Cytokines and HIF-1α as dysregulation factors of migration and differentiation of monocyte progenitor cells of endotheliocytes in the pathogenesis of ischemic cardiomyopathy

Background. Angiogenic endothelial dysfunction and progenitor endothelial cells (EPCs) in ischemic cardiomyopathy (ICMP) have not been studied enough.The aim. To establish the nature of changes in the cytokine profile and HIF-1α in blood and bone marrow associated with impaired differentiation of monocytic progenitor cells of endotheliocytes (CD14+VEGFR2+) in the bone marrow and their migration into the blood in patients with coronary heart disease (CHD), suffering and not suffering from ICMP.Materials and methods. A single-stage, single-centre, observational case-control study was conducted involving 74 patients with CHD, suffering and not suffering from ICMP (30 and 44 people, respectively), and 25 healthy donors. In patients with CHD, bone marrow was obtained during coronary bypass surgery, peripheral blood – before surgery. Healthy donors were taken peripheral blood. The number of CD14+VEGFR2+ in bone marrow and blood was determined by flow cytometry; the concentration of IL-6, TNF-α, M-CSF, GM-CSF, MCP-1 and HIF-1α – by the method of enzyme immunoassay.Results. A high content of CD14+VEGFR2+ cells in the blood of patients with CHD without cardiomyopathy was established relative to patients with ICMP against the background of a comparable number of these cells in myeloid tissue. Regardless of the presence of ICMP in the blood, patients with CHD showed an excess of TNF-α, a normal concentration of IL-6, GM-CSF, HIF-1α and a deficiency of M-CSF, and in the bone marrow supernatant, the concentration of IL-6 and TNF-α exceeded that in the blood plasma (the level of GM-CSF – only in patients without cardiomyopathy). With ICMP, the normal concentration of MCP-1 was determined in the blood plasma, and with CHD without cardiomyopathy, its elevated content was determined.Conclusion. The formation of ICMP is accompanied by insufficient activation of EPCs migration with the CD14+VEGFR2+ phenotype in blood without disruption of their differentiation in the bone marrow, which associated with the absence of an increase in the concentration of MCP-1 in blood plasma and not associated with the plasma content of M-CSF, GM-CSF, HIF-1α, IL-6 and TNF-α

Interleukins 4 and 6 as factors of modulation of subpopulation composition of blood monocytes in patients with ischemic cardiomyopathy

Aim. To evaluate the ratio of the fractions of classical, intermediate, non-classical and transitional monocytes in correlation with the concentration of interleukins 4 and 6 in the blood of patients with ischemic cardiomyopathy. Methods. 18 patients with ischemic cardiomyopathy (17 men and 1 woman) aged 47-66 years with circulatory insufficiency of functional class II-III according to the classification of heart failure of the New York Heart Association, were examined. The control group included 14 healthy donors matched by gender and age to patients with ischemic cardiomyopathy without any diseases of cardiovascular system and other systems in an exacerbation stage. In blood of the patients with ischemic cardiomyopathy, the relative content of classical (CD14++CD16-), intermediate (CD14++CD16+), non-classical (CD14+CD16+) and transitional (CD14+CD16-) monocytes was assessed by flow cytometry and the concentration of interleukins 4 and 6 by enzyme-linked immunosorbent assay (ELISA). Results. It was shown that the number of non-classical monocytes in the blood of patients with ischemic cardiomyopathy was 2 times lower than normal (5.05 % [4.08; 6.58] and 10.07 % [9.34; 13.84], respectively, p < 0.01), as well as the concentration of interleukin-4 (0.02 pg/ml [0; 0.04] and 0.15 pg/ml [0.05; 0.65], respectively, p < 0.05). The number of classical monocytes in the blood of patients had a tendency to decrease, and the proportion of intermediate monocytes and the concentration of interleukin-6, on the contrary, were slightly higher than in healthy individuals, and were interdependent (r = 0.61; p < 0.05). The relative content of transitional monocytes in the blood was comparable with that of healthy donors. Conclusions. The subpopulation composition of blood monocytes in patients with ischemic cardiomyopathy is characterized by a deficiency of the fraction of non-classical monocytes with protective properties against endothelium, and interleukin-4 in the blood with a certain increase in the content of interleukin-6 and the number of intermediate cells with ability to cooperate with T-lymphocytes, which predisposes to diffuse atheromatosis of small coronary arteries and diffuse hypoxic myocardial damage in ischemic cardiomyopathy

Expression of CD80 and HLA-DR molecules on blood monocytes in patients with pulmonary tuberculosis

