Трансплантация пластов мезенхимальных прогениторных клеток сердца для васкуляризации миокарда после инфаркта

Purpose. To develop a method of producing tissue-engineered constructs (TECs) on the basis of resident mesenchymal progenitor cells (MPC) of the human heart and to assess the effect of TECs transplantation on regenerative processes in the heart using a model of myocardial infarction in rats.Materials and methods. Human resident MPCs were isolated from the right atrial auricle of CAD patients. A similar protocol was used to obtain MPCs from Wistar rats. The MPC immunophenotype was determined by cytofluorometry. Corresponding TECs were obtained on the basis of MPC sheets of human and rats' hearts. Myocardial infarction in rats was induced by ligation of the anterior descending coronary artery followed by TEC transplantation. Euthanasia was performed 30 days after the transplantation. Histological examination of the implant and vascularization cells, morphometric analysis, tracking of the MPC differentiation ability, determination of the content of growth factors by solid-phase ELISA were carried out. Statistical evaluation of the significance of differences was performed using the Statistica 8.0 software package.Results. The analysis of the obtained cell constructs showed that they consisted of several layers of cells interacting with each other by means of connexin 43 and were characterized by good cell viability as a part TECs. The number of vessels in the peri-infarction area under the transplant from the MPC was significantly higher than that in the reference group with signs of differentiation of cardiac MPCs transplanted into endothelial vascular cells.The increased vascularization was combined with an increase in the area of viable myocardial sites and a decrease in LV cavity dilation. Analysis of the cardiac MPC secretion products showed that they produce the most important growth factors and cytokines that regulate angiogenesis and migration of stem cells.Conclusion. The strategy of using epicardial TEC transplantation based on MPC sheets seems to be a rational approach for effective delivery of viable stem/progenitor cells to the damaged myocardium. The use of TEC helps to reduce or temporarily eliminate the effect of factors that contribute to progressive heart dysfunction by local paracrine exposure and activation of the revascularization processes in the affected zone.Цель. Разработать способ получения тканеинженерных конструкций (ТИК), на основе резидентных мезенхимальных прогениторных клеток (МПК) сердца человека и оценить влияние трансплантации ТИК на регенеративные процессы в сердце на модели инфаркта миокарда крысы.Материалы и методы. Резидентные МПК человека выделяли из ушка правого предсердия пациентов с ИБС. По аналогичному протоколу выделяли МПК крысы линии Wistar. Методом проточной цитофлуориметрии определяли иммунофенотип МПК. На основе пластов МПК сердца человека и крыс получали соответствующие ТИК. Инфаркт миокарда у крыс был индуцирован путем перевязки передней нисходящей коронарной артерии, после чего проводили трансплантацию ТИК. Через 30 дней после трансплантации выполняли эвтаназию. Проводили гистологическую оценку состояния клеток имплантата и васкуляризации, морфометрический анализ, трекинг дифференцировочной способности МПК, определение содержания ростовых факторов методом твердофазного ИФА. Статистическую оценку достоверности различий проводили с использованием программного пакета Statistica 8.0.Результаты. Анализ полученных клеточных конструкций показал, что они состоят из нескольких слоев клеток, взаимодействующих между собой при помощи коннексин–43, и характеризуются хорошей жизнеспособностью клеток в составе ТИК. Количество сосудов в периинфарктной области под трансплантатом из МПК было значительно больше, чем в контрольной группе, с признаками дифференцировки трансплантированных МПК сердца в эндотелиальные клетки сосудов.Увеличение васкуляризации сочеталось с увеличением площади участков жизнеспособного миокарда, уменьшением дилатации полости ЛЖ. Анализ продуктов секреции МПК сердца показал, что они продуцируют важнейшие факторы роста и цитокины, регулирующие ангиогенез и миграцию стволовых клеток.Заключение. Стратегия использования эпикардиальной трансплантации ТИК на основе пластов из МПК представляется рациональным подходом для эффективной доставки жизнеспособных стволовых/прогениторных клеток в поврежденный миокард. Применение ТИК способствует уменьшению или временному исключению действия факторов, способствующих прогрессирующей дисфункции сердца, путем локального паракринного воздействия и активации процессов реваскуляризации зоны повреждения

