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Dilated cardiomyopathy (DCM) is a primary myocardial disease, leading to heart failure and excessive risk of sudden cardiac death with rather poorly understood pathophysiology. In 2015, Parvari's group identified a recessive mutation in the autophagy regulator, PLEKHM2 gene, in a family with severe recessive DCM and left ventricular non-compaction (LVNC). Fibroblasts isolated from these patients exhibited abnormal subcellular distribution of endosomes, Golgi apparatus, lysosomes and had impaired autophagy flux. To better understand the effect of mutated PLEKHM2 on cardiac tissue, we generated and characterized induced pluripotent stem cells-derived cardiomyocytes (iPSC-CMs) from two patients and a healthy control from the same family. The patient iPSC-CMs showed low expression levels of genes encoding for contractile functional proteins (α and ß-myosin heavy chains and 2v and 2a-myosin light chains), structural proteins integral to heart contraction (Troponin C, T and I) and proteins participating in Ca2+ pumping action (SERCA2 and Calsequestrin 2) compared to their levels in control iPSC-derived CMs. Furthermore, the sarcomeres of the patient iPSC-CMs were less oriented and aligned compared to control cells and generated slowly beating foci with lower intracellular calcium amplitude and abnormal calcium transient kinetics, measured by IonOptix system and MuscleMotion software. Autophagy in patient's iPSC-CMs was impaired as determined from a decrease in the accumulation of autophagosomes in response to chloroquine and rapamycin treatment, compared to control iPSC-CMs. Impairment in autophagy together with the deficiency in the expression of NKX2.5, MHC, MLC, Troponins and CASQ2 genes, which are related to contraction-relaxation coupling and intracellular Ca2+ signaling, may contribute to the defective function of the patient CMs and possibly affect cell maturation and cardiac failure with time.
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Cardiomiopatia Dilatada , Células-Tronco Pluripotentes Induzidas , Humanos , Cálcio/metabolismo , Cálcio/farmacologia , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/metabolismo , Diferenciação Celular , Mutação , Miócitos Cardíacos/metabolismoRESUMO
Pleckstrin Homology And RUN Domain Containing M2 (PLEKHM2) [delAG] mutation causes dilated cardiomyopathy with left ventricular non-compaction (DCM-LVNC), resulting in a premature death of PLEKHM2[delAG] individuals due to heart failure. PLEKHM2 is a factor involved in autophagy, a master regulator of cellular homeostasis, decomposing pathogens, proteins and other cellular components. Autophagy is mainly carried out by the lysosome, containing degradation enzymes, and by the autophagosome, which engulfs substances marked for decomposition. PLEKHM2 promotes lysosomal movement toward the cell periphery. Autophagic dysregulation is associated with neurodegenerative diseases' pathogenesis. Thus, modulation of autophagy holds considerable potential as a therapeutic target for such disorders. We hypothesized that PLEKHM2 is involved in neuronal development and function, and that mutated PLEKHM2 (PLEKHM2[delAG]) neurons will present impaired functions. Here, we studied PLEKHM2-related abnormalities in induced pluripotent stem cell (iPSC)-derived motor neurons (iMNs) as a neuronal model. PLEKHM2[delAG] iMN cultures had healthy control-like differentiation potential but exhibited reduced autophagic activity. Electrophysiological measurements revealed that PLEKHM2[delAG] iMN cultures displayed delayed functional maturation and more frequent and unsynchronized activity. This was associated with increased size and a more perinuclear lysosome cellular distribution. Thus, our results suggest that PLEKHM2 is involved in the functional development of neurons through the regulation of autophagic flux.
