Spiny mice (Acomys cahirinus) exhibit the ability to regenerate the damaged myocardium and functional indices of the heart. demonstrated in various models of cardiac pathologies. Cardioregenerative potential of Acomys is associated with partial preservation of the neonatal phenotype of cardiac tissue in adult animals. The electrophysiology and mechanisms regulating the heart rhythm in Acomys are extremely poorly understood. The EEG was recorded in vivo and bioelectrical activity of the supraventricular myocardium was evaluated and mapped in spiny mice. Pacemaker-type action potentials were detected in a significant part of the right atrium, and primary activation involved approximately 41

The cardiac perivascular niche is a cellular microenvironment of a blood vessel. The principles of niche regulation are still poorly understood. We studied the effect of TGFβ1 on cells forming the cardiac perivascular niche using 3D cell culture (cardiospheres). Cardiospheres contained progenitor (c-Kit), endothelial (CD31), and mural (αSMA) cells, basement membrane proteins (laminin) and extracellular matrix proteins (collagen I, fibronectin). TGFβ1 treatment decreased the length of CD31+ microvasculature, VE cadherin protein level, and proportion of NG2+ cells, and increased proportion of αSMA+ cells and transgelin/SM22α protein level. We supposed that this effect is related to the stabilizing function of TGFβ1 on vascular cells: decreased endothelial cell proliferation, as shown for HUVEC, and activation of mural cell differentiation.

Thermogenic adipocytes have potential utility for the development of approaches to treat type 2 diabetes and obesity-associated diseases. Although several reports have proved the positive effect of beige and brown adipocyte transplantation in obese mice, translation to human cell therapy needs improvement. Here, we describe the application of CRISPR activation (CRISPRa) technology for generating safe and efficient adipose-tissue-engineered constructs with enhanced mitochondrial uncoupling protein 1 (UCP1) expression. We designed the CRISPRa system for the activation of UCP1 gene expression. CRISPRa-UCP1 was delivered into mature adipocytes by a baculovirus vector. Modified adipocytes were transplanted in C57BL/6 mice, followed by analysis of grafts, inflammation and systemic glucose metabolism. Staining of grafts on day 8 after transplantation shows them to contain UCP1-positive adipocytes. Following transplantation, adipocytes remain in grafts and exhibit expression of PGC1α transcription factor and hormone sensitive lipase (HSL). Transplantation of CRISPRa-UCP1-modified adipocytes does not influence glucose metabolism or inflammation in recipient mice. We show the utility and safety of baculovirus vectors for CRISPRa-based thermogenic gene activation. Our findings suggest a means of improving existing cell therapy approaches using baculovirus vectors and CRISPRa for modification and transplantation of non-immunogenic adipocytes.

ЦЕЛЬ: исследовать влияние бариатрической операции (БО) на жизнеспособность и пролиферацию мезенхимальных стволовых клеток жировой ткани (МСК ЖТ) пациентов с сахарным диабетом 2 типа (СД2). МАТЕРИАЛЫ И МЕТОДЫ: МСК были получены из подкожной ЖТ пациентов с СД2 до бариатрической операции и спустя полгода после бариатрической операции (пациенты с ИМТ>35 кг/м2, длительность ожирения более 10 лет). Выделение клеток проводили согласно стандартному протоколу с использованием коллагеназы 1 типа. Жизнеспособность клеток оценивали с помощью колориметрического МТТ-теста с предварительной инкубацией клеток в среде с различным содержанием сыворотки – 1% и 10% эмбриональной бычьей сыворотки (FBS). Экспрессию ядерного антигена пролиферирующих клеток (PCNA) определяли с помощью иммуноцитохимического окрашивания и иммуноблоттинга. РЕЗУЛЬТАТЫ: иммуноцитохимическое окрашивание показало, что стимуляция клеток сывороткой демонстрирует более чем в два раза увеличенное содержание PCNA-положительных клеток среди МСК, полученных после бариатрической операции по сравнению с МСК до бариатрической операции. Аналогичные результаты наблюдаются при анализе экспрессии PCNA методом иммуноблоттинга – стимуляция сывороткой способствует большей экспрессии PCNA в клетках пациентов после бариатрической операции. При этом МТТ-тест продемонстрировал увеличение жизнеспособности МСК после бариатрической операции, вне зависимости от процентного содержания сыворотки в среде. ВЫВОДЫ: мезенхимальные стволовые клетки, полученные после бариатрической операции из подкожной жировой ткани пациентов с сахарным диабетом 2 типа, обладают увеличенным пролиферативным потенциалом и жизнеспособностью по сравнению с МСК пациентов до бариатрической операции.

