Hypoxia-Inducible Factor-1 in Physiological and Pathophysiological Angiogenesis: Applications and Therapies

The cardiovascular system ensures the delivery of oxygen and nutrients to all cells, tissues, and organs. Under extended exposure to reduced oxygen levels, cells are able to survive through the transcriptional activation of a series of genes that participate in angiogenesis, glucose metabolism, and cell proliferation. The oxygen-sensitive transcriptional activator HIF-1 (hypoxia-inducible factor-1) is a key transcriptional mediator of the response to hypoxic conditions. The HIF-1 pathway was found to be a master regulator of angiogenesis. Whether the process is physiological or pathological, HIF-1 seems to participate in vasculature formation by synergistic correlations with other proangiogenic factors such as VEGF (vascular endothelial growth factor), PlGF (placental growth factor), or angiopoietins. Considering the important contributions of HIF-1 in angiogenesis and vasculogenesis, it should be considered a promising target for treating ischaemic diseases or cancer. In this review, we discuss the roles of HIF-1 in both physiological/pathophysiological angiogenesis and potential strategies for clinical therapy

Effect of miR-195 inhibition on human skeletal muscle-derived stem/progenitor cells

BACKGROUND: Application of a circulating miR-195 inhibitor could be a helping factor in in vitro model of human skeletal muscle- derived stem/progenitor cells (SkMDS/PCs). Previously, miR-195 expression has been reported to be a negative factor for myogenesis. AIMS: The aim of the study was to obtain anti-apoptotic and anti-aging effects in in vitro cultured myoblasts and to improve their ability to form myotubes by suppressing miR-195 expression. METHODS: Human wild-type (WT) SkMDS/PC cells incubated with control (nonspecific) miRNA inhibitor and miR-195-inhibited SkMDS/PCs were studied. Functional assays (myotube formation and cell ageing), antioxidant, and myogenic gene expression analyses were performed at two time points, at the 7th and 11th cell passages. RESULTS: Myotube formation was found to be almost 2-fold higher in the miR-195-inhibited SkMDS/PCs population (p<0.05) compared to WT cells. miR-195 inhibition did not appear to affect cell ageing or rejuvenate human SkMDS/PCs. Antioxidant (SOD3 and FOXO) gene expression was augmented in the miR-195-inhibited SkMDS/PCs population, but no positive effect on the remaining antioxidant genes (SOD1, SOD2, and catalase) was observed. A significant increase in MyoD gene expression with a concomitant decrease in MyoG (p<0.05) was further documented in miR-195-inhibited SkMDS/PCs compared to WT cells (11th cell passage). CONCLUSIONS: The performed studies may lead to the preconditioning of myogenic stem cells to extend their potential for pro-regenerative activity. miR-195 inhibitor may serve as conditioning factor augmenting selective antioxidant genes expression and proliferative potential of SkMDS/PCs, but not having an impact on cell aging and/ or apoptosis

Poprawa właściwości regeneracyjnych komórek macierzystych pochodzenia miogennego poprzez zastosowanie modyfikowanych genetycznie mioblastów w terapii serca po zawale