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Células-Tronco Pluripotentes Induzidas , Humanos , Autofagia/genética , Autofagossomos/metabolismo , Lisossomos/metabolismo , Neurônios MotoresRESUMO
INTRODUCTION: Acetaminophen (APAP) intoxication is a major cause of acute liver failure. Alginate, an anionic polysaccharide, was previously shown as a macroporous scaffold, to reduce liver inflammation and sustain hepatic synthetic function, when implanted on liver remnant after extended partial hepatectomy. In the recent study we wanted to examine in a model of APAP intoxication the potential of a specially formulated alginate solution to prevent APAP toxicity. METHODS: Three alginate solutions from low (30-50â¯kDa, VLVG), medium (100â¯kDa, LVG54) and high (150â¯kDa, LVG150) molecular weights were examined. Mice were orally administered with the alginate solution before, with and after APAP administration and were compared to control mice which received vehicle only. All mice were euthanized 24â¯h after APAP administration. Liver enzyme, blood APAP, IL-6 and liver histology including Ki-67 proliferation, IgG necrosis and nitrotyrosine staining were studied. RESULTS: VLVG- treated mice presented low ALT levels while 20-40 fold increase was demonstrated in control mice. The effect of LVG solutions was marginal. Accordingly, liver histology was normal with no hepatocytes proliferation in the VLVG group while massive centrilobular necrosis, increased nitrotyrosine staining and high proliferation appeared in livers of control mice. APAP blood levels were comparable in the two groups. Treatment with VLVG was associated with prevention of increase of IL-6 serum levels. CONCLUSION: VLVG, a novel alginate solution, alleviated the liver toxicity and inhibited oncotic necrosis and related immune-mediated damage. VLVG may serve as a novel hepato-protector and prevent drug induced liver injury.
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Acetaminofen/toxicidade , Alginatos/uso terapêutico , Analgésicos não Narcóticos/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Substâncias Protetoras/uso terapêutico , Acetaminofen/sangue , Administração Oral , Alanina Transaminase/sangue , Alginatos/farmacologia , Analgésicos não Narcóticos/sangue , Animais , Aspartato Aminotransferases/sangue , Doença Hepática Induzida por Substâncias e Drogas/sangue , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Modelos Animais de Doenças , Humanos , Interleucina-6/sangue , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Necrose/sangue , Necrose/induzido quimicamente , Necrose/prevenção & controle , Estresse Oxidativo/efeitos dos fármacos , Substâncias Protetoras/farmacologiaRESUMO
Nano- and microscale topographical cues have become recognized as major regulators of cell growth, migration, and phenotype. In tissue engineering, the complex and anisotropic architecture of culture platforms is aimed to imitate the high degree of spatial organization of the extracellular matrix and basement membrane components. Here, we developed a method of creating a novel, magnetically aligned, three-dimensional (3D) tissue culture matrix with three distinct classes of anisotropy-surface topography, microstructure, and physical properties. Alginate-stabilized magnetic nanoparticles (MNPs) were added to a cross-linked alginate solution, and an external magnetic field of about 2400 G was applied during freezing to form the aligned macroporous scaffold structure. The resultant scaffold exhibited anisotropic topographic features on the submicron scale, the directionality of the pore shape, and increased scaffold stiffness in the direction of magnetic alignment. These scaffold features were modulated by an alteration in the impregnated MNP size and concentration, as quantified by electron microscopy, advanced image processing analyses, and rheological methods. Mouse myoblasts (C2C12) cultured on the magnetically aligned scaffolds, demonstrated co-oriented morphology in the direction of the magnetic alignment. In summary, magnetic alignment introduces several degrees of anisotropy in the scaffold structure, providing diverse mechanical cues that can affect seeded cells and further tissue development. Multiscale anisotropy together with the capability of the MNP-containing alginate scaffolds to undergo reversible shape deformation in an oscillating magnetic field creates interesting opportunities for multifarious stimulation of cells and functional tissue development.
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MicroRNA-based therapy that targets cardiac macrophages holds great potential for treatment of myocardial infarction (MI). Here, we explored whether boosting the miRNA-21 transcript level in macrophage-enriched areas of the infarcted heart could switch their phenotype from pro-inflammatory to reparative, thus promoting resolution of inflammation and improving cardiac healing. We employed laser capture microdissection (LCM) to spatially monitor the response to this treatment in the macrophage-enriched zones. MiRNA-21 mimic was delivered to cardiac macrophages post MI by nanoparticles (NPs), spontaneously assembled due to the complexation of hyaluronan-sulfate with the nucleic acid mediated by calcium ion bridges, yielding slightly anionic NPs with a mean diameter of 130 nm. Following intravenous administration, the miRNA-21 NPs were targeted to cardiac macrophages at the infarct zone, elicited their phenotype switch from pro-inflammatory to reparative, promoted angiogenesis, and reduced hypertrophy, fibrosis and cell apoptosis in the remote myocardium. Our work thus presents a new therapeutic strategy to manipulate macrophage phenotype using nanoparticle delivery of miRNA-21 with a potential for use to attenuate post-MI remodeling and heart failure.