Активность аутофагии в клетках эпикарда при развитии острого перикардита

Pericarditis is a group of polyetiological diseases often associated with emergence of life– threatening conditions. Poor knowledge of underlying cellular mechanisms and lack of relevant approaches to investigation of pericarditis result in major challenges in diagnosis and treatment.The aim of this work was to identify changes in the activity of autophagy in epicardial cells in acute pericarditis.Materials and methods. Acute pericarditis in mice was induced by intrapericardial injection of Freund's adjuvant in the study group (n=15). The control group included animals receiving either intrapericardial injection of phosphate-buffered saline (PBS) (n=15), or sham surgery without injections (n=7). On Days 3 or 5 after surgery the animals were euthanized under isoflurane anesthesia. Immunofluorescence staining of cardiac tissue cryo-sections and immunoblotting were used to assess the intensity of inflammation and autophagy in the epicardium.Results. Inflammation and other signs of acute pericarditis resulting in thickening of some epicardial areas were found: 68+9% in the control (after PBS injection) and 124+22% after Freund's adjuvant injection (p=0.009); other signs included cellular infiltration of epicardium and multiple adhesions. The epicardial layer exhibited signs of mesothelial cells reorganization with 11-fold increase of autophagy markers LC3 II/LC3 I ratio: 0.07+0.02 in the control group (after PBS injection) and 0.84+0.07 - in acute pericarditis (p=0.04), and accumulation of collagen fibers.Conclusion. Development of acute pericarditis is accompanied by activation of epicardial mesothelial cells, intensified autophagy and development of fibrous changes in epicacardial/ subepicardial areas.Перикардит – это группа полиэтилогичных заболеваний, которые часто ассоциированы с развитием жизнеугрожающих состояний. Существенные сложности при их диагностике и лечении в значительной степени обусловлены ограниченным пониманием клеточных механизмов развития перикардита и отсутствием релевантных подходов при его изучении.Цель данной работы: выявление изменения активности аутофагии в клетках эпикарда при остром перикардите.Материалы и методы. Острый перикардит в сердце мышей моделировали путем интраперикардиального введения 50 мкл адъюванта Фрейнда (n=15). Контрольным животным интраперкардиально вводили 50 мкл раствора фосфатно-солевого буфера (ФСБ) (n=15) или выполняли операции без интраперикардиального введения какого-либо препарата (ложнооперированные животные, n=7). На 3-й или 5-й день от проведения хирургической операции после ингаляционной наркотизации изофлюраном производили эвтаназию животных. Активность воспаления в зоне эпикарда и выраженность аутофагии исследовали с помощью иммунофлуоресцентных методов окрашивания криосрезов сердца и иммуноблотинга.Результаты. Обнаружили развитие воспалительной реакции и появление признаков острого перикардита, ассоциированного с утолщением зоны эпикарда: 68+9% в контроле (после введения ФСБ) и 124+22% после введения адъюванта Фрейнда, p=0,009, его полиморфно-клеточной инфильтрацией и формированием множественных спаек. В составе эпикардиального слоя наблюдали признаки реорганизации клеток мезотелия, 11-кратное повышение соотношения в них маркеров аутофагии LC3 II/LC3 I: 0,07+0,02 в контроле (после введения ФСБ) и 0,84+0,07 при остром перикардите, р=0,04, а также аккумуляцию коллагеновых волокон.Заключение. Развитие острого перикардита сопровождается активацией клеток эпикардиального мезотелия, повышением выраженности аутофагии и развитием фиброзных изменений в зоне эпикарда/субэпикарда. Изучение возможности модуляции аутофагии с целью воздействия на развитие острого перикардита является предметом дальнейших исследований

Эпикардиальная трансплантация пластов из мезенхимальных стромальных клеток жировой клетчатки способствует активации эпикарда и стимулирует ангиогенез при инфаркте миокарда (экспериментальное исследование)

