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1.
Bioact Mater ; 16: 433-450, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35415291

RESUMO

Small-diameter vascular grafts fabricated from synthetic biodegradable polymers exhibit beneficial mechanical properties but often face poor regenerative potential. Different tissue engineering approaches have been employed to improve tissue regeneration in vascular grafts, but there remains a requirement for a new generation of synthetic grafts that can orchestrate the host response to achieve robust vascular regeneration. Vascular stem/progenitor cells (SPCs) are mostly found in quiescent niches but can be activated in response to injury and participate in endothelium and smooth muscle regeneration during neo-artery formation. Here, we developed a functional vascular graft by surface immobilization of stem cell antigen-1 (Sca-1) antibody on an electrospun poly(ε-caprolactone) graft (PCL-Sca-1 Ab). PCL-Sca-1 Ab promoted capture and retainment of Sca-1+ SPCs in vitro. In rat abdominal aorta replacement models, PCL-Sca-1 Ab stimulated in vivo recruitment of Sca-1+ SPCs, and drove SPCs differentiation towards vascular cell lineages. The origin of infiltrated Sca-1+ SPCs was further investigated using a bone marrow transplantation mouse model, which revealed that Sca-1+ SPCs originating from the resident tissues and bone marrow contributed to rapid vascular regeneration of vascular grafts. Our data indicated that PCL-Sca-1 Ab vascular grafts may serve as a useful strategy to develop next generation cell-free vascular grafts.

2.
Adv Mater ; : e2201736, 2022 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-35487518

RESUMO

An abundant number of nanomaterials have been discovered to possess enzyme-like catalytic activity, termed nanozymes. It was identified that a variety of internal and external factors influence the catalytic activity of nanozymes. However, there is a lack of essential methodologies to uncover the hidden mechanisms between nanozyme features and enzyme-like activity. Here we demonstrated a data-driven approach that utilized machine learning algorithms to understand particle-property relationships, allowing for classification and quantitative predictions of enzyme-like activity exhibited by nanozymes. High consistency between predicted outputs and the observations was confirmed by accuracy (90.6%) and R2 (up to 0.80). Furthermore, sensitive analysis of the models revealed the central roles of transition metals in determining nanozyme activity. As an example, the models were successfully applied to predict or design desirable nanozymes by uncovering the hidden relationship between different periods of transition metals and their enzyme-like performance. This study offers a promising strategy to develop nanozymes with desirable catalytic activity and demonstrates the potential of machine learning within the field of material science. This article is protected by copyright. All rights reserved.

3.
Adv Sci (Weinh) ; : e2105408, 2022 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-35319828

RESUMO

Acute myocardial infarction (MI) is the leading cause of death worldwide. Exogenous delivery of nitric oxide (NO) to the infarcted myocardium has proven to be an effective strategy for treating MI due to the multiple physiological functions of NO. However, reperfusion of blood flow to the ischemic tissues is accompanied by the overproduction of toxic reactive oxygen species (ROS), which can further exacerbate tissue damage and compromise the therapeutic efficacy. Here, an injectable hydrogel is synthesized from the chitosan modified by boronate-protected diazeniumdiolate (CS-B-NO) that can release NO in response to ROS stimulation and thereby modulate ROS/NO disequilibrium after ischemia/reperfusion (I/R) injury. Furthermore, administration of CS-B-NO efficiently attenuated cardiac damage and adverse cardiac remodeling, promoted repair of the heart, and ameliorated cardiac function, unlike a hydrogel that only released NO, in a mouse model of myocardial I/R injury. Mechanistically, regulation of the ROS/NO balance activated the antioxidant defense system and protected against oxidative stress induced by I/R injury via adaptive regulation of the Nrf2-Keap1 pathway. Inflammation is then reduced by inhibition of the activation of NF-κB signaling. Collectively, these results show that this dual-function hydrogel may be a promising candidate for the protection of tissues and organs after I/R injury.