Комбинированная генная терапия сердечно-сосудистых заболеваний является многообещающим инструментом для потенциальной трансляции в клиническую практику. В данной работе мы изучали действие плазмиды HGF/VEGF на восстановление ишемизированной нижней конечности в условиях высокожировой диеты и метаболических нарушений, свойственных ожирению. МАТЕРИАЛЫ И МЕТОДЫ: в эксперимент отбирали самцов мышей линии C57BL/6J, которые содержа- лись 10 недель в условиях низко- и высокожировой диет (НЖД и ВЖД соответственно). На 10 неделе про- водили оценку метаболического статуса животных с помощью глюкозо- и инсулинотолерантного тестов. Также на 10 неделе выполняли операцию по индукции ишемии нижней конечности с последующей оценкой подкожного кровотока путем лазерного допплеровского сканирования сосудов на 7, 14 и 21 дни после операции. На 13 неделе проводили повторную оценку метаболического статуса животных с последующим анализом ткани большеберцовой мышцы. РЕЗУЛЬТАТЫ: на 10 неделе ВЖД группа демонстрировала нарушенную толерантность к глюкозе. Введение плазмиды HGF/VEGF ухудшало толерантность к глюкозе в группе ВЖД, но не в группе НЖД. Восстановление кровотока не изменялось при введении плазмиды HGF/VEGF как в НЖД, так и в ВЖД условиях. Генная терапия в условиях НЖД, но не ВЖД, увеличивала площадь некроза мышцы и ее воспалительную инфильтрацию. Тем не менее, HGF/VEGF плазмида усиливала нейритогенез в восстанавливающейся мышце, увеличивала число митохондрий и удельную экспрессию комплексов электрон-транспортной цепи. ВЫВОДЫ: данное исследование демонстрирует существенную роль диетарных условий в доклинических испытаниях невирусных генно-терапевтических препаратов. HGF/VEGF плазмида демонстрирует новый механизм участия в регенерации ишемизированной скелетной мышцы путем регуляции иннервации и биоэнергетики мышцы.

BACKGROUND:Combined non-viral gene therapy (GT) of ischemia and cardiovascular disease is a promising tool for potential clinical translation. In previous studies our group has developed combined gene therapy by vascular endothelial growth factor 165 (VEGF165) + hepatocyte growth factor (HGF). Our recent works have demonstrated that a bicistronic pDNA that carries both human HGF and VEGF165 coding sequences has a potential for clinical application in peripheral artery disease (PAD). The present study aimed to test HGF/VEGF combined plasmid efficacy in ischemic skeletal muscle comorbid with predominant complications of PAD-impaired glucose tolerance and type 2 diabetes mellitus (T2DM).METHODS:Male C57BL mice were housed on low-fat (LFD) or high-fat diet (HFD) for 10 weeks and metabolic parameters including FBG level, ITT, and GTT were evaluated. Hindlimb ischemia induction and plasmid administration were performed at 10 weeks with 3 weeks for post-surgical follow-up. Limb blood flow was assessed by laser Doppler scanning at 7, 14, and 21 days after ischemia induction. The necrotic area of m.tibialis anterior, macrophage infiltration, angio- and neuritogenesis were evaluated in tissue sections. The mitochondrial status of skeletal muscle (total mitochondria content, ETC proteins content) was assessed by Western blotting of muscle lysates.RESULTS:At 10 weeks, the HFD group demonstrated impaired glucose tolerance in comparison with the LFD group. HGF/VEGF plasmid injection aggravated glucose intolerance in HFD conditions. Blood flow recovery was not changed by HGF/VEGF plasmid injection either in LFD or HFD conditions. GT in LFD, but not in HFD conditions, enlarged the necrotic area and CD68+ cells infiltration. However, HGF/VEGF plasmid enhanced neuritogenesis and enlarged NF200+ area on muscle sections. In HFD conditions, HGF/VEGF plasmid injection significantly increased mitochondria content and ETC proteins content.CONCLUSIONS:The current study demonstrated a significant role of dietary conditions in pre-clinical testing of non-viral GT drugs. HGF/VEGF combined plasmid demonstrated a novel aspect of potential participation in ischemic skeletal muscle regeneration, through regulation of innervation and bioenergetics of muscle. The obtained results made HGF/VEGF combined plasmid a very promising tool for PAD therapy in impaired glucose tolerance conditions.

