Involvement of Urokinase-Type Plasminogen Activator Receptor in the Formation of a Profibrotic Microenvironment in the Epicardial Region

The study of the mechanisms of development and progression of fibrosis is one of the key directions of modern cardiology. Our work suggests that the urokinase receptor (uPAR) is involved in the regulation of mesothelial cell activity and epicardial fibrosis development, which, when interacting with specific ligands and intermediate proteins, can activate intracellular signaling, trigger the cascade of proteolytic reactions, including local plasmin formation and activation of matrix metalloproteinases, providing matrix remodeling.Objective: to perform a comparative study of fibrogenic activity of the epicardium in the hearts of uPAR-/- and wild-type animals and evaluate the effect of cardiac microenvironment factors on the migration activity of epicardial mesothelial cells.Material and methods. We used histological and immunofluorescent staining, microarray analysis of proinflammatory cytokine levels, and a method for assessing the migratory properties of epicardial cells.Results. Results. We found that compared to wild-type animals, uPAR-/- animals show significant thickening of the epicardial area (2.46+0.77 (uPAR-/- mice) and 1.02+0.17 (Wt mice) relative units, P=0.033) accompanied by accumulation of extracellular matrix proteins. Deficiency of uPAR gene leads to formation of proinflammatory microenvironment in the heart (increased levels of proinflammatory factors such as IL-1, IL-13, IL-17, RANTES and MIP1), increased migratory activity of epicardial mesothelial cells, accumulation of TCF21+fibroblast/myofibroblast precursors (29.8+13.7 (uPAR-/- mouse) and 3.03+0.8 (Wt mouse) cells per visual field,P=0.02), as well as development of subepicardial fibrosis.Conclusion. These findings suggest that uPAR is a promising candidate for the developing targeted agents to prevent the development and progression of cardiac fibrosis

Клеточные пласты на основе кардиальных прогениторных клеток продуцируют проангиогенные факторы роста и оказывают локальное стимулирующее воздействие на формирование капилляров после инфаркта миокарда

Background.      The application of tissue-engineered constructs that simulate the natural microenvironment of cells, maintain their viability and functional properties, is a new promising route for the treatment of ischemic diseases. However, the mechanisms that ensure the effectiveness of this type of treatment and the principles of choosing the optimal population of progenitor cells remain poorly understood.        Aim. To study the profile of secretion of proangiogenic growth factors of cardiosphere-derived cell sheet (CS), and to study the effect of their transplantation on postinfarction myocardial vascularization.            Methods. Assembly of cardiosphere-derived cell sheets were performed on thermosensitive culture plates. Characterization of cell sheets was performed using immunofluorescence staining and a commercial kit for the determination of proangiogenic factors “Mouse Angiogenesis Antibody Array”. The evaluation of the angiogenic properties of the cell graft in vivo was carried out using a rat myocardial infarction model.              Results. It was found that the cardiosphere-derived cell sheet secrete factors involved in the regulation of vasculo-/angiogenesis. At the same time, the cultivation of cell sheets under hypoxic conditions (3% O2) led to an increase in the secretion of proangigenic factors VEGF and pIgF, fGf-1, FGF-2, endothelin-1, as well as MMP-9, which is involved in extracellular matrix remodeling. Cell sheet transplantation on the epicardial surface of the heart after myocardial infarction ensures cell viability and local increase in capillarization of the damaged area. Conclusion. Thus, the application of cardiosphere-derived cell sheets, which have proangiogenic properties and ability to maintain post transplantation cell survival, can be considered as a promising approach for the development of new methods of therapy for heart diseasesАктуальность. Использование тканеинженерных конструкций, моделирующих естественное микроокружение клеток, поддерживающих их жизнеспособность и функциональные свойства, является новым перспективным направлением лечения заболеваний ишемической природы. Однако механизмы, обеспечивающие эффективность такого вида лечения и условия выбора оптимальной популяции прогениторных клеток, остаются малоизученными.Цель. Исследовать профиль секреции проангиогенных факторов роста клетками кардиосфер, формирующими клеточные пласты (КП), и влияние трансплантации конструкции на восстановление капилляров после инфаркта.Материалы и методы               . Для создания КП на основе клеток кардиосфер использованы чашки с термочувствительным покрытием. Характеристику КП выполняли с помощью иммунофлуоресцентного окрашивания и коммерческого набора для определения проангиогенных факторов Mouse Angiogenesis Antibody Array (R&D, США). Оценку ангиогенных свойств клеточного графта in vivo проводили на модели инфаркта миокарда у крыс.Результаты. Обнаружено, что сформированные КП секретируют факторы, участвующие в регуляции васкуло- и ангиогенеза. При этом культивирование КП в условиях умеренной гипоксии (3% O2) приводило к повышению секреции проангиогенных факторов VEGF и PIGF, FGF1, FGF2, эндотелина 1, а также MMP9, регулирующей уровень VEGF и участвующей в ремоделировании внеклеточного матрикса. Трансплантация КП на эпикардиальную поверхность сердца после инфаркта способствует сохранению жизнеспособности клеток и локальному повышению васкуляризации зоны повреждения.Заключение. Использование КП на основе клеток кардиосфер, обладающих проангиогенными свойствами и возможностью поддерживать посттрансплантационную выживаемость клеток, может рассматриваться в качестве перспективного подхода для разработки новых средств лечения заболеваний сердца