4.
Sci Adv ; 8(11): eabl3888, 2022 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-35294246

RESUMO

There is a lack in clinically-suitable vascular grafts. Biotubes, prepared using in vivo tissue engineering, show potential for vascular regeneration. However, their mechanical strength is typically poor. Inspired by architectural design of steel fiber reinforcement of concrete for tunnel construction, poly(ε-caprolactone) (PCL) fiber skeletons (PSs) were fabricated by melt-spinning and heat treatment. The PSs were subcutaneously embedded to induce the assembly of host cells and extracellular matrix to obtain PS-reinforced biotubes (PBs). Heat-treated medium-fiber-angle PB (hMPB) demonstrated superior performance when evaluated by in vitro mechanical testing and following implantation in rat abdominal artery replacement models. hMPBs were further evaluated in canine peripheral arterial replacement and sheep arteriovenous graft models. Overall, hMPB demonstrated appropriate mechanics, puncture resistance, rapid hemostasis, vascular regeneration, and long-term patency, without incidence of luminal expansion or intimal hyperplasia. These optimized hMPB properties show promise as an alternatives to autologous vessels in clinical applications.

5.
Adv Mater ; 34(14): e2110352, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35107869

RESUMO

Restoration of sufficient blood supply for the treatment of ischemia remains a significant scientific and clinical challenge. Here, a cell-like nanoparticle delivery technology is introduced that is capable of recapitulating multiple cell functions for the spatiotemporal triggering of vascular regeneration. Specifically, a copper-containing protein is successfully prepared using a recombinant protein scaffold based on a de novo design strategy, which facilitates the timely release of nitric oxide and improved accumulation of particles within ischemic tissues. Through closely mimicking physiological cues, the authors demonstrate the benefits of bioactive factors secreted from hypoxic stem cells on promoting angiogenesis. Following this cell-mimicking manner, artificial hybrid nanosized cells (Hynocell) are constructed by integrating the hypoxic stem cell secretome into nanoparticles with surface coatings of cell membranes fused with copper-containing protein. The Hynocell, hybridized with different cell-derived components, provides synergistic effects on targeting ischemic tissues and promoting vascular regeneration in acute hindlimb ischemia and acute myocardial infarction models. This study offers new insights into the utilization of nanotechnology to potentiate the development of cell-free therapeutics.


Assuntos
Biomimética , Neovascularização Fisiológica , Animais , Cobre , Membro Posterior/irrigação sanguínea , Isquemia/terapia
6.
Theranostics ; 12(3): 1132-1147, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35154478

RESUMO

Rationale: Employing in situ bioorthogonal catalysis within subcellular organelles, such as lysosomes, remains a challenge. Lysosomal membranes pose an intracellular barrier for drug sequestration, thereby greatly limiting drug accumulation and concentrations at intended targets. Here, we provide a proof-of-concept report of a nanozyme-based strategy that mediates in situ bioorthogonal uncaging reactions within lysosomes, followed by lysosomal escape and the release of uncaged drugs into the cytoplasm. Methods: A model system composed of a protein-based nanozyme platform (based on the transition metals Co, Fe, Mn, Rh, Ir, Pt, Au, Ru and Pd) and caged compound fluorophores was designed to screen for nanozyme/protecting group pairings. The optimized nanozyme/protecting group pairing was then selected for utilization in the design of anti-cancer pro-drugs and drug delivery systems. Results: Our screening system identified Pd nanozymes that mimic mutant P450BM3 activity and specifically cleave propargylic ether groups. We found that the intrinsic peroxidase-like activity of Pd nanozymes induced the production of free radicals under acid conditions, resulting in lysosomal membrane leakage of uncaged molecules into the cytoplasm. Using a multienzyme synergistic approach, our Pd nanozymes achieved in situ bioorthogonal catalysis and nanozyme-mediated lysosomal membrane leakage, which were successfully applied to the design of model pro-drugs for anti-cancer therapy. The extension of our nanozyme system to the construction of a liposome-based "all-in-one" delivery system offers promise for realizing efficacious in vivo tumor-targeted therapies. Conclusions: This strategy shows a promising new direction by utilizing nanotechnology for drug development through in situ catalyzing bioorthogonal chemistry within specific subcellular organelles.