Aim: to evaluate the impact of tissue-engineered structures (TES) transplantation based on mesenchymal stromal cell (MSC) sheets in myocardial infarction on the activation of the epicardial cell pool and vascularization of the damaged zone.Materials and methods. Mesenchymal stromal cells were obtained from samples of subcutaneous fat of Wistar rats and C57Bl/6 mice. Tissue engineering structures were obtained by culturing cell sheets on thermosensitive plates (Nunc Dishes with UpCell Surface). Transplantation of TESs was performed after myocardial infarction modeling in rats by ligation of the anterior descending coronary artery. Transplant cells and damaged zones were assessed using immunofluorescent staining of myocardial cryosections. The impact of MSC secretion products on the migration activity of epicardial cells in vitro was evaluated using the explant culture method.Results. MSCs in TESs after transplantation remain viable and induce activation of the epicardial cell pool and local increase of the damaged zone vascularization. The in vitro experiments showed that the conditioned environment of MSCs stimulates the migratory activity of epicardial cells and initiates the formation of activated Wt1/POD1 precursor cells.Conclusion. TES transplantation on the basis of MSC sheets seems to be a promising approach for effective delivery of viable cells into myocardium to activate the epicardial cellular niche and reparative angiogenesis.Цель исследования: оценить влияние трансплантации тканеинженерных конструкций (ТИК) на основе пластов мезенхимальных стромальных клеток (МСК) при инфаркте миокарда на активацию эпикардиального пула клеток и васкуляризацию зоны повреждения.Материалы и методы. МСК получили из образцов подкожной жировой клетчатки крыс линии Wistar и мышей линии C57Bl/6. ТИК получили путем культивирования пластов клеток на чашках с термочувствительным покрытием (Nunc Dishes with UpCell Surface). Трансплантацию ТИК проводили после моделирования инфаркта миокарда у крысы путем перевязки передней нисходящей коронарной артерии. Оценку состояния клеток трансплантата и зоны повреждения проводили с использованием иммунофлуоресцентного окрашивания криосрезов миокарда. Для оценки влияния продуктов секреции МСК на миграционную активность клеток эпикарда in vitro использовали метод эксплантной культуры.Результаты. МСК в составе ТИК после трансплантации сохраняют жизнеспособность и вызывают активацию эпикардиального пула клеток и локальное повышение васкуляризации зоны повреждения. Эксперименты in vitro показали, что кондиционированная среда МСК оказывает стимулирующее воздействие на миграционную активность клеток эпикарда и вызывает образование активированных Wt1/POD1 клеток-предшественниц.Заключение. Трансплантация ТИК на основе пластов МСК представляется рациональным подходом для эффективной доставки жизнеспособных клеток в миокард с целью активирующего воздействия на эпикардиальную клеточную нишу и репаративный ангиогенез

Combined Transfer of Human VEGF165 and HGF Genes Renders Potent Angiogenic Effect in Ischemic Skeletal Muscle

Increased interest in development of combined gene therapy emerges from results of recent clinical trials that indicate good safety yet unexpected low efficacy of “single-gene” administration. Multiple studies showed that vascular endothelial growth factor 165 aminoacid form (VEGF165) and hepatocyte growth factor (HGF) can be used for induction of angiogenesis in ischemic myocardium and skeletal muscle. Gene transfer system composed of a novel cytomegalovirus-based (CMV) plasmid vector and codon-optimized human VEGF165 and HGF genes combined with intramuscular low-voltage electroporation was developed and tested in vitro and in vivo. Studies in HEK293T cell culture, murine skeletal muscle explants and ELISA of tissue homogenates showed efficacy of constructed plasmids. Functional activity of angiogenic proteins secreted by HEK293T after transfection by induction of tube formation in human umbilical vein endothelial cell (HUVEC) culture. HUVEC cells were used for in vitro experiments to assay the putative signaling pathways to be responsible for combined administration effect one of which could be the ERK1/2 pathway. In vivo tests of VEGF165 and HGF genes co-transfer were conceived in mouse model of hind limb ischemia. Intramuscular administration of plasmid encoding either VEGF165 or HGF gene resulted in increased perfusion compared to empty vector administration. Mice injected with a mixture of two plasmids (VEGF165+HGF) showed significant increase in perfusion compared to single plasmid injection. These findings were supported by increased CD31+ capillary and SMA+ vessel density in animals that received combined VEGF165 and HGF gene therapy compared to single gene therapy. Results of the study suggest that co-transfer of VEGF and HGF genes renders a robust angiogenic effect in ischemic skeletal muscle and may present interest as a potential therapeutic combination for treatment of ischemic disorders

Transplantation of Cardiac Mesenchymal Progenitor Cell Sheets for Myocardial Vascularization after an Infarction (Experimental Study)