3D сфероиды - клеточная модель для изучения воздействия гипоксии на эпикардиальное микроокружение

Fundamental research in recent years has allowed us to reassess the molecular and cellular mechanisms of cardiac ontogenesis and its repair after damage. The epicardium, the outer, tightly adjoining layer of the cardiac wall formed by epicardial mesothelial cells, collagen and elastic fibers, has gained special relevance as an important participant of reparative processes. Better insight into poorly understood epicardial function is challenged due to anatomical issues and lack of relevant cellular models.The aim of this study was to develop a spheroid 3D model of the epicardial microenvironment and determine responses of spheroids to hypoxia.Materials and methods. Spheroids were harvested in V-shaped culture dishes with a low adhesion coating. Immunofluorescent staining of cryosections, histological methods and real-time PCR were used for characterization of cultured spheroids.Results. We demonstrated that cultivation of cells under low adhesion conditions in V-shaped culture dishes resulted in the formation of spheroids with an average size of 136+21 µm and cell viability rates of over 98%. The cells in the spheroids cultured under normoxic conditions formed tight junctions and were characterized by a low level of proliferation and the ability to synthesize extracellular matrix proteins. Under hypoxia cells in the spheroids showed partial loss of intercellular contacts, acquired a spindle shape, started to express HIF1a, SNAIL, COL1Al and accumulate collagen. All these features demonstrated the activation of mesothelial(endothelial)-mesenchymal transition strongly resembling epicardial cellular responses to ischemia in vivo.Conclusion. An epicardial spheroid cell culture model suitable for study cellular responses to hypoxic environment was developed. This model can be used to clarify mechanisms regulating epicardial microenvironment and test new targeted candidate drugs.Фундаментальные исследования последних лет позволили переосмыслить молекулярные и клеточные механизмы онтогенеза сердца и его репарации после повреждения. Особую актуальность приобретает изучение эпикарда — наружного, плотно примыкающего к миокарду слоя сердечной стенки, образованного гетерогенной популяцией клеток эпикардиального мезотелия, коллагеновыми и эластическими волокнами, являющегося важным участником репаративных процессов. Изучение эпикарда затруднено в связи с анатомическими ограничениями и отсутствием релевантных клеточных моделей.Цель исследования. Разработка 3D модели эпикардиального микроокружения и оценка влияния гипоксии на ее характеристики.Материал и методы. Сборку сфероидов проводили в V-образных культуральных чашках с низкоадгезионным покрытием. Характеристику сфероидов выполняли с использованием иммунофлуоресцентного окрашивания криосрезов, гистологических методов, ПЦР в реальном времени.Результаты. Культивирование клеток в низкоадгезионных условиях в V-образных культуральных чашках ведет к формированию сфероидов, имеющих размер 136±21 мкм и показатели жизнеспособности клеток более 98%. Клетки в составе сфероидов, культивированных в условиях нормоксии, образовывали плотные межклеточные контакты, характеризовались низким уровнем пролиферации и способностью синтезировать белки внеклеточного матрикса. В условиях гипоксии клетки сфероидов частично утрачивали межклеточные контакты, приобретали веретенообразную форму, экспрессировали HIF1a, SNAI1, ACTA2, FN1, COL1A1 и накапливали коллаген, что указывает на признаки активации мезотелиально-мезенхимального перехода и сходные черты с клеточным ответом эпикарда на острое ишемическое повреждение in vivo.Заключение. На основе клеточного сфероида разработали и охарактеризовали модель эпикарда, которая может реализовать клеточный ответ на воздействие гипоксического стимула и быть использована для изучения механизмов регуляции эпикардиального микроокружения, тестирования лекарственных препаратов направленного действия