Assuntos
Neoplasias , Pró-Fármacos , Catálise , Humanos , Lisossomos
7.
Methods Mol Biol ; 2303: 453-468, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34626400

RESUMO

The ubiquitous extracellular glycosaminoglycan hyaluronan (HA) is a polymer composed of repeated disaccharide units of alternating D-glucuronic acid and D-N-acetylglucosamine residues linked via alternating ß-1,4 and ß-1,3 glycosidic bonds. Emerging data continue to reveal functions attributable to HA in a variety of physiological and pathological contexts. Defining the mechanisms regulating expression of the human hyaluronan synthase (HAS) genes that encode the corresponding HA-synthesizing HAS enzymes is therefore important in the context of HA biology in health and disease. We describe here methods to analyze transcriptional regulation of the HAS and HAS2-antisense RNA 1 genes. Elucidation of mechanisms of HA interaction with receptors such as the cell surface molecule CD44 is also key to understanding HA function. To this end, we provide protocols for fluorescent recovery after photobleaching analysis of CD44 membrane dynamics in the process of fibroblast to myofibroblast differentiation, a phenotypic transition that is common to the pathology of fibrosis of large organs such as the liver and kidney.


Assuntos
Diferenciação Celular , Regulação da Expressão Gênica , Miofibroblastos , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Hialuronan Sintases/genética , Hialuronan Sintases/metabolismo , Ácido Hialurônico , Miofibroblastos/metabolismo
8.
Methods Mol Biol ; 2375: 153-164, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34591306

RESUMO

Electrospinning has become a popular polymer processing technique for application in vascular tissue engineering due to its unique capability to fabricate porous vascular grafts with fibrous morphology closely mimicking the natural extracellular matrix (ECMs). However, the inherently small pore sizes of electrospun vascular grafts often inhibit cell infiltration and impede vascular regeneration. Here we describe an effective and controllable method to increase the pore size of electrospun poly(ε-caprolactone) (PCL) vascular graft. With this method, composite grafts are prepared by turning on or off electrospraying of poly(ethylene oxide) (PEO) microparticles during the process of electrospinning PCL fibers. The PEO microparticles are used as a porogen agent and can be subsequently selectively removed to create a porogenic layer within the electrospun PCL grafts. Three types of porogenic PCL grafts were constructed using this method. The porogenic layer was either the inner layer, the middle one, or the outer one.


Assuntos
Tecidos Suporte , Óxido de Etileno , Poliésteres , Polietilenoglicóis , Engenharia Tecidual
9.
Materials (Basel) ; 14(23)2021 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-34885623

RESUMO

Bile duct injury (BDI) and bile tract diseases are regarded as prominent challenges in hepatobiliary surgery due to the risk of severe complications. Hepatobiliary, pancreatic, and gastrointestinal surgery can inadvertently cause iatrogenic BDI. The commonly utilized clinical treatment of BDI is biliary-enteric anastomosis. However, removal of the Oddi sphincter, which serves as a valve control over the unidirectional flow of bile to the intestine, can result in complications such as reflux cholangitis, restenosis of the bile duct, and cholangiocarcinoma. Tissue engineering and biomaterials offer alternative approaches for BDI treatment. Reconstruction of mechanically functional and biomimetic structures to replace bile ducts aims to promote the ingrowth of bile duct cells and realize tissue regeneration of bile ducts. Current research on artificial bile ducts has remained within preclinical animal model experiments. As more research shows artificial bile duct replacements achieving effective mechanical and functional prevention of biliary peritonitis caused by bile leakage or obstructive jaundice after bile duct reconstruction, clinical translation of tissue-engineered bile ducts has become a theoretical possibility. This literature review provides a comprehensive collection of published works in relation to three tissue engineering approaches for biomimetic bile duct construction: mechanical support from scaffold materials, cell seeding methods, and the incorporation of biologically active factors to identify the advancements and current limitations of materials and methods for the development of effective artificial bile ducts that promote tissue regeneration.