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Ácido Hialurônico/análogos & derivados , MicroRNAs/administração & dosagem , Infarto do Miocárdio/terapia , Nanopartículas/química , Animais , Feminino , Técnicas de Transferência de Genes , Terapia Genética , Microdissecção e Captura a Laser , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos Endogâmicos C57BL , MicroRNAs/química , MicroRNAs/genética , MicroRNAs/uso terapêutico , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Miocárdio/metabolismo , Miocárdio/patologiaRESUMO
Tissue development and homeostasis are dependent upon the concerted synthesis, maintenance, and degradation of extracellular matrix (ECM) molecules. Cardiac fibrosis is now recognized as a primary contributor to incidence of heart failure, particularly heart failure with preserved ejection fraction, wherein cardiac filling in diastole is compromised. Periostin is a cell-associated protein involved in cell fate determination, proliferation, tumorigenesis, and inflammatory responses. As a non-structural component of the ECM, secreted 90 kDa periostin is emerging as an important matricellular factor in cardiac mesenchymal tissue development. In addition, periostin's role as a mediator in cell-matrix crosstalk has also garnered attention for its association with fibroproliferative diseases in the myocardium, and for its association with TGF-ß/BMP signaling. This review summarizes the phylogenetic history of periostin, its role in cardiac development, and the major signaling pathways influencing its expression in cardiovascular pathology. Further, we provide a synthesis of the current literature to distinguish the multiple roles of periostin in cardiac health, development and disease. As periostin may be targeted for therapeutic treatment of cardiac fibrosis, these insights may shed light on the putative timing for application of periostin-specific therapies.
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Doenças Cardiovasculares/metabolismo , Moléculas de Adesão Celular/metabolismo , Valvas Cardíacas/embriologia , Animais , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/genética , Matriz Extracelular/metabolismo , Coração/fisiologia , Humanos , Mesoderma/metabolismo , Família Multigênica , Domínios Proteicos , RegeneraçãoRESUMO
Osteochondral defects (OCDs) are conditions affecting both cartilage and the underlying bone. Since cartilage is not spontaneously regenerated, our group has recently developed a strategy of injecting bioactive alginate hydrogel into the defect for promoting endogenous regeneration of cartilage via presentation of affinity-bound transforming growth factor ß1 (TGF-ß1). As in vivo model systems often provide only limited insights as for the mechanism behind regeneration processes, here we describe a novel flow bioreactor for the in vitro modeling of the OCD microenvironment, designed to promote cell recruitment from the simulated bone marrow compartment into the hydrogel, under physiological flow conditions. Computational fluid dynamics modeling confirmed that the bioreactor operates in a relevant slow-flowing regime. Using a chemotaxis assay, it was shown that TGF-ß1 does not affect human mesenchymal stem cell (hMSC) chemotaxis in 2D culture. Accessible through live imaging, the bioreactor enabled monitoring and discrimination between erosion rates and profiles of different alginate hydrogel compositions, using green fluorescent protein-expressing cells. Mathematical modeling of the erosion front progress kinetics predicted the erosion rate in the bioreactor up to 7 days postoperation. Using quantitative real-time polymerase chain reaction of early chondrogenic markers, the onset of chondrogenic differentiation in hMSCs was detected after 7 days in the bioreactor. In conclusion, the designed bioreactor presents multiple attributes, making it an optimal device for mechanistical studies, serving as an investigational tool for the screening of other biomaterial-based, tissue engineering strategies.