ДИФФЕРЕНЦИАЛЬНАЯ ДИАГНОСТИКА МЕДИАСТИНАЛЬНОЙ ЛИМФАДЕНОПАТИИ ПРИ ЛИМФОМЕ И САРКОИДОЗЕ С ПОМОЩЬЮ ДИФФУЗИОННО-ВЗВЕШЕННОЙ МАГНИТНО-РЕЗОНАНСНОЙ ТОМОГРАФИИ

The aim of the study. Investigation of the potential of diffusion-weighted magnetic resonance imaging (DW-MRI) with apparent diffusion coefficient (ADC) evaluation for differential diagnosis of mediastinal lymphadenopathy in lymphoma and sarcoidosis.Materials and methods. The study included 15 patients with sarcoidosis and 7 patients with lymphoma. In each patient ADC values of 3 largest lymph nodes were measured.Results. Differences of mean ADC values in lymphoma (1,01±0,3×10-3mm2/s) and sarcoidosis (1,51±0,25×10-3mm2/s) were significant, p<0,0001. Optimal cut-point value for ADC of ≤1,1×10-3mm2/s differentiated lymphoma from sarcoidosis with sensitivity of 76,2%, specificity of 97,8% and accuracy of 90,9%, the area under the ROC-curve was 0,89.Conclusion. Preliminary results of the study have demonstrated the potential of DW-MRI in differential diagnosis of lymphoma and sarcoidosis and expediency of the further study.Цель. Изучение возможностей диффузионно-взвешенной магнитно-резонансной томографии (ДВ-МРТ) грудной клетки с оценкой измеряемого коэффициента диффузии (ИКД) в дифференциальной диагностике медиастинальной лимфаденопатии при лимфоме и  саркоидозе.Материалы и методы. В исследование включены 15 пациентов с саркоидозом и 7  пациентов с лимфомой. У каждого пациента значения ИКД измерены в трех наиболее  крупных лимфатических узлах.Результаты. Выявлены значимые отличия средних значений ИКД при лимфоме (1,01±0,3×10-3 мм2/с) и саркоидозе (1,51±0,25×10-3 мм2/с), p<0,0001. При использовании порогового значения ИКД ≤1,1×10-3 мм2/с чувствительность, специфичность и точность метода в дифференциальной диагностике лимфомы и саркоидоза составили 76,2; 97,8 и  90,9% соответственно, площадь под ROC-кривой — 0,89.Заключение. Предварительные результаты показали высокую информативность ДВ-МРТ в дифференциальной диагностике лимфомы и саркоидоза и целесообразность продолжения исследования

Evaluation of the Efficiency of Ultra-Early Endovascular Embolization of Cerebral Aneurisms in Subarachnoid Hemorrage

The review discusses the effectiveness of endovascular embolization of intracranial aneurysms in subarachnoid hemorrhage (SAH) in the first 24 hours after rupture. The need for this study is dictated by the medical and social significance of the disease, in particular, the high degree of mortality and disability in SAH of aneurysmal origin, including in young patients. The choice of embolization timing for SAH remains a subject of discussion in the professional community since the introduction of the technique into clinical practice. Performing embolization on the first day after the onset of the  disease, the so-called “ultra-early embolization” has a potentially positive effect on the outcomes of SAH, reducing the number of deaths and increasing the number of favorable outcomes. The main advantage of this tactic is to prevent repeated rupture of the aneurysm, the probability of which is highest in the first day of  the disease, as well as the possibility of starting intensive therapy for SAH at the earliest possible time

Intramyocardial Injection of Plasmid Encoding Platelet Growth Factor Increases Epicardial-Mediated Post Infarction Myocardial Vascularization (Experimental Study)

Increasing incidence of ischemic diseases and limited resources for their treatment stimulate increased interest in studying the mechanisms of vascularization and finding new approaches for its promotion. One of these approaches is gene therapy aimed at activating the epicardium to produce the vascular precursor cells and microenvironment for the «assembly» of de novo vessels.The aim is to investigate the possibility of activating epicardial cells and post infarction cardiac vascularization by injecting a genetic construct encoding PDGFBB.Material and methods. A model of experimental myocardial infarction in a rat with subsequent intramyocardial injection of normal saline solution, control plasmid and plasmid encoding PDGFBB was used. The study of PDGFBB effect on epicardial cell activity was performed on the ex vivo model, as well as in vitro mesothelial cell culture.Results. Post infarction injection of plasmid encoding PDGFBB increases the density of the vascular network in the peri-infarct area as well as migration of pericytes to the injured zone. PDGFBB promotes activation of epicardial cell pool and expression of smooth muscle cell markers in them (shown on the ex vivo model), as well as stimulates activation of epithelial-mesenchymal transition (in vitro).Conclusion. Intramyocardial injection of a genetic construct encoding PDGFBB after an experimental myocardial infarction stimulated vascularization of the peri-infarction zone, which may have been partially due to the activation of the epicardial cell pool