10.
Front Bioeng Biotechnol ; 9: 770121, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34917597

RESUMO

Artificial small-diameter vascular grafts (SDVG) fabricated from synthetic biodegradable polymers, such as poly(ε-caprolactone) (PCL), exhibit beneficial mechanical properties but are often faced with issues impacting their long-term graft success. Nitric oxide (NO) is an important physiological gasotransmitter with multiple roles in orchestrating vascular tissue function and regeneration. We fabricated a functional vascular graft by electrospinning of nitrate-functionalized poly(ε-caprolactone) that could release NO in a sustained manner via stepwise biotransformation in vivo. Nitrate-functionalized SDVG (PCL/NO) maintained patency following abdominal arterial replacement in rats. PCL/NO promoted cell infiltration at 3-months post-transplantation. In contrast, unmodified PCL SDVG showed slow cell in-growth and increased incidence of neointima formation. PCL/NO demonstrated improved endothelial cell (EC) alignment and luminal coverage, and more defined vascular smooth muscle cell (VSMC) layer, compared to unmodified PCL SDVG. In addition, release of NO stimulated Sca-1+ vascular progenitor cells (VPCs) to differentiate and contribute to rapid luminal endothelialization. Furthermore, PCL/NO inhibited the differentiation of VPCs into osteopontin-positive cells, thereby preventing vascular calcification. Overall, PCL/NO demonstrated enhanced cell ingrowth, EC monolayer formation and VSMC layer regeneration; whilst inhibiting calcified plaque formation. Our results suggested that PCL/NO could serve as promising candidates for improved and long-term success of SDVG implants.

11.
Adv Mater ; 33(39): e2103128, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34350648

RESUMO

Biomimetic design of nanomaterials with enzyme-like characteristics has emerged as a promising method for the generation of novel therapeutics. However, synthesis of nanomaterials while maintaining a high degree of control over both geometry and valency poses a prominent challenge. Herein, the authors introduce a nanomaterial-based synthetic biology strategy for accurate and quantitative tailoring of high-ordered nanostructures that uses a "bottom-up" hierarchical incorporation of protein building blocks. The assembled nano-oligomers possessed tunable protein motifs and multivalent binding domains, which facilitated prolonged blood circulation time, accumulation within tumor cells through direct targeting of cell receptors, and deep tumor tissue penetration via a transcytosis mechanism. Using these protein/protein nano-oligomers as scaffolds, the authors created a new series of artificial nano-scaled metalloenzymes (nanozymes) by the in situ incorporation of metal nanoclusters within the cavity of the protein nanocages. Nanozymes were capable of mimicking peroxidase-like activity and generated cytotoxic free radicals. Compared to nanozyme alone, the systemic delivery of oligomeric nanozymes demonstrated significantly enhanced therapeutic and anti-tumor benefits. This study shows a new insight into nanotechnology by taking advantage of synthetic biotechnology.


Assuntos
Materiais Biomiméticos/química , Metaloproteínas/química , Nanoestruturas/química , Animais , Apoptose/efeitos dos fármacos , Materiais Biomiméticos/metabolismo , Materiais Biomiméticos/farmacologia , Materiais Biomiméticos/uso terapêutico , Linhagem Celular Tumoral , Ferritinas/química , Humanos , Metais/química , Camundongos , Camundongos Nus , Neoplasias/tratamento farmacológico , Neoplasias/mortalidade , Neoplasias/patologia , Polietilenoglicóis/química , Espécies Reativas de Oxigênio/metabolismo , Taxa de Sobrevida , Distribuição Tecidual , Transplante Heterólogo
12.
Biomolecules ; 11(8)2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34439762

RESUMO

Myofibroblasts are contractile, α-smooth muscle actin-positive cells with multiple roles in pathophysiological processes. Myofibroblasts mediate wound contractions, but their persistent presence in tissues is central to driving fibrosis, making them attractive cell targets for the development of therapeutic treatments. However, due to shared cellular markers with several other phenotypes, the specific targeting of myofibroblasts has long presented a scientific and clinical challenge. In recent years, myofibroblasts have drawn much attention among scientific research communities from multiple disciplines and specialisations. As further research uncovers the characterisations of myofibroblast formation, function, and regulation, the realisation of novel interventional routes for myofibroblasts within pathologies has emerged. The research community is approaching the means to finally target these cells, to prevent fibrosis, accelerate scarless wound healing, and attenuate associated disease-processes in clinical settings. This comprehensive review article describes the myofibroblast cell phenotype, their origins, and their diverse physiological and pathological functionality. Special attention has been given to mechanisms and molecular pathways governing myofibroblast differentiation, and updates in molecular interventions.