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Doenças das Cartilagens/terapia , Cartilagem Articular/fisiologia , Hidrogel de Polietilenoglicol-Dimetacrilato/administração & dosagem , Microscopia Intravital/métodos , Modelos Teóricos , Reatores Biológicos , Células Cultivadas , Quimiotaxia , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/fisiologia , Regeneração , Fator de Crescimento Transformador beta/metabolismoRESUMO
Controlled delivery of heparin-binding (HB) proteins represents a challenge in regenerative medicine strategies. Here, we describe the features of novel nanoparticles (NPs), spontaneously coassembled due to affinity interactions between HB proteins and the semisynthetic anionic polysaccharide, alginate-sulfate. The NPs efficiently encapsulated and protected the proteins from proteolysis. Injection of a combination of NPs encapsulating multiple therapeutic growth factors promoted effective and long-term tissue repair in animal models of severe ischemia (murine model of hindlimb ischemia and acute myocardial infarction in rats). This simple yet efficient NP fabrication method is amenable for clinical use.
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Portadores de Fármacos/administração & dosagem , Heparina/administração & dosagem , Nanopartículas/administração & dosagem , Medicina Regenerativa , Alginatos/química , Animais , Materiais Biocompatíveis/química , Modelos Animais de Doenças , Portadores de Fármacos/química , Ácido Glucurônico/química , Heparina/metabolismo , Ácidos Hexurônicos/química , Membro Posterior/patologia , Isquemia/tratamento farmacológico , Infarto do Miocárdio/tratamento farmacológico , Nanopartículas/química , Ligação Proteica , Sulfatos/químicaRESUMO
BACKGROUND AIMS: Clinical trials have documented beneficial effects of mesenchymal stromal cells from bone marrow and adipose tissue (ASCs) as treatment in patients with ischemic heart disease. However, retention of transplanted cells is poor. One potential way to increase cell retention is to inject the cells in an in situ cross-linked alginate hydrogel. METHODS: ASCs from abdominal human tissue were embedded in alginate hydrogel and alginate hydrogel modified with Arg-Gly-Asp motifs (RGD-alginate) and cultured for 1 week. Cell viability, phenotype, immunogenicity and paracrine activity were determined by confocal microscopy, dendritic cell co-culture, flow cytometry, reverse transcriptase quantitative polymerase chain reaction, Luminex multiplex, and lymphocyte proliferation experiments. RESULTS: ASCs performed equally well in alginate and RGD-alginate. After 1 week of alginate culture, cell viability was >93%. Mesenchymal markers CD90 and CD29 were reduced compared with International Society for Cellular Therapy criteria. Cells sedimented from the alginates during cultivation regained the typical level of these markers, and trilineage differentiation was performed by standard protocols. Hepatocyte growth factor mRNA was increased in ASCs cultivated in alginates compared with monolayer controls. Alginates and alginates containing ASCs did not induce dendritic cell maturation. ASCs in alginate responded like controls to interferon-gamma stimulation (licensing), and alginate culture increased the ability of ASCs to inhibit lymphocyte proliferation. DISCUSSION: ASCs remain viable in alginates; they transiently change phenotype in alginate hydrogel but regain the phenotype of monolayer controls upon release. Cells maintain their paracrine potential while in alginates; the combination of ASCs and alginate is non-immunogenic and, in fact, immunosuppressive.