Purpose. To develop a method of producing tissue-engineered constructs (TECs) on the basis of resident mesenchymal progenitor cells (MPC) of the human heart and to assess the effect of TECs transplantation on regenerative processes in the heart using a model of myocardial infarction in rats.Materials and methods. Human resident MPCs were isolated from the right atrial auricle of CAD patients. A similar protocol was used to obtain MPCs from Wistar rats. The MPC immunophenotype was determined by cytofluorometry. Corresponding TECs were obtained on the basis of MPC sheets of human and rats' hearts. Myocardial infarction in rats was induced by ligation of the anterior descending coronary artery followed by TEC transplantation. Euthanasia was performed 30 days after the transplantation. Histological examination of the implant and vascularization cells, morphometric analysis, tracking of the MPC differentiation ability, determination of the content of growth factors by solid-phase ELISA were carried out. Statistical evaluation of the significance of differences was performed using the Statistica 8.0 software package.Results. The analysis of the obtained cell constructs showed that they consisted of several layers of cells interacting with each other by means of connexin 43 and were characterized by good cell viability as a part TECs. The number of vessels in the peri-infarction area under the transplant from the MPC was significantly higher than that in the reference group with signs of differentiation of cardiac MPCs transplanted into endothelial vascular cells.The increased vascularization was combined with an increase in the area of viable myocardial sites and a decrease in LV cavity dilation. Analysis of the cardiac MPC secretion products showed that they produce the most important growth factors and cytokines that regulate angiogenesis and migration of stem cells.Conclusion. The strategy of using epicardial TEC transplantation based on MPC sheets seems to be a rational approach for effective delivery of viable stem/progenitor cells to the damaged myocardium. The use of TEC helps to reduce or temporarily eliminate the effect of factors that contribute to progressive heart dysfunction by local paracrine exposure and activation of the revascularization processes in the affected zone

A Novel Thromboplastin-Based Rat Model of Ischemic Stroke

The thromboembolic ischemia model is one of the most applicable for studying ischemic stroke in humans. The aim of this study was to develop a novel thromboembolic stroke model, allowing, by affordable tools, to reproduce cerebral infarction in rats. In the experimental group, the left common carotid artery, external carotid artery, and pterygopalatine branch of maxillary artery were ligated. A blood clot that was previously formed (during a 20 min period, in a catheter and syringe, by mixing with a thromboplastin solution and CaCl2) was injected into the left internal carotid artery. After 10 min, the catheter was removed, and the incision was sutured. The neurological status of the animals was evaluated using a 20-point scale. Histological examination of brain tissue was performed 6, 24, 72 h, and 6 days post-stroke. All groups showed motor and behavioral disturbances 24 h after surgery, which persisted throughout the study period. A histological examination revealed necrotic foci of varying severity in the cortex and subcortical regions of the ipsilateral hemisphere, for all experimental groups. A decrease in the density of hippocampal pyramidal neurons was revealed. Compared with existing models, the proposed ischemic stroke model significantly reduces surgical time, does not require an expensive operating microscope, and consistently reproduces brain infarction in the area of the middle cerebral artery supply

Epicardial Transplantation of Adipose Mesenchymal Stromal Cell Sheets Promotes Epicardial Activation and Stimulates Angiogenesis in Myocardial Infarction (Experimental Study)

Aim: to evaluate the impact of tissue-engineered structures (TES) transplantation based on mesenchymal stromal cell (MSC) sheets in myocardial infarction on the activation of the epicardial cell pool and vascularization of the damaged zone.Materials and methods. Mesenchymal stromal cells were obtained from samples of subcutaneous fat of Wistar rats and C57Bl/6 mice. Tissue engineering structures were obtained by culturing cell sheets on thermosensitive plates (Nunc Dishes with UpCell Surface). Transplantation of TESs was performed after myocardial infarction modeling in rats by ligation of the anterior descending coronary artery. Transplant cells and damaged zones were assessed using immunofluorescent staining of myocardial cryosections. The impact of MSC secretion products on the migration activity of epicardial cells in vitro was evaluated using the explant culture method.Results. MSCs in TESs after transplantation remain viable and induce activation of the epicardial cell pool and local increase of the damaged zone vascularization. The in vitro experiments showed that the conditioned environment of MSCs stimulates the migratory activity of epicardial cells and initiates the formation of activated Wt1/POD1 precursor cells.Conclusion. TES transplantation on the basis of MSC sheets seems to be a promising approach for effective delivery of viable cells into myocardium to activate the epicardial cellular niche and reparative angiogenesis