Assuntos
Fibrose/terapia , Miofibroblastos/metabolismo , Pele/patologia , Actinas/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Cicatriz , Receptores ErbB/metabolismo , Humanos , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/metabolismo , Ligantes , Camundongos , Fenótipo , Transdução de Sinais/fisiologia , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Proteínas Wnt/metabolismo , Cicatrização , beta Catenina/metabolismo
13.
J Biol Chem ; 297(3): 100987, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34364871

RESUMO

Progressive fibrosis leads to loss of organ function and affects many organs as a result of excessive extracellular matrix production. The ubiquitous matrix polysaccharide hyaluronan (HA) is central to this through association with its primary receptor, CD44, which exists as standard CD44 (CD44s) or multiple splice variants. Mediators such as profibrotic transforming growth factor (TGF)-ß1 and proinflammatory interleukin (IL)-1ß are widely associated with fibrotic progression. TGF-ß1 induces myofibroblast differentiation, while IL-1ß induces a proinflammatory fibroblast phenotype that promotes fibroblast binding to monocyte/macrophages. CD44 expression is essential for both responses. Potential CD44 splice variants involved, however, are unidentified. The TGF-ß1-activated CD44/epidermal growth factor receptor complex induces differentiation of metastatic cells through interactions with the matrix metalloproteinase inducer, CD147. This study aimed to determine the CD44 variants involved in TGF-ß1- and IL-1ß-mediated responses and to investigate the potential profibrotic role of CD147. Using immunocytochemistry and quantitative PCR, standard CD44s were shown to be essential for both TGF-ß1-induced fibroblast/myofibroblast differentiation and IL-1ß-induced monocyte binding. Co-immunoprecipitation identified that CD147 associated with CD44s. Using CD147-siRNA and confocal microscopy, we also determined that incorporation of the myofibroblast marker, αSMA, into F-actin stress fibers was prevented in the absence of CD147 and myofibroblast-dependent collagen gel contraction was inhibited. CD147 did not associate with HA, but removal of HA prevented the association of CD44s with CD147 at points of cell-cell contact. Taken together, our data suggest that CD44s/CD147 colocalization is essential in regulating the mechanical tension required for the αSMA incorporation into F-actin stress fibers that regulates myofibroblast phenotype.


Assuntos
Basigina/fisiologia , Diferenciação Celular/fisiologia , Receptores de Hialuronatos/fisiologia , Miofibroblastos/citologia , Fator de Crescimento Transformador beta1/fisiologia , Basigina/metabolismo , Humanos , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/metabolismo , Interleucina-1beta/fisiologia , Miofibroblastos/metabolismo
14.
Shock ; 56(6): 1028-1039, 2021 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-34313253

RESUMO

BACKGROUND: Crush syndrome (CS) is the most common cause of deaths following earthquakes and other disasters. The pathogenesis of CS has yet to be fully elucidated. Thus, clinical choice of ideal drug treatments for CS remains deficient. METHODS AND RESULTS: In this study, we first evaluated the relation between extrusion force and the severities of CS. Rats were exposed to different extrusion forces: 1 kg, 3 kg, 5 kg, and 8 kg, respectively. Survival rates, crushed muscle tissue edema, serum biochemical parameters, and histopathological staining were used to assess severity. Our results showed that there were no statistical differences in survival rate or changes in thigh circumference among the different extrusion forces groups. However, serum levels of potassium, creatine kinase, blood urea nitrogen, creatinine, and myoglobin were elevated at 12- and 24-h post-decompression in 5 kg and 8 kg groups, compared with 1 kg and 3 kg groups. Histopathological staining demonstrated that the degree of organ damage to kidney, muscle, and lung tissues correlated with increasing extrusion force. We next analyzed changes in serum protein profiles in 3 kg or 5 kg extrusion pressure groups. A total of 76 proteins (20 upregulated, 56 downregulated) were found to be altered at all three time points (0, 12, and 72 h) post-decompression, compared with the control group. Three common upregulated proteins alpha-1-acid glycoprotein (α1-AGP), neutrophil gelatinase-associated lipocalin (NGAL), and Haptoglobin were selected for validation of increased expression. α1-AGP was explored as a treatment for CS-induced acute kidney injury (AKI). Intraperitoneal injection of α1-AGP protected kidneys from CS-induced AKI by regulating TNF-α and IL-6 production, attenuating neutrophil recruitment, and reducing renal cell apoptosis. CONCLUSION: Our findings demonstrated that the severity of crush injury is causally related to extrusion pressure and increase in blood serum markers. Our identification of the biomarker and treatment candidate, α1-AGP, suggests its implication in predicting the severity of CS and its use as a mediator of CS-induced AKI, respectively.