Background: Modern therapies of post infarcted heart failure are focused on perfusion improvement of the injured myocardium. This effect can be achieved by, among other means, implanting stem cells which could be genetically modified with factors inducing the formation of new blood vessels in the post infarction scar area. Combined stem cell and gene therapy seems to be a promising strategy to heal an impaired myocardium. The creation of new blood vessels can be indirectly stimulated via factors inducing vascular endothelial growth factor synthesis, for example endothelial nitric oxide synthase (eNOS). The product of this enzyme, nitric oxide, is a molecule that can influence numerous physiological activities; it can contribute to vasodilation, stimulation of endothelial cell growth, prevention of platelet aggregation and leukocyte adhesion to the endothelium.Aim: To verify the pro-angiogenic and regenerative potential of human primary myoblasts and murine myoblast cell line C2C12 transiently transfected with eNOS gene.Methods: Stem cells (either human or murine) were maintained in standard in vitro conditions. Next, both types of myoblasts were modified using electroporation and lipofection (human and murine cells), respectively. The efficacy of the transfection method was evaluated using flow cytometry. The concentration of eNOS protein was measured by ELISA immunoassay. The biological properties of modified cells were assessed using an MTT proliferation test and DAPI cell cycle analysis. To verify the influence of oxidative stress on myoblasts, cytometric tests using Annexin V and propidium iodide were applied. To check possible alterations in myogenic gene expression of stem cells transduced by genetic modification, the myogenic regulatory factors were evaluated by real-time PCR. The function of genetic modification was confirmed by a HUVEC capillary sprouting test using myoblasts supernatants.Results: Electroporation turned out to be an efficient transfection method. High amounts of secreted protein were obtained (in the range 2,000 pg/mL) in both cell types studied. Moreover, the functionality of gene overexpression product was confirmed in capillary development assay. Human myoblasts did not exhibit any changes in cell cycle; however, eNOS transfected murine myoblasts revealed a statistically significant reduction in cell cycle ratio compared to controls (p < 0.001). In the case of myogenic gene expression, a decrease in Myogenin level was only detected in the human transfected myoblast population (p < 0.05).Conclusions: The results of our study may suggest that transplantation of myoblasts overexpressing eNOS could be promising for cell therapy in regenerating the post infarction heart.Wstęp: Nowoczesne terapie regeneracyjne stosowane w leczeniu serca po zawale skupiają się głównie na poprawie perfuzji w obszarze uszkodzonego miokardium. Efekt ten można osiągnąć m.in. poprzez wszczepienie w obszar blizny pozawałowej komórek macierzystych zmodyfikowanych czynnikami indukującymi powstawanie naczyń krwionośnych. Połączenie dwóch rodzajów terapii: komórkowej i genowej stanowi obiecującą metodę leczenia skutków zawału serca. Zapoczątkowanie tworzenia naczyń krwionośnych może się odbywać w sposób pośredni, poprzez uczestnictwo enzymu eNOS w stymulacji powstawania dużej ilości czynnika VEGF, będącego głównym induktorem angiogenezy. Dodatkowo produkt enzymu eNOS — tlenek azotu jest cząsteczką, która ma duże znaczenie dla fizjologii organizmu; przyczynia się m.in. do rozszerzania naczyń krwionośnych, stymuluje komórki śródbłonka, zapobiega agregacji płytek krwi i adhezji leukocytów w naczyniach.Cel: Głównym celem pracy było sprawdzenie potencjału regeneracyjnego i właściwości proangiogennych zmodyfikowanych genetycznie komórek macierzystych mięśni szkieletowych, zarówno pierwotnej zawiesiny ludzkiej, jak i mysich komórek linii C2C12 za pomocą konstrukcji genowej zawierającej sekwencję kodującą dla genu eNOS.Metody: Komórki macierzyste (ludzkie i mysie) były hodowane w standardowych warunkach in vitro. Następnie obydwa typy komórek zostały zmodyfikowane za pomocą elektroporacji i lipofekcji (ta ostatnia dla komórek mysich). Wydajność transfekcji sprawdzono za pomocą cytometrii przepływowej. Stężenie białka eNOS oznaczono testem immunoenzymatycznym ELISA. Podstawowe funkcje biologiczne zmodyfikowanych komórek zostały ocenione przy użyciu testu proliferacyjnego MTT oraz DAPI do analizy cyklu komórkowego. Wpływ stresu oksydacyjnego na przeżywalność komórek zbadano cytometrycznie, wykorzystując aneksynę V i jodek propidyny. Aby ocenić, czy ekspresja genów szlaku miogennego nie została zaburzona przez wniesioną modyfikację genetyczną, przeprowadzono reakcję PCR w czasie rzeczywistym. Funkcjonalność zastosowanej modyfikacji genetycznej sprawdzono za pomocą testu proliferacji komórek HUVEC z użyciem mediów zebranych znad poszczególnych populacji komórek po hodowli.Wyniki: Elektroporacja okazała się wydajną metodą transfekcji pierwotnych komórek ludzkich. Otrzymano wysokie stężenie białka eNOS w przypadku obu badanych rodzajów komórek: mysich i ludzkich (> 2000 pg/ml). Funkcjonalność modyfikacji potwierdzono w teście tworzenia kapilar przez komórki HUVEC. Ludzkie mioblasty nie wykazały żadnych istotnych zmian w cyklu komórkowym, natomiast komórki mysie wykazały istotny statystycznie spadek we współczynniku proliferacji (na poziomie istotności p < 0,001). W profilu ekspresji genów miogennych istotny statystycznie okazał się jednie spadek ekspresji miogeniny w przypadku mioblastów ludzkich (p < 0,05). Przeżywalność komórek modyfikowanych nie odbiegała znacząco od wyników uzyskanych w przypadku komórek ‘natywnych’.Wnioski: Wyniki badań wskazują na słuszność zastosowania modyfikacji genetycznej komórek macierzystych z perspektywicznym planem użycia genu eNOS w badaniach przedklinicznych na modelu mysim. Taka terapia może się okazać w przyszłości obiecującym sposobem regeneracji serca po zawale

