациентов; формирование стриктуры уретры – у 1 (1,1 %) больного. Поздние лучевые осложнения I степени со стороны нижних отделов желудочно-кишечного тракта отмечены у 8 (9,1 %), II степени – у 6 (6,8 %) больных.Заключение. Предварительные результаты исследования свидетельствуют об удовлетворительной переносимости СЛТ больными РПЖ. Уровень развития острых лучевых реакций и осложнений со стороны критических органов вполне приемлем, позволяет сохранить высокое качество жизни пациентов и не превышает опубликованные данные