Murine peritoneal macrophages isolated from the lavage fluid after administration of thioglycolate and concanavalin A are presented by two populations of cells of different diameters. Polarization of macrophages into a proinflammatory (M1) phenotype is accompanied by an increase in number of small cells. Macrophages obtained after administration of thioglycolate demonstrate higher tendency to anti-inflammatory (M2) phenotype, while macrophages isolated after administration of concanavalin A are committed in the proinflammatory direction. Lactate level is increased in M1 macrophages in comparison with M2 cells, which indicates predominance of glycolytic metabolism. Macrophages obtained after administration of concanavalin A have reduced mitochondrial potential, which reflects a tendency to apoptosis. Autophagy activation and inhibition neutralize the differences in pro- and anti-inflammatory properties of polarized macrophages obtained after thioglycolate administration, but have less pronounced effect on macrophages obtained after administration concanavalin A. Autophagy inhibitor increases mitochondrial potential in non-polarized macrophages obtained after administration of concanavalin A. These results demonstrate divergent properties of macrophages obtained after administration of glycolate and concanavalin A due to the difference in the mechanisms of experimental peritonitis.

Cell therapy of the post-infarcted myocardium is still far from clinical use. Poor survival of transplanted cells, insufficient regeneration, and replacement of the damaged tissue limit the potential of currently available cell-based techniques. In this study, we generated a multilayered construct from adipose-derived mesenchymal stromal cells (MSCs) modified to secrete stem cell factor, SCF. In a rat model of myocardium infarction, we show that transplantation of SCF producing cell sheet induced activation of the epicardium and promoted the accumulation of c-kit positive cells in ischemic muscle. Morphometry showed the reduction of infarct size (16%) and a left ventricle expansion index (0.12) in the treatment group compared to controls (24–28%; 0.17–0.32). The ratio of viable myocardium was more than 1.5-fold higher, reaching 49% compared to the control (28%) or unmodified cell sheet group (30%). Finally, by day 30 after myocardium infarction, SCF-producing cell sheet transplantation increased left ventricle ejection fraction from 37% in the control sham-operated group to 53%. Our results suggest that, combining the genetic modification of MSCs and their assembly into a multilayered construct, we can provide prolonged pleiotropic effects to the damaged heart, induce endogenous regenerative processes, and improve cardiac function.

Therapeutic angiogenesis is a promising strategy for relief of ischemic conditions, and gene delivery was used to stimulate blood vessels’ formation and growth. We have previously shown that intramuscular injection of a mixture containing plasmids encoding vascular endothelial growth factor (VEGF)165 and hepatocyte growth factor (HGF) leads to restoration of blood flow in mouse ischemic limb, and efficacy of combined delivery was superior to each plasmid administered alone. In this work, we evaluated different approaches for co-expression of HGF and VEGF165 genes in a panel of candidate plasmid DNAs (pDNAs) with internal ribosome entry sites (IRESs), a bidirectional promoter or two independent promoters for each gene of interest. Studies in HEK293T culture showed that all plasmids provided synthesis of HGF and VEGF165 proteins and stimulated capillary formation by human umbilical vein endothelial cells (HUVEC), indicating the biological potency of expressed factors. Tests in skeletal muscle explants showed a dramatic difference and most plasmids failed to express HGF and VEGF165 in a significant quantity. However, a bicistronic plasmid with two independent promoters (cytomegalovirus (CMV) for HGF and chicken b-actin (CAG) for VEGF165) provided expression of both grow factors in skeletal muscle at an equimolar ratio. Efficacy tests of bicistronic plasmid were performed in a mouse model of hind limb ischemia. Intramuscular administration of plasmid induced significant restoration of perfusion compared to an empty vector and saline. These findings were supported by increased CD31+ capillary density in animals that received pHGF/VEGF. Overall, our study reports a first-in-class candidate gene therapy drug to deliver two pivotal angiogenic growth factors (HGF and VEGF165) with properties that provide basis for future development of treatment for an unmet medical need—peripheral artery disease and associated limb ischemia.