Co-Transplantation of Bone Marrow-MSCs and Myogenic Stem/Progenitor Cells from Adult Donors Improves Muscle Function of Patients with Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a genetic disorder associated with a progressive deficiency of dystrophin that leads to skeletal muscle degeneration. In this study, we tested the hypothesis that a co-transplantation of two stem/progenitor cell populations, namely bone marrow-derived mesenchymal stem cells (BM-MSCs) and skeletal muscle-derived stem/progenitor cells (SM-SPCs), directly into the dystrophic muscle can improve the skeletal muscle function of DMD patients. Three patients diagnosed with DMD, confirmed by the dystrophin gene mutation, were enrolled into a study approved by the local Bioethics Committee (no. 79/2015). Stem/progenitor cells collected from bone marrow and skeletal muscles of related healthy donors, based on HLA matched antigens, were expanded in a closed MC3 cell culture system. A simultaneous co-transplantation of BM-MSCs and SM-SPCs was performed directly into the biceps brachii (two patients) and gastrocnemius (one patient). During a six-month follow-up, the patients were examined with electromyography (EMG) and monitored for blood kinase creatine level. Muscle biopsies were examined with histology and assessed for dystrophin at the mRNA and protein level. A panel of 27 cytokines was analysed with multiplex ELISA. We did not observe any adverse effects after the intramuscular administration of cells. The efficacy of BM-MSC and SM-SPC application was confirmed through an EMG assessment by an increase in motor unit parameters, especially in terms of duration, amplitude range, area, and size index. The beneficial effect of cellular therapy was confirmed by a decrease in creatine kinase levels and a normalised profile of pro-inflammatory cytokines. BM-MSCs may support the pro-regenerative potential of SM-SPCs thanks to their trophic, paracrine, and immunomodulatory activity. Both applied cell populations may fuse with degenerating skeletal muscle fibres in situ, facilitating skeletal muscle recovery. However, further studies are required to optimise the dose and timing of stem/progenitor cell delivery

pNiPAM-Nanoparticle-Based Antiapoptotic Approach for Pro-Regenerative Capacity of Skeletal Myogenic Cells

The biocompatibility of pNiPAM (Poly N-isopropylacrylamide) copolymers has been examined and they did not exert any cytotoxic effects. Their properties and vulnerable temperature characteristics make them candidates for use in medical applications. We synthesized a well-characterized nanoparticles-based cargo system that would effectively deliver a biological agent to human skeletal myogenic cells (SkMCs); among other aspects, a downregulating apoptotic pathway potentially responsible for poor regeneration of myocardium. We confirmed the size of the pNiPAM based spheres at around 100 nm and the nanomeric shape of nanoparticles (NP) obtained. We confirmed that 33 °C is the adequate temperature for phase transition. We performed the dynamics of cargo release. A small amount of examined protein was detected at 10 min after reaching LCTS (lower critical solution temperature). The presented results of the test with BSA (bovine serum albumin) and doxorubicin loaded into nanoparticles showed a similar release profile for both substances. SkMCs incubated with NP loaded with antiapoptotic agent, BCB (Bax channel blocker), significantly diminished cell apoptosis (p < 0.01). Moreover, the lowest apoptotic level was detected in SkMCs treated with camptothecin and simultaneously incubated with pNiPAMs loaded with BCB. Application of nanoparticles loaded with BCB or subjected to BCB alone did not, however, diminish the amount of apparently necrotic cells