Assuntos
Injúria Renal Aguda/sangue , Injúria Renal Aguda/diagnóstico , Injúria Renal Aguda/terapia , Orosomucoide/análise , Injúria Renal Aguda/etiologia , Animais , Biomarcadores/sangue , Síndrome de Esmagamento/complicações , Masculino , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Índice de Gravidade de Doença
15.
Biomaterials ; 271: 120746, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33725586

RESUMO

Cellular transition to hypoxia following tissue injury, has been shown to improve angiogenesis and regeneration in multiple tissues. To take advantage of this, many hypoxia-mimicking scaffolds have been prepared, yet the oxygen access state of implanted artificial small-diameter vascular grafts (SDVGs) has not been investigated. Therefore, the oxygen access state of electrospun PCL grafts implanted into rat abdominal arteries was assessed. The regions proximal to the lumen and abluminal surfaces of the graft walls were normoxic and only the interior of the graft walls was hypoxic. In light of this differential oxygen access state of the implanted grafts and the critical role of vascular regeneration on SDVG implantation success, we investigated whether modification of SDVGs with HIF-1α stabilizer dimethyloxalylglycine (DMOG) could achieve hypoxia-mimicking responses resulting in improving vascular regeneration throughout the entirety of the graft wall. Therefore, DMOG-loaded PCL grafts were fabricated by electrospinning, to support the sustained release of DMOG over two weeks. In vitro experiments indicated that DMOG-loaded PCL mats had significant biological advantages, including: promotion of human umbilical vein endothelial cells (HUVECs) proliferation, migration and production of pro-angiogenic factors; and the stimulation of M2 macrophage polarization, which in-turn promoted macrophage regulation of HUVECs migration and smooth muscle cells (SMCs) contractile phenotype. These beneficial effects were downstream of HIF-1α stabilization in HUVECs and macrophages in normoxic conditions. Our results indicated that DMOG-loaded PCL grafts improved endothelialization, contractile SMCs regeneration, vascularization and modulated the inflammatory reaction of grafts in abdominal artery replacement models, thus promoting vascular regeneration.


Assuntos
Bioprótese , Enxerto Vascular , Animais , Prótese Vascular , Hipóxia , Ratos , Regeneração
16.
Int J Mol Sci ; 22(4)2021 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-33557232

RESUMO

Fibrosis is characterized by excessive production of disorganized collagen- and fibronectin-rich extracellular matrices (ECMs) and is driven by the persistence of myofibroblasts within tissues. A key protein contributing to myofibroblast differentiation is extra domain A fibronectin (EDA-FN). We sought to target and interfere with interactions between EDA-FN and its integrin receptors to effectively inhibit profibrotic activity and myofibroblast formation. Molecular docking was used to assist in the design of a blocking polypeptide (antifibrotic 38-amino-acid polypeptide, AF38Pep) for specific inhibition of EDA-FN associations with the fibroblast-expressed integrins α4ß1 and α4ß7. Blocking peptides were designed and evaluated in silico before synthesis, confirmation of binding specificity, and evaluation in vitro. We identified the high-affinity EDA-FN C-C' loop binding cleft within integrins α4ß1 and α4ß7. The polypeptide with the highest predicted binding affinity, AF38Pep, was synthesized and could achieve specific binding to myofibroblast fibronectin-rich ECM and EDA-FN C-C' loop peptides. AF38Pep demonstrated potent myofibroblast inhibitory activity at 10 µg/mL and was not cytotoxic. Treatment with AF38Pep prevented integrin α4ß1-mediated focal adhesion kinase (FAK) activation and early signaling through extracellular-signal-regulated kinases 1 and 2 (ERK1/2), attenuated the expression of pro-matrix metalloproteinase 9 (MMP9) and pro-MMP2, and inhibited collagen synthesis and deposition. Immunocytochemistry staining revealed an inhibition of α-smooth muscle actin (α-SMA) incorporation into actin stress fibers and attenuated cell contraction. Increases in the expression of mRNA associated with fibrosis and downstream from integrin signaling were inhibited by treatment with AF38Pep. Our study suggested that AF38Pep could successfully interfere with EDA-FN C-C' loop-specific integrin interactions and could act as an effective inhibitor of fibroblast of myofibroblast differentiation.