We examined expression pattern of CD80 and HLA-DR pro-inflammatory molecules on the monocytes in patients with pulmonary tuberculosis (TB), depending on the clinical form of the disease and susceptibility of the pathogen to anti-tuberculosis drugs. The study involved forty-five patients with newly diagnosed pulmonary TB (25 men and 20 women aged 18 to 55 years, average age — 44.0±12.4 years). The control group included 15 healthy donors with similar socio-demographic characteristics as in TB patients. Venous blood was used as biomaterial for assays. Studies of the monocyte immunophenotype were carried out by flow cytometry of whole blood cells using Cytoflex flow cytometer (Beckman Coulter, USA) with specific monoclonal antibodies (eBioscience, USA). We determined the content of cells expressing surface markers of monocytes, i.e., CD14, CD45, CD80, and HLA-DR. The results of this study were evaluated using SPSS Statistics 17.0 standard software package and Microsoft Excel. In the course of the study, we have suggested a working hypothesis that the monocytes in TB patients, still being in circulation, can express activation markers during their migration to inflammation focus, especially CD80 and HLA-DR molecules. Analysis of the total CD14+ monocyte number showed its decrease in all forms and variants of clinical course of pulmonary tuberculosis compared with the control group. Assessment of pro-inflammatory markers expressed on CD14 positive monocytes, i.e., HLA-DR activation marker and CD80 co-stimulatory molecule, showed that the number of monocytes with HLA-DR expression in all TB patients was higher than in healthy donors. HLA- DR expression on CD14+ monocytes in the group of patients with infiltrative TB proved to be 15% higher than in patients with disseminated TB. The expression of CD80 on CD14+ monocytes in TB patients showed no differences between the groups and varied within the normal range. Hence, an imbalance within monocyte population in patients with pulmonary tuberculosis, regardless of its clinical form and drug sensitivity of the pathogen is developed, due to decrease in total number of CD14+ cells, along with increased relative number of monocytes expressing HLA-DR activation marker (pro-inflammatory phenotype). Meanwhile, expression of the CD80 co-stimulatory molecule on monocytes was within normal values

Дифференциация и субпопуляционный состав VEGFR2+ моноцитов крови и костного мозга при ишемической кардиомиопатии