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Tecido Adiposo/citologia , Alginatos/administração & dosagem , Hidrogel de Polietilenoglicol-Dimetacrilato/administração & dosagem , Transplante de Células-Tronco Mesenquimais/métodos , Inclusão do Tecido/métodos , Adipócitos/química , Adulto , Idoso , Idoso de 80 Anos ou mais , Alginatos/química , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Técnicas de Cocultura , Feminino , Ácido Glucurônico/administração & dosagem , Ácido Glucurônico/química , Fator de Crescimento de Hepatócito/genética , Fator de Crescimento de Hepatócito/metabolismo , Ácidos Hexurônicos/administração & dosagem , Ácidos Hexurônicos/química , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Imunomodulação , Interferon gama/metabolismo , Masculino , Células-Tronco Mesenquimais/citologia , Pessoa de Meia-Idade , Oligopeptídeos/química , RNA Mensageiro/genética , Adulto JovemRESUMO
Nanoparticles (NPs) have great potential to increase the diagnostic capacity of many imaging modalities. MRI is currently regarded as the method of choice for the imaging of deep tissues, and metal ions, such as calcium ions (Ca(2+)), are essential ingredients for life. Despite the tremendous importance of Ca(2+) for the well-being of living systems, the noninvasive determination of the changes in Ca(2+) levels in general, and extracellular Ca(2+) levels in particular, in deep tissues remains a challenge. Here, we describe the preparation and contrast mechanism of a flexible easy to prepare and selective superparamagnetic iron oxide (SPIO) NPs for the noninvasive determination of changes in extracellular Ca(2+) levels using conventional MRI. We show that SPIO NPs coated with monodisperse and purified alginate, having a specific molecular weight, provide a tool to selectively determine Ca(2+) concentrations in the range of 250 µm to 2.5 mm, even in the presence of competitive ions. The alginate-coated magnetic NPs (MNPs) aggregate in the presence of Ca(2+) , which, in turn, affects the T2 relaxation of the water protons in their vicinity. The new alginate-coated SPIO NP formulations, which have no effect on cell viability for 24 h, allow the detection of Ca(2+) levels secreted from ischemic cell cultures and the qualitative examination of the change in extracellular Ca(2+) levels in vivo. These results demonstrate that alginate-coated MNPs can be used, at least qualitatively, as a platform for the noninvasive MRI determination of extracellular Ca(2+) levels in myriad in vitro and in vivo biomedical applications.
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Alginatos/química , Cálcio/análise , Imageamento por Ressonância Magnética/métodos , Nanopartículas de Magnetita/química , Animais , Sobrevivência Celular , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Luz , Masculino , Camundongos Endogâmicos C57BL , Ratos Wistar , Espalhamento de Radiação , Processamento de Sinais Assistido por Computador , Fatores de Tempo , ÁguaRESUMO
Cardiac tissue engineering offers new possibilities for the functional and structural restoration of damaged or lost heart tissue by applying cardiac patches created in vitro. Engineering such functional cardiac patches is a complex mission, involving material design on the nano- and microscale as well as the application of biological cues and stimulation patterns to promote cell survival and organization into a functional cardiac tissue. Herein, we present a novel strategy for creating a functional cardiac patch by combining the use of a macroporous alginate scaffold impregnated with magnetically responsive nanoparticles (MNPs) and the application of external magnetic stimulation. Neonatal rat cardiac cells seeded within the magnetically responsive scaffolds and stimulated by an alternating magnetic field of 5 Hz developed into matured myocardial tissue characterized by anisotropically organized striated cardiac fibers, which preserved its features for longer times than non-stimulated constructs. A greater activation of AKT phosphorylation in cardiac cell constructs after applying a short-term (20 min) external magnetic field indicated the efficacy of magnetic stimulation to actuate at a distance and provided a possible mechanism for its action. Our results point to a synergistic effect of magnetic field stimulation together with nanoparticulate features of the scaffold surface as providing the regenerating environment for cardiac cells driving their organization into functionally mature tissue.
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Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Engenharia Tecidual/métodos , Alicerces Teciduais , Animais , Animais Recém-Nascidos , Adesão Celular , Células Cultivadas , Óxido Ferroso-Férrico/química , Campos Magnéticos , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Alicerces Teciduais/químicaRESUMO
Polysaccharides have emerged as important functional materials because of their unique properties such as biocompatibility, biodegradability, and availability of reactive sites for chemical modifications to optimize their properties. The overwhelming majority of the methods to modify polysaccharides employ random chemical modifications, which often improve certain properties while compromising others. On the other hand, the employed methods for selective modifications often require excess of coupling partners, long reaction times and are limited in their scope and wide applicability. To circumvent these drawbacks, aniline-catalyzed oxime formation is developed for selective modification of a variety of polysaccharides through their reducing end. Notably, it is found that for efficient oxime formation, different conditions are required depending on the composition of the specific polysaccharide. It is also shown how our strategy can be applied to improve the physical and functional properties of alginate hydrogels, which are widely used in tissue engineering and regenerative medicine applications. While the randomly and selectively modified alginate exhibits similar viscoelastic properties, the latter forms significantly more stable hydrogel and superior cell adhesive and functional properties. Our results show that the developed conjugation reaction is robust and should open new opportunities for preparing polysaccharide-based functional materials with unique properties.