Assuntos
Desenho de Fármacos , Fibroblastos/efeitos dos fármacos , Fibronectinas/metabolismo , Fibrose/tratamento farmacológico , Integrinas/metabolismo , Miofibroblastos/efeitos dos fármacos , Peptídeos/farmacologia , Sítios de Ligação , Diferenciação Celular , Matriz Extracelular/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibronectinas/química , Fibrose/metabolismo , Fibrose/patologia , Humanos , Integrinas/química , Pulmão/citologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Simulação de Acoplamento Molecular , Miofibroblastos/citologia , Miofibroblastos/metabolismo , Ligação Proteica , Domínios Proteicos , Transdução de Sinais
17.
Adv Mater ; 33(9): e2006570, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33480459

RESUMO

Development of enzyme mimics for the scavenging of excessive mitochondrial superoxide (O2 •- ) can serve as an effective strategy in the treatment of many diseases. Here, protein reconstruction technology and nanotechnology is taken advantage of to biomimetically create an artificial hybrid nanozyme. These nanozymes consist of ferritin-heavy-chain-based protein as the enzyme scaffold and a metal nanoparticle core as the enzyme active center. This artificial cascade nanozyme possesses superoxide dismutase- and catalase-like activities and also targets mitochondria by overcoming multiple biological barriers. Using cardiac ischemia-reperfusion animal models, the protective advantages of the hybrid nanozymes are demonstrated in vivo during mitochondrial oxidative injury and in the recovery of heart functionality following infarction via systemic delivery and localized release from adhesive hydrogels (i.e., cardiac patch), respectively. This study illustrates a de novo design strategy in the development of enzyme mimics and provides a promising therapeutic option for alleviating oxidative damage in regenerative medicine.


Assuntos
Materiais Biomiméticos/química , Ferritinas/química , Sequestradores de Radicais Livres/química , Compostos de Manganês/química , Nanopartículas Metálicas/química , Mitocôndrias/metabolismo , Óxidos/química , Superóxidos/química , Aminoácidos/química , Animais , Materiais Biomiméticos/metabolismo , Catalase/química , Catalase/metabolismo , Catálise , Permeabilidade da Membrana Celular , Ferritinas/metabolismo , Coração , Humanos , Hidrogéis , Camundongos , Modelos Animais , Superóxido Dismutase/química , Superóxido Dismutase/metabolismo , Cicatrização
18.
J Am Soc Nephrol ; 31(10): 2292-2311, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32769144

RESUMO

BACKGROUND: Progressive fibrosis is the underlying pathophysiological process of CKD, and targeted prevention or reversal of the profibrotic cell phenotype is an important goal in developing therapeutics for CKD. Nanoparticles offer new ways to deliver antifibrotic therapies to damaged tissues and resident cells to limit manifestation of the profibrotic phenotype. METHODS: We focused on delivering plasmid DNA expressing bone morphogenetic protein 7 (BMP7) or hepatocyte growth factor (HGF)-NK1 (HGF/NK1) by encapsulation within chitosan nanoparticles coated with hyaluronan, to safely administer multifunctional nanoparticles containing the plasmid DNA to the kidneys for localized and sustained expression of antifibrotic factors. We characterized and evaluated nanoparticles in vitro for biocompatibility and antifibrotic function. To assess antifibrotic activity in vivo, we used noninvasive delivery to unilateral ureteral obstruction mouse models of CKD. RESULTS: Synthesis of hyaluronan-coated chitosan nanoparticles containing plasmid DNA expressing either BMP7 or NGF/NKI resulted in consistently sized nanoparticles, which-following endocytosis driven by CD44+ cells-promoted cellular growth and inhibited fibrotic gene expression in vitro. Intravenous tail injection of these nanoparticles resulted in approximately 40%-45% of gene uptake in kidneys in vivo. The nanoparticles attenuated the development of fibrosis and rescued renal function in unilateral ureteral obstruction mouse models of CKD. Gene delivery of BMP7 reversed the progression of fibrosis and regenerated tubules, whereas delivery of HGF/NK1 halted CKD progression by eliminating collagen fiber deposition. CONCLUSIONS: Nanoparticle delivery of HGF/NK1 conveyed potent antifibrotic and proregenerative effects. Overall, this research provided the proof of concept on which to base future investigations for enhanced targeting and transfection of therapeutic genes to kidney tissues, and an avenue toward treatment of CKD.