Aim. To identify disturbances of differentiation and subpopulation composition of VEGFR2+ cells in the blood and bone marrow associated with the features of the cytokine profile in the blood and bone marrow in patients with coronary artery disease (CAD) with and without ischemic cardiomyopathy (ICM).Materials and methods. The study included 74 patients with СAD with and without ICM (30 and 44 people, respectively) and 18 healthy donors. In all patients with СAD, peripheral blood sampling was performed immediately before coronary artery bypass grafting, and bone marrow samples were taken during the surgery via a sternal incision. In the healthy donors, only peripheral blood sampling was performed. In the bone marrow and blood samples, the number of VEGFR2+ cells (CD14+VEGFR2+ cells) and their immunophenotypes CD14++CD16-VEGFR2+, CD14++CD16+VEGFR2+, CD14+CD16++VEGFR2+, and CD14+CD16-VEGFR2+ was determined by flow cytometry. Using enzyme-linked immunosorbent assay, the levels of VЕGF-А, TNFα, M-CSF, and IL-13, as well as the content of MCP-1 (only in the blood) and the M-CSF / IL-13 ratio (only in the bone marrow) were determined.Results. The content of CD14+VEGFR2+ cells in the blood of CAD patients with and without ICM was higher than normal values due to the greater number of CD14++CD16-VEGFR2+, CD14++CD16+VEGFR2+, and CD14+CD16++VEGFR2+. In the bone marrow of the patients with ICM, the content of CD14++CD16-VEGFR2+, CD14+CD16++VEGFR2+, and CD14+CD16-VEGFR2+ was lower than in patients with CAD without ICM, and the number of CD14++CD16+VEGFR2+ cells corresponded to that in the controls. Regardless of the presence of ICM in CAD, a high concentration of TNFα and normal levels of VEGF-A and IL-13 were observed in the blood. In CAD without ICM, an excess of MCP-1 and deficiency of M-CSF were revealed in the blood. In the bone marrow, the levels of VEGF-A, TNFα, M-CSF, and IL-13 were comparable between the groups of patients against the background of a decrease in the M-CSF / IL-13 ratio in the patients with ICM.Conclusion. Unlike CAD without cardiomyopathy, in ICM, no excess of VEGFR2+ cells and MCP-1 in the blood is observed, which hinders active migration of CD14+CD16++VEGFR2+ cells from the myeloid tissue, and a decrease in the M-CSF / IL-13 ratio in the bone marrow disrupts differentiation of other forms of VEGFR2+ cells, preventing vascular repair.Цель: установить нарушения дифференцировки и субпопуляционного состава VEGFR2+ моноцитов в крови и костном мозге во взаимосвязи с особенностями цитокинового профиля крови и костного мозга у больных ишемической болезнью сердца (ИБС), страдающих и не страдающих ишемической кардиомиопатией (ИКМП).Материалы и методы. В исследование вошли 74 больных ИБС, страдающих и не страдающих ИКМП (30 и 44 человека соответственно), и 18 здоровых доноров. У всех больных ИБС забор периферической крови производился непосредственно перед операцией коронарного шунтирования, а костного мозга – из разреза грудины во время операции. У здоровых доноров забирали только периферическую кровь.  В костном мозге и крови методом проточной цитофлуориметрии определяли численность VEGFR2+ моноцитов (CD14+VЕGFR2+ клеток) и их иммунофенотипов CD14++CD16-VEGFR2+, CD14++CD16+VEGFR2+, CD14+CD16++VEGFR2+, CD14+CD16-VEGFR2+, методом иммуноферментного анализа регистрировали концентрацию VЕGF-А, TNFα, M-CSF, IL-13, а также содержание MCP-1 (только в крови) и соотношение M-CSF/IL-13 (только в костном мозге).Результаты. Содержание CD14+VEGFR2+ клеток в крови у больных ИБС без кардиомиопатии и с ИКМП было выше нормы из-за большей численности CD14++CD16-VEGFR2+, CD14++CD16+VEGFR2+ и CD14+CD16++VEGFR2+ форм. В костном мозге у больных ИКМП содержание CD14++CD16-VEGFR2+, CD14+CD16++VEGFR2+ и CD14+CD16-VEGFR2+ форм было ниже, чем у больных ИБС без кардиомиопатии, а количество CD14++CD16+VEGFR2+ клеток соответствовало их числу в группе сравнения. Вне зависимости от наличия ИКМП при ИБС в крови отмечалась высокая концентрация TNFα, нормальный уровень VEGF-А и IL-13; при ИБС без кардиомиопатии – избыток МСР-1 и дефицит M-CSF в крови. В костном мозге концентрация VЕGF-А, TNFα, M-CSF, IL-13 была сопоставимой между группами больных на фоне снижения M-CSF/IL-13 у пациентов с ИКМП.Заключение. В отличие от ИБС без кардиомиопатии при ИКМП не формируется избыток VEGFR2+ моноцитов и МСР-1 в крови, что затрудняет активную миграцию CD14+CD16++VEGFR2+ клеток из миелоидной ткани, а снижение M-CSF/IL-13 в костном мозге нарушает дифференцировку остальных форм VEGFR2+ моноцитов, препятствуя репарации сосудов

Production of angiogenesis mediators and the structure of the vascular wall in the heart in ischemic cardiomyopathy

Background. In the pathogenesis of ischemic cardiomyopathy (ICMP), angiopoiesis remains unexplored.The aim. To describe the vasculature of the heart and the imbalance of angiogenesis mediators in the coronary circulation in association with the number of endothelial progenitor cells (EPC) and desquamated endothelial cells (DEC) in the blood of patients with coronary heart disease (CHD), suffering and not suffering from ICMP.Methods. Fifty-two patients with CHD (30  patients with ICMP, 22  patients without  ICMP), 15  healthy donors were examined. The content of EPC (CD14+CD34+VEGFR2+) in the blood from the cubital vein and DEC (CD45–CD146+) in the blood from the coronary sinus and the cubital vein was determined by flow cytometry. The concentrations of VEGF-A (vascular endothelial growth factor A), PDGF (platelet-derived growth factor), and SDF-1 (stromal cell-derived factor 1) in blood plasma were recorded using immunofluorescence assay; the angiopoietin-2, MMP-9 (matrix metallopeptidase 9) were recorded using enzyme immunoassay. In myocardial biopsies the specific area of vessels and the expression of αSMA (smooth muscle alpha-actin) were determined by morphometric and immunohistochemical methods.Results. In the peripheral blood of patients with CHD, regardless of the presence of ICMP, the DEC content exceeded the physiological level, and the VEGF-A, PDGF, angiopoietin-2, and MMP-9 corresponded to the norm. In CHD patients without cardiomyopathy, there was an excess of SDF-1 and EPC in the blood from the cubital vein, and in ICMP, their physiological significance was noted. In the coronary blood flow in patients with CHD without cardiomyopathy, an increase in the concentration of PDGF was found, which was not determined in patients with ICMP, who had an increased content of DEC, angiopoietin-2 and MMP-9. The specific area of the vessels in the patients of the two groups was comparable; the expression of αSMA in ICMP was 6.2 times lower than in patients with CHD without cardiomyopathy.Conclusion. The development of ICMP is accompanied by impaired maturation of vessels in the myocardium, associated with the absence of a compensatory reaction of activation of cellular and humoral factors of angiogenesis