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Herein we investigated a new strategy for the modulation of cardiac macrophages to a reparative state, at a predetermined time after myocardial infarction (MI), in aim to promote resolution of inflammation and elicit infarct repair. The strategy employed intravenous injections of phosphatidylserine (PS)-presenting liposomes, mimicking the anti-inflammatory effects of apoptotic cells. Following PS-liposome uptake by macrophages in vitro and in vivo, the cells secreted high levels of anti-inflammatory cytokines [transforming growth factor ß (TGFß) and interleukin 10 (IL-10)] and upregulated the expression of the mannose receptor--CD206, concomitant with downregulation of proinflammatory markers, such as tumor necrosis factor α (TNFα) and the surface marker CD86. In a rat model of acute MI, targeting of PS-presenting liposomes to infarct macrophages after injection via the femoral vein was demonstrated by magnetic resonance imaging (MRI). The treatment promoted angiogenesis, the preservation of small scars, and prevented ventricular dilatation and remodeling. This strategy represents a unique and accessible approach for myocardial infarct repair.
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Lipossomos , Macrófagos/fisiologia , Infarto do Miocárdio/patologia , Miocárdio/patologia , Fosfatidilserinas/administração & dosagem , Animais , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Imuno-Histoquímica , Interleucina-10/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Ratos , Ratos Sprague-Dawley , Fator de Crescimento Transformador beta/metabolismoRESUMO
In recent years, extrusion-based three-dimensional (3D) bioprinting is employed for engineering cardiac patches (CP) due to its ability to assemble complex structures from hydrogel-based bioinks. However, the cell viability in such CPs is low due to shear forces applied on the cells in the bioink, inducing cellular apoptosis. Herein, we investigated whether the incorporation of extracellular vesicles (EVs) in the bioink, engineered to continually deliver the cell survival factor miR-199a-3p would increase the viability within the CP. EVs from THP-1-derived activated macrophages (MΦ) were isolated and characterized by nanoparticle tracking analysis (NTA), cryogenic electron microscopy (cryo-TEM), and Western blot analysis. MiR-199a-3p mimic was loaded into EVs by electroporation after optimization of applied voltage and pulses. Functionality of the engineered EVs was assessed in neonatal rat cardiomyocyte (NRCM) monolayers using immunostaining for the proliferation markers ki67 and Aurora B kinase. To examine the effect of engineered EVs on 3D-bioprinted CP viability, the EVs were added to the bioink, consisting of alginate-RGD, gelatin, and NRCM. Metabolic activity and expression levels of activated-caspase 3 for apoptosis of the 3D-bioprinted CP were evaluated after 5 days. Electroporation (850 V with 5 pulses) was found to be optimal for miR loading; miR-199a-3p levels in EVs increased fivefold compared to simple incubation, with a loading efficiency of 21.0%. EV size and integrity were maintained under these conditions. Cellular uptake of engineered EVs by NRCM was validated, as 58% of cTnT+ cells internalized EVs after 24 h. The engineered EVs induced CM proliferation, increasing the ratio of cell-cycle re-entry of cTnT+ cells by 30% (Ki67) and midbodies+ cell ratio by twofold (Aurora B) compared with the controls. The inclusion of engineered EVs in bioink yielded CP with threefold greater cell viability compared to bioink with no EVs. The prolonged effect of EVs was evident as the CP exhibited elevated metabolic activities after 5 days, with less apoptotic cells compared to CP with no EVs. The addition of miR-199a-3p-loaded EVs to the bioink improved the viability of 3D-printed CP and is expected to contribute to their integration in vivo.