Assuntos
Antifibrinolíticos/administração & dosagem , Proteína Morfogenética Óssea 7/genética , Técnicas de Transferência de Genes , Fator de Crescimento de Hepatócito/genética , Nanopartículas Multifuncionais , Insuficiência Renal Crônica/terapia , Animais , Técnicas de Cultura de Células , Quitosana , Modelos Animais de Doenças , Ácido Hialurônico , Camundongos , Polímeros
19.
ACS Nano ; 14(9): 12133-12147, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32790341

RESUMO

Extracellular vesicles (EVs) derived from mesenchymal stem cells (MSC-EVs) have been recognized as a promising cell-free therapy for acute kidney injury (AKI), which avoids safety concerns associated with direct cell engraftment. However, low stability and retention of MSC-EVs have limited their therapeutic efficacy. RGD (Arg-Gly-Asp) peptide binds strongly to integrins, which have been identified on the surface of MSC-EV membranes; yet RGD has not been applied to EV scaffolds to enhance and prolong bioavailability. Here, we developed RGD hydrogels, which we hypothesized could augment MSC-EV efficacy in the treatment of AKI models. In vivo tracking of the labeled EVs revealed that RGD hydrogels increased retention and stability of EVs. Integrin gene knockdown experiments confirmed that EV-hydrogel interaction was mediated by RGD-integrin binding. Upon intrarenal injection into mouse AKI models, EV-RGD hydrogels provided superior rescuing effects to renal function, attenuated histopathological damage, decreased tubular injury, and promoted cell proliferation in early phases of AKI. RGD hydrogels also augmented antifibrotic effects of MSC-EVs in chronic stages. Further analysis revealed that the presence of microRNA let-7a-5p in MSC-EVs served as the mechanism contributing to the reduced cell apoptosis and elevated cell autophagy in AKI. In conclusion, RGD hydrogels facilitated MSC-derived let-7a-5p-containing EVs, improving reparative potential against AKI. This study developed an RGD scaffold to increase the EV integrin-mediated loading and in turn improved therapeutic efficacy in renal repair; therefore this strategy shed light on MSC-EV application as a cell-free treatment for potentiated efficiency.


Assuntos
Vesículas Extracelulares , Nanofibras , Animais , Arginina , Ácido Aspártico , Glicina , Rim , Camundongos , Oligopeptídeos
20.
Sci Adv ; 6(19): eaaz8011, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32494716

RESUMO

Development of new approaches to biomimetically reconstruct vasculature networks remains challenging in regenerative medicine. We introduce a particle-based artificial stem cell spheroid (ASSP) technology that recapitulates paracrine functions of three-dimensional (3D) SSPs for vasculature regeneration. Specifically, we used a facile method to induce the aggregation of stem cells into 3D spheroids, which benefited from hypoxia microenvironment-driven and enhanced secretion of proangiogenic bioactive factors. Furthermore, we artificially reconstructed 3D spheroids (i.e., ASSP) by integration of SSP-secreted factors into micro-/nanoparticles with cell membrane-derived surface coatings. The easily controllable sizes of the ASSP particles provided superior revascularization effects on the ischemic tissues in hindlimb ischemia models through local administration of ASSP microparticles and in myocardial infarction models via the systemic delivery of ASSP nanoparticles. The strategy offers a promising therapeutic option for ischemic tissue regeneration and addresses issues faced by the bottlenecked development in the delivery of stem cell therapies.

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