Potential use of superparamagnetic iron oxide nanoparticles for in vitro and in vivo bioimaging of human myoblasts

Abstract Myocardial infarction (MI) is one of the most frequent causes of death in industrialized countries. Stem cells therapy seems to be very promising for regenerative medicine. Skeletal myoblasts transplantation into postinfarction scar has been shown to be effective in the failing heart but shows limitations such, e.g. cell retention and survival. We synthesized and investigated superparamagnetic iron oxide nanoparticles (SPIONs) as an agent for direct cell labeling, which can be used for stem cells imaging. High quality, monodisperse and biocompatible DMSA-coated SPIONs were obtained with thermal decomposition and subsequent ligand exchange reaction. SPIONs’ presence within myoblasts was confirmed by Prussian Blue staining and inductively coupled plasma mass spectrometry (ICP-MS). SPIONs’ influence on tested cells was studied by their proliferation, ageing, differentiation potential and ROS production. Cytotoxicity of obtained nanoparticles and myoblast associated apoptosis were also tested, as well as iron-related and coating-related genes expression. We examined SPIONs’ impact on overexpression of two pro-angiogenic factors introduced via myoblast electroporation method. Proposed SPION-labeling was sufficient to visualize firefly luciferase-modified and SPION-labeled cells with magnetic resonance imaging (MRI) combined with bioluminescence imaging (BLI) in vivo. The obtained results demonstrated a limited SPIONs’ influence on treated skeletal myoblasts, not interfering with basic cell functions

Multiparametric Evaluation of Post-MI Small Animal Models Using Metabolic ([18F]FDG) and Perfusion-Based (SYN1) Heart Viability Tracers

Cardiovascular diseases (CVD), with myocardial infarction (MI) being one of the crucial components, wreak havoc in developed countries. Advanced imaging technologies are required to obtain quick and widely available diagnostic data. This paper describes a multimodal approach to in vivo perfusion imaging using the novel SYN1 tracer based on the fluorine-18 isotope. The NOD-SCID mice were injected intravenously with SYN1 or [18F] fluorodeoxyglucose ([18F]-FDG) radiotracers after induction of the MI. In all studies, the positron emission tomography–computed tomography (PET/CT) technique was used. To obtain hemodynamic data, mice were subjected to magnetic resonance imaging (MRI). Finally, the biodistribution of the SYN1 compound was performed using Wistar rat model. SYN1 showed normal accumulation in mouse and rat hearts, and MI hearts correctly indicated impaired cardiac segments when compared to [18F]-FDG uptake. In vivo PET/CT and MRI studies showed statistical convergence in terms of the size of the necrotic zone and cardiac function. This was further supported with RNAseq molecular analyses to correlate the candidate function genes’ expression, with Serpinb1c, Tnc and Nupr1, with Trem2 and Aldolase B functional correlations showing statistical significance in both SYN1 and [18F]-FDG. Our manuscript presents a new fluorine-18-based perfusion radiotracer for PET/CT imaging that may have importance in clinical applications. Future research should focus on confirmation of the data elucidated here to prepare SYN1 for first-in-human trials

Supplementary_table - The impact of in vitro cell culture duration on the maturation of human cardiomyocytes derived from induced pluripotent stem cells of myogenic origin

<p>Supplementary_table for The impact of in vitro cell culture duration on the maturation of human cardiomyocytes derived from induced pluripotent stem cells of myogenic origin by Jarosław Lewandowski, Natalia Rozwadowska, Tomasz J Kolanowski, Agnieszka Malcher, Agnieszka Zimna, Anna Rugowska, Katarzyna Fiedorowicz, Wojciech Łabędź, Łukasz Kubaszewski, Katarzyna Chojnacka, Katarzyna Bednarek-Rajewska, Przemysław Majewski, and Maciej Kurpisz in Cell Transplantation</p