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Nanomedicine offers great potential for the treatment of cardiovascular disease and particulate systems have the capacity to markedly improve bioavailability of therapeutics. The delivery of pro-angiogenic hepatocyte growth factor (HGF) and pro-survival and pro-myogenic insulin-like growth factor (IGF-1) encapsulated in Alginate-Sulfate nanoparticles (AlgS-NP) might improve left ventricular (LV) functional recovery after myocardial infarction (MI). In a porcine ischemia-reperfusion model, MI is induced by 75 min balloon occlusion of the mid-left anterior descending coronary artery followed by reperfusion. After 1 week, pigs (n = 12) with marked LV-dysfunction (LV ejection fraction, LVEF < 45%) are randomized to fusion imaging-guided intramyocardial injections of 8 mg AlgS-NP prepared with 200 µg HGF and IGF-1 (HGF/IGF1-NP) or PBS (Control). Intramyocardial injection is safe and pharmacokinetic studies of Cy5-labeled NP confirm superior cardiac retention compared to intracoronary infusion. Seven weeks after intramyocardial-injection of HGF/IGF1-NP, infarct size, measured using magnetic resonance imaging, is significantly smaller than in controls and is associated with increased coronary flow reserve. Importantly, HGF/IGF1-NP-treated pigs show significantly increased LVEF accompanied by improved myocardial remodeling. These findings demonstrate the feasibility and efficacy of using AlgS-NP as a delivery system for growth factors and offer the prospect of innovative treatment for refractory ischemic cardiomyopathy.
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Infarto do Miocárdio , Traumatismo por Reperfusão Miocárdica , Nanopartículas , Animais , Modelos Animais de Doenças , Fator de Crescimento de Hepatócito , Fator de Crescimento Insulin-Like I , Sulfatos , SuínosRESUMO
Sustained drug-release systems prolong the retention of therapeutic drugs within target tissues to alleviate the need for repeated drug administration. Two major caveats of the current systems are that the release rate and the timing cannot be predicted or fine-tuned because they rely on uncontrolled environmental conditions and that the system must be redesigned for each drug and treatment regime because the drug is bound via interactions that are specific to its structure and composition. We present a controlled and universal sustained drug-release system, which comprises minute spherical particles in which a therapeutic protein is affinity-bound to alginate sulfate (AlgS) through one or more short heparin-binding peptide (HBP) sequence repeats. Employing post-myocardial infarction (MI) heart remodeling as a case study, we show that the release of C9-a matrix metalloproteinase-9 (MMP-9) inhibitor protein that we easily bound to AlgS by adding one, two, or three HBP repeats to its sequence-can be directly controlled by modifying the number of HBP repeats. In an in vivo study, we directly injected AlgS particles, which were bound to C9 through three HBP repeats, into the left ventricular myocardium of mice following MI. We found that the particles substantially reduced post-MI remodeling, attesting to the sustained, local release of the drug within the tissue. As the number of HBP repeats controls the rate of drug release from the AlgS particles, and since C9 can be easily replaced with almost any protein, our tunable sustained-release system can readily accommodate a wide range of protein-based treatments.
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Metaloproteinase 9 da Matriz , Infarto do Miocárdio , Camundongos , Animais , Metaloproteinase 9 da Matriz/metabolismo , Preparações de Ação Retardada/uso terapêutico , Remodelação Ventricular , Função Ventricular Esquerda/fisiologia , Infarto do Miocárdio/terapia , Miocárdio/metabolismoRESUMO
The recent progress made in the bioengineering of cardiac patches offers a new therapeutic modality for regenerating the myocardium after myocardial infarction (MI). We present here a strategy for the engineering of a cardiac patch with mature vasculature by heterotopic transplantation onto the omentum. The patch was constructed by seeding neonatal cardiac cells with a mixture of prosurvival and angiogenic factors into an alginate scaffold capable of factor binding and sustained release. After 48 h in culture, the patch was vascularized for 7 days on the omentum, then explanted and transplanted onto infarcted rat hearts, 7 days after MI induction. When evaluated 28 days later, the vascularized cardiac patch showed structural and electrical integration into host myocardium. Moreover, the vascularized patch induced thicker scars, prevented further dilatation of the chamber and ventricular dysfunction. Thus, our study provides evidence that grafting prevascularized cardiac patch into infarct can improve cardiac function after MI.