Цитокины как индукторы постперфузионной системной воспалительной реакции у кардиохирургических больных с различной продолжительностью коронарной патологии

Aim. The changes of blood cytokine profile in patients with ischemic heart disease (IHD) with different development rates and formation of systemic inflammatory response (SIR) after coronary artery bypass grafting by using cardiopulmonary bypass (CPB) are analyzed in this article.Materials and methods. The patients with slowly progressive of IHD (20 patients) and rapidly progressive of IHD (20 patients) were examined. The concentration of interleukine (IL) 1β, IL-1ra, IL-4, IL-6, IL-8 and tumor necrosis factor (TNF) α in blood plasma were evaluated by ELISA at patients with IHD before surgery and at 6 and 24 h after surgery.The results of the study showed that concentration of IL-1β, IL-6, IL-8, TNF-α and IL-1ra in blood plasma increases in patients with IHD of both groups before surgery. The concentration of IL-4 in the blood saved in the normal range before the operation in the case of slow disease progression, but maximum increase in content of proinflammatory (TNF-α, IL-6) and anti-inflammatory (IL-4, IL-1ra) cytokines in the blood and the IL-1ra/IL-1β ratio was detected in a rapidly developing of IHD. It was noticed that after coronary artery bypass grafting in patients with long history case of IHD the content of IL-1β, IL-8, TNF-α, IL-1ra, IL-4 increased with a normalization of the IL-6 concentration in the blood; in patients with a short period of IHD increase of IL-1 concentration and high content of IL-6 are combined with remaining unchanged level of IL1β, IL-8, IL-4 and negative dynamics of the TNF-α concentration in the blood. Thus, the operation in the СРВ in the case of IHD with prolonged course induces the formation of SIR, typical for acute inflammation, and coordinated anti-inflammatory response, and in the case of short period of coronary disease progress this operation causes SIR, characteristic for chronic inflammation, and uncoordinated anti-inflammatory response. Цель исследования – определить характер изменений цитокинового профиля крови при формировании системной воспалительной реакции (СВР) у больных ишемической болезнью сердца (ИБС) с разными темпами развития заболевания после коронарного шунтирования с применением искусственного кровообращения (ИК).Материал и методы. Обследованы больные с медленно прогрессирующей ИБС (20 человек) и быстро прогрессирующей ИБС (20 человек). У больных ИБС до операции и через 6 и 24 ч после оперативного вмешательства оценивали концентрацию цитокинов – интерлейкина (IL)-1β, IL-1ra, IL-4, IL-6, IL-8 и фактора некроза опухоли (TNF) α в плазме крови методом иммуноферментного анализа.Результаты исследования показали, что до операции у больных ИБС обеих групп отмечалось повышение плазменной концентрации IL-1β, IL-6, IL-8, TNF-α и IL-1ra. При этом в случае медленного прогрессирования заболевания концентрация IL-4 в крови сохранялась в пределах нормы, в то время как при быстро развивающейся ИБС обнаруживалось максимально выраженное увеличение содержания провоспалительных (TNF-α, IL-6) и противовоспалительных (IL-4, IL-1rа) цитокинов в крови и соотношения IL-1rа/IL-1β. После коронарного шунтирования у больных ИБС с длительным анамнезом заболевания отмечалось увеличение содержания IL-1β, IL-8, TNF-α, IL-1rа, IL-4 при нормализации концентрации IL-6 в крови; у пациентов с коротким периодом развития ИБС – рост концентрации IL-1rа, высокое содержание IL-6 при сохраняющемся без изменений уровне IL-1β, IL-8, IL-4 и отрицательной динамике концентрации TNF-α в плазме крови.Таким образом, при длительном течении ИБС выполнение операции в условиях ИК вызывает формирование СВР, свойственной острому воспалению, и координированный противовоспалительный ответ, а при коротком периоде коронарной болезни – СВР, характерную для хронического воспаления, и некоординированный противовоспалительный ответ.