Angiogenic and pleiotropic effects of VEGF165 and HGF combined gene therapy in a rat model of myocardial infarction

<div><p>Since development of plasmid gene therapy for therapeutic angiogenesis by J. Isner this approach was an attractive option for ischemic diseases affecting large cohorts of patients. However, first placebo-controlled clinical trials showed its limited efficacy questioning further advance to practice. Thus, combined methods using delivery of several angiogenic factors got into spotlight as a way to improve outcomes. This study provides experimental proof of concept for a combined approach using simultaneous delivery of VEGF165 and HGF genes to alleviate consequences of myocardial infarction (MI). However, recent studies suggested that angiogenic growth factors have pleiotropic effects that may contribute to outcome so we expanded focus of our work to investigate potential mechanisms underlying action of VEGF165, HGF and their combination in MI. Briefly, Wistar rats underwent coronary artery ligation followed by injection of plasmid bearing VEGF165 or HGF or mixture of these. Histological assessment showed decreased size of post-MI fibrosis in both—VEGF165- or HGF-treated animals yet most prominent reduction of collagen deposition was observed in VEGF165+HGF group. Combined delivery group rats were the only to show significant increase of left ventricle (LV) wall thickness. We also found dilatation index improved in HGF or VEGF165+HGF treated animals. These effects were partially supported by our findings of c-kit+ cardiac stem cell number increase in all treated animals compared to negative control. Sporadic Ki-67+ mature cardiomyocytes were found in peri-infarct area throughout study groups with comparable effects of VEGF165, HGF and their combination. Assessment of vascular density in peri-infarct area showed efficacy of both–VEGF165 and HGF while combination of growth factors showed maximum increase of CD31+ capillary density. To our surprise arteriogenic response was limited in HGF-treated animals while VEGF165 showed potent positive influence on a-SMA+ blood vessel density. The latter hinted to evaluate infiltration of monocytes as they are known to modulate arteriogenic response in myocardium. We found that monocyte infiltration was driven by VEGF165 and reduced by HGF resulting in alleviation of VEGF-stimulated monocyte taxis after combined delivery of these 2 factors. Changes of monocyte infiltration were concordant with a-SMA+ arteriole density so we tested influence of VEGF165 or HGF on endothelial cells (EC) that mediate angiogenesis and inflammatory response. In a series of <i>in vitro</i> experiments we found that VEGF165 and HGF regulate production of inflammatory chemokines by human EC. In particular MCP-1 levels changed after treatment by recombinant VEGF, HGF or their combination and were concordant with NF-κB activation and monocyte infiltration in corresponding groups <i>in vivo</i>. We also found that both–VEGF165 and HGF upregulated IL-8 production by EC while their combination showed additive type of response reaching peak values. These changes were HIF-2 dependent and siRNA-mediated knockdown of HIF-2α abolished effects of VEGF165 and HGF on IL-8 production. To conclude, our study supports combined gene therapy by VEGF165 and HGF to treat MI and highlights neglected role of pleiotropic effects of angiogenic growth factors that may define efficacy via regulation of inflammatory response and endothelial function.</p></div

Quantitative analysis of c-kit+ CSC and Ki-67+ cardiomyocyte density.

<p>(A) Representative images of sections co-stained for c-kit/troponin-I (upper panel) and Ki-67 (lower panel); (B) Statistical analysis of c-kit+ CSC and Ki-67+ cardiomyocyte counts. n = 4-5/group, data presented as Mean±S.D.; *p<0.05 vs “pC4W” negative control (Mann-Whitney’s U-test). No significant differences were found in comparison of “VEGF+HGF” vs. “VEGF” or “HGF” groups.</p

Evaluation of angio- and arteriogenesis after gene delivery of VEGF165 and HGF.

<p>A—Representative images of peri-infacrt area in cross section of myocardium after immunofluorescent visualization of CD31 and α-SMA (upper row of images in ×200 magnification emphasizes capillary morphology, lower row in ×100 magnification shows representative examples of arteriole-sized vessels); B—Results of vascular density analysis in groups treated by VEGF165, HGF or combined gene delivery (data presented as Mean±S.E.M.). Mann-Whitney U-test (n = 4-5/group): *p<0.05 vs. “pC4W” negative control; **p<0.01 vs. “pC4W”, “HGF” or “VEGF+HGF” groups; N.S.–non-significant (p>0.05 vs. “pC4W” negative control).</p