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Técnicas de Cultura de Células/métodos , Diferenciação Celular , Transplante de Coração/métodos , Infarto do Miocárdio/cirurgia , Omento/irrigação sanguínea , Omento/cirurgia , Engenharia Tecidual/métodos , Animais , Células Cultivadas , Eletrocardiografia , Sobrevivência de Enxerto , Masculino , Microscopia Eletrônica de Varredura , Infarto do Miocárdio/fisiopatologia , Neovascularização Fisiológica , Omento/citologia , Ratos , Ratos Sprague-Dawley , Transplante Heterotópico , Resultado do TratamentoRESUMO
Cardiac tissue engineering has emerged as a promising strategy to treat infarcted cardiac tissues by replacing the injured region with an ex vivo fabricated functional cardiac patch. Nevertheless, integration of the transplanted patch with the host tissue is still a burden, limiting its clinical application. Here, a bi-functional, 3D bio-printed cardiac patch (CP) design is proposed, composed of a cell-laden compartment at its core and an extracellular vesicle (EV)-laden compartment at its shell for better integration of the CP with the host tissue. Alginate-based bioink solutions were developed for each compartment and characterized rheologically, examined for printability and their effect on residing cells or EVs. The resulting 3D bio-printed CP was examined for its mechanical stiffness, showing an elastic modulus between 4-5 kPa at day 1 post-printing, suitable for transplantation. Affinity binding of EVs to alginate sulfate (AlgS) was validated, exhibiting dissociation constant values similar to those of EVs with heparin. The incorporation of AlgS-EVs complexes within the shell bioink sustained EV release from the CP, with 88% cumulative release compared with 92% without AlgS by day 4. AlgS also prolonged the release profile by an additional 2 days, lasting 11 days overall. This CP design comprises great potential at promoting more efficient patch assimilation with the host.
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Achievement of a high dose of drug in the tumor while minimizing its systemic side effects is one of the important features of an improved drug delivery system. Thus, developing responsive carriers for site-specific delivery of chemotherapeutic agents has become a main goal of research efforts. One of the known hallmarks of cancerous tumors is hypoxia, which offers a target for selective drug delivery. The stimuli-sensitive micellar system developed by us, (PEG-azobenzene-PEI-DOPE (PAPD) has proven to be effective in vitro. The proposed construct developed, PAPD, contains an azobenzene group as a hypoxia-sensitive moiety that triggers the shedding of the PEG layer from the nanoparticle surface under conditions of hypoxia to improve cellular uptake. Using microfluidics, we show significantly improved cellular association and penetration under hypoxia in both single cells and in a 3D tumor model. Employing an in vivo model, we demonstrate slower tumor growth that did not induce systemic side effects, including weight loss in an experimental animal model, when compared to the free drug treatment. This complex-in-nature but simple-in-design system for the simultaneous delivery of siRNA to silence the P-glycoprotein and doxorubicin with active tumor targeting and proven therapeutic efficacy represents a universal platform for the delivery of other hydrophobic chemotherapeutic agents and siRNA molecules which can be further modified.
Assuntos
Doxorrubicina , Hipóxia , Animais , Linhagem Celular Tumoral , Doxorrubicina/uso terapêutico , Micelas , RNA Interferente Pequeno/genéticaRESUMO
After myocardial infarction (MI), the heart's reparative response to the ischemic insult and the related loss of cardiomyocytes involves cardiac fibrosis, in which the damaged tissue is replaced with a fibrous scar. Although the scar is essential to prevent ventricular wall rupture in the infarction zone, it expands over time to remote, non-infarct areas, significantly increasing the extent of fibrosis and markedly altering cardiac structure. Cardiac function in this scenario deteriorates, thereby increasing the probability of heart failure and the risk of death. Recent works have suggested that the matricellular protein periostin, known to be involved in fibrosis, is a candidate therapeutic target for the regulation of MI-induced fibrosis and remodeling. Different strategies for the genetic manipulation of periostin have been proposed previously, yet those works did not properly address the time dependency between periostin activity and cardiac fibrosis. Our study aimed to fill that gap in knowledge and fully elucidate the explicit timing of cellular periostin upregulation in the infarcted heart to enable the safer and more effective post-MI targeting of periostin-producing cells. Surgical MI was performed in C57BL/6J and BALB/c mice by ligation of the left anterior descending coronary artery. Flow cytometry analyses of cells derived from the infarcted hearts and quantitative real-time PCR of the total cellular RNA revealed that periostin expression increased during days 2-7 and peaked on day 7 post-infarct, regardless of mouse strain. The established timeline for cellular periostin expression in the post-MI heart is a significant milestone toward the development of optimal periostin-targeted gene therapy.