Targeted gene delivery in tumor xenografts by the combination of ultrasound-targeted microbubble destruction and polyethylenimine to inhibit survivin gene expression and induce apoptosis

<p>Abstract</p> <p>Background</p> <p>Noninvasive and tissue-specific technologies of gene transfection would be valuable in clinical gene therapy. This present study was designed to determine whether it could enhance gene transfection <it>in vivo </it>by the combination of ultrasound-targeted microbubble destruction (UTMD) with polyethylenimine (PEI) in tumor xenografts, and illuminate the effects of gene silencing and apoptosis induction with short hairpin RNA (shRNA) interference therapy targeting human survivin by this novel technique.</p> <p>Methods</p> <p>Two different expression vectors (pCMV-LUC and pSIREN) were incubated with PEI to prepare cationic complexes (PEI/DNA) and confirmed by the gel retardation assay. Human cervical carcinoma (Hela) tumors were planted subcutaneously in both flanks of nude mice. Tumor-bearing mice were administered by tail vein with PBS, plasmid, plasmid and SonoVue microbubble, PEI/DNA and SonoVue microbubble. One tumor was exposed to ultrasound irradiation, while the other served as control. The feasibility of targeted delivery and tissue specificity facilitated by UTMD and PEI were investigated. Moreover, immunohistochemistry analyses about gene silencing and apoptosis induction were detected.</p> <p>Results</p> <p>Electrophoresis experiment revealed that PEI could condense DNA efficiently. The application of UTMD significantly increases the tissue transfection. Both expression vectors showed that gene expressions were present in all sections of tumors that received ultrasound exposure but not in control tumors. More importantly, the increases in transgene expression were related to UTMD with the presence of PEI significantly. Silencing of the survivin gene could induce apoptosis effectively by downregulating survivin and bcl-2 expression, also cause up-regulation of bax and caspase-3 expression.</p> <p>Conclusions</p> <p>This noninvasive, novel combination of UTMD with PEI could enhance targeted gene delivery and gene expression in tumor xenografts at intravenous administration effectively without causing any apparently adverse effect, and might be a promising candidate for gene therapy. Silencing of survivin gene expression with shRNA could be facilitated by this non-viral technique, and lead to significant cell apoptosis.</p

Роль IL-23 в развитии Th17-лимфоцитов у пациентов с туберкулезом легких

The objective: to evaluate the role of IL-23 in the development of Th17 lymphocytes in patients with different clinical and pathogenetic forms of pulmonary tuberculosis.Subjects and Methods. 165 pulmonary tuberculosis patients were examined. Venous blood was used for tests. Mononuclear leukocytes were isolated by centrifugation and monocytes were extracted and transformed into dendritic cells. The concentration of IL-23 in the supernatants of culture suspensions of dendritic cells was determined by ELISA. Immunophenotyping of Th17 lymphocytes (CD4+CD161+IL-17A+ cells) was performed by flow cytometry. Real-time PCR was used to determine the expression of the RORC2 transcription factor gene in lymphocytes.Results. In patients with infiltrative drug susceptible and drug resistant pulmonary tuberculosis against the background of normal production of IL-23 by dendritic cells, an increase in blood level of Th17 lymphocytes and the level of mRNA of the RORC2 transcription factor gene was registered. The course of disseminated pulmonary tuberculosis (regardless of drug susceptibility and resistance) is associated with pronounced decrease in the concentration of IL-23 in vitro and the absence of response from Th17 lymphocytes.Цель исследования: оценить роль IL-23 в развитии Th17-лимфоцитов у пациентов с различными клинико-патогенетическими вариантами туберкулеза легких.Материалы и методы. Обследовано 165 пациентов с туберкулезом легких (ТБЛ). Материалом для исследования являлась венозная кровь. Центрифугированием выделяли мононуклеарные лейкоциты и осуществляли экстракцию моноцитов и их трансформацию в дендритные клетки. Твердофазным иммуноферментным методом определяли концентрацию IL-23 в супернатантах культуральных суспензий дендритных клеток. Методом проточной цитофлуориметрии проводили иммунофенотипирование Th17-лимфоцитов (CD4+CD161+IL-17A+ клеток). Методом ПЦР в режиме реального времени определяли экспрессию гена транскрипционного фактора RORC2 в лимфоцитах.Результаты. У пациентов с инфильтративным лекарственно-чувствительным и лекарственно-устойчивым ТБЛ на фоне нормопродукции IL-23 дендритными клетками регистрируется повышение содержания Th17-лимфоцитов в крови и уровня мРНК гена транскрипционного фактора этих клеток – RORC2. Течение диссеминированного ТБЛ (вне зависимости от лекарственной чувствительности возбудителя) сопровождается выраженным снижением концентрации IL-23 in vitro и отсутствием реакции со стороны Th17-лимфоцитов