In heart attack, FSTL-1 is actively secreted by cardiomyocytes, accelerates growth of heart myofibrils and stimulates of vascular endothelial growth factor expression. The aim of this work was to investigate the effect of Etoxidol on synthesis of FSTL-1 in rats after myocardial infarction. The experiments were performed on Wistar rats weighing 250-350 g with simulated myocardial infarction or intact (group 5). Animals of control groups (groups 1, 2) were treated with saline for 7 and 14 days; Ethoxidol (24 mg/kg) was injected to animals of experimental groups (group 3, 4) (the daily dose was 6.36 mg/animal) for 6 or 14 days. The injection volume was 0.2 ml. At the beginning and at the end of the study plasma concentrations of FSTL-1 were determined by the ELISA method. Myocardial FSTL-1 gene expression was determined by real-time PCR. At the end of the experiments, the hearts were also used for histochemical analysis. To determine the size of the scar formed after the modeled heart attack, we used the classic Mallory staining method. The results show that the development of experimental acute myocardial infarction is accompanied by a significant increase in FSTL-1 expression in the heart, which was detected on the 7th day and stored increased by 14 days after a heart attack. After therapy with Ethoxidol, a tendency to a decrease in the expression of FSTL-1 by the 14th day was observed; it coincided with the dynamics of the plasma protein FSTL-1 level. It can be assumed that the downregulation trend in the FSTL-1 expression is associated with a more effective repair process after a heart attack, since FSTL-1 increases precisely in response to myocardial damage and decreases when the incentives for its expression from damaged heart tissue are reduced. Indirectly, this assumption is confirmed by the detected tendency to reduce the size of post-infarction fibrosis in the treatment with Ethoxidol. The results indicate the ability of Ethoxidol to influence FSTL-1 synthesis of in rats after myocardial infarction.

A stimulating effect of a combination of hepatocyte growth factor (HGF) and glial neurotrophic factor (GDNF) on the growth of neurites in the spinal ganglion model was demonstrated. The mechanism of neurite growth in the spinal ganglion model is associated with transactivation of HGF c-met receptor in the presence of both HGF and GDNF. The combination of HGF and GDNF significantly activated mitogenic signaling cascade mediated by protein kinases ERK1/2, which can be a mechanism for increasing the number of neurites. Our findings can be used for developing effective methods for restoring impaired peripheral nerve function after traumatic and ischemic injury using a combination of GDNF and HGF.

Cell therapy remains a promising approach for the treatment of cardiovascular diseases. In this regard, the contemporary trend is the development of methods to overcome low cell viability and enhance their regenerative potential. In the present study, we evaluated the therapeutic potential of gene-modified adipose-derived stromal cells (ADSC) that overexpress hepatocyte growth factor (HGF) in a mice hind limb ischemia model. Angiogenic and neuroprotective effects were assessed following ADSC transplantation in suspension or in the form of cell sheet. We found superior blood flow restoration, tissue vascularization and innervation, and fibrosis reduction after transplantation of HGF-producing ADSC sheet compared to other groups. We suggest that the observed effects are determined by pleiotropic effects of HGF, along with the multifactorial paracrine action of ADSC which remain viable and functionally active within the engineered cell construct. Thus, we demonstrated the high therapeutic potential of the utilized approach for skeletal muscle recovery after ischemic damage associated with complex tissue degenerative effects.

The paper provides current data on some proteins of the TGF- p family which are potentially capable of exerting a protective effect in diseases of the heart, lungs, placenta, gonads, and pancreas. The study investigated the anti-inflammatory properties of follistatin-like protein-1 (FSTL-1), one of the proteins of this family, at the cellular level. It was demonstrated that FSTL-1 is responsible for heart muscle regeneration in mammals through activation of angiogenic factors. Despite the fact that this protein plays a key role in myocardial regeneration, its concentration in the epicardium decreases immediately after a heart attack, which hampers effective self-repair of the heart. The paper summarises the results of studies of the efficacy of intravenous administration of FSTL-1 in rats with myocardial infarction. However, the administration of a foreign protein can cause allergic reactions, therefore a drug that induces FSTL-1 secretion was chosen instead.The aim of the study was to provide experimental substantiation of the possibility of exogenous regulation of FSTL-1 secretion.Materials and methods: FSTL-1 concentration in rat plasma was assessed by enzyme immunoassay before and after treatment with the antioxidant drug ethyl methyl hydroxypyridine malate. The antioxidant was administered to 15 healthy male Wistar rats subcutaneously 3 times a day at a dose of 6 mg/day for 14 days. A fasting blood sample was obtained on the first day before administration of the drug and on day 15.Results: after the period of treatment with ethyl methyl hydroxypyridine malate the concentration of FSTL-1 in the plasma of the laboratory rats increased significantly (p = 0.0011) to reach 0.92 ± 0.11 ng/mL as compared to the initial concentration of 0.48 ± 0.04 ng/mL.Conclusion: the study provided experimental evidence for new properties of ethyl methyl hydroxypyridine malate, i.e. induction of FSTL-1 in healthy rats. Further studies are encouraged to assess potential use of this drug as an inductor of FSTL-1 in myocardial ischemia.