ABSTRACT
Nanoplastics (NPs) have become common worldwide and attracted increasing attention due to their serious toxic effects. Owing to their higher surface area and volume ratios and ability to easily enter tissues, NPs impose more serious toxic effects than microplastics. However, the effect of NP exposure on vascular stenosis remains unclear. To measure the effects of polystyrene NP (PS-NP) exposure on vascular toxicity, we conducted analyses of blood biochemical parameters, pathological histology, high-throughput sequencing, and bioinformatics. Red fluorescent PS-NPs (100 nm) were effectively uptake by mouse vascular arterial tissue. The uptake of PS-NPs resulted in vascular toxicity, including alterations in lipid metabolism and thickening of the arterial wall. Based on PIWI-interacting RNA (piRNA) sequencing, 1547 and 132 differentially expressed piRNAs (DEpiRNAs) were detected in the PS-NP treatment group after 180 and 30 days, including 787 and 86 upregulated and 760 and 46 downregulated compared with the control group, respectively. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses indicated that the target genes of DEpiRNAs were mostly involved in cell growth and cell motility-related signaling, such as the MAPK signaling pathway. This is the first study to highlight the alteration in piRNA levels in mouse vascular arterial tissue after PS-NP exposure. This study adds to the knowledge regarding the regulatory mechanism of pathological changes induced by PS-NP exposure.
Subject(s)
Nanoparticles , Water Pollutants, Chemical , Animals , Mice , Piwi-Interacting RNA , Constriction, Pathologic , Microplastics , Polystyrenes/toxicity , PlasticsABSTRACT
Owing to their potential adverse health effects, global contamination by microplastics (MPs) has attracted increased scientific and societal concerns. However, in vivo studies on MP toxicity, along with its effects and underlying mechanisms, remain limited. We recently found that non-coding RNA (ncRNAs) contribute to MP-mediated vascular toxicity. Moreover, previous studies have identified N6-methyladenosine (m6A) modifications in ncRNAs as influencing factors in cardiovascular disease. However, whether and how m6A modifications in ncRNAs are affected by MP-induced cardiotoxicity remain unknown. Herein, we profiled differentially expressed ncRNAs and their related m6A modification profiles in MP-exposed myocardial tissue using RNA sequencing (RNA-seq) and methylated RNA immunoprecipitation sequencing (MeRIP-seq). First, we observed that MPs accumulated in different organs and upregulated apoptosis in the heart, liver, spleen, and kidney cells. Furthermore, total m6A and METTL3 levels increased in the myocardium after exposure to MPs. RNA-seq results revealed that 392 lncRNAs and 302 circRNAs were differentially expressed in MP-treated mouse myocardium compared to the control group. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses showed that these altered lncRNAs and circRNAs were closely associated with endocytosis, cellular senescence, and cell cycle signaling pathways, which may cause cardiotoxicity. Furthermore, MeRIP-seq data showed different distributions and abundances of m6A modifications in lncRNAs and circRNAs. Additionally, we identified differentially m6A methylated lncRNAs and circRNAs through conjoint analysis of the two high-throughput sequencing datasets and found that both m6A modifications and the expression of circ-Arfgef2 and lncG3bp2 were upregulated after exposure to MPs. This suggests that MP-induced m6A modifications in ncRNAs are involved in cardiotoxicity. Our findings contribute to a better understanding of MP-induced cardiotoxicity and new molecular targets for treating cardiac injury.
ABSTRACT
Extensive inflammation of endothelial cells (ECs) facilitates atherosclerotic lesion formation. Circular RNA (circRNA) participates in atherosclerosis (AS)-related inflammation responses; however, whether and how circ_0086296 regulates atherosclerotic inflammation and lesions have not been investigated. Microarray analysis, quantitative real-time polymerase chain reaction, and fluorescence in situ hybridization assay were performed to detect the expression and location of hsa_circ_0086296 in human carotid artery plaques, aorta of atherosclerotic mice, and human umbilical vein endothelial cells (HUVECs). Sanger sequencing was used to verify the loop structure of circ_0086296. The relationship among circ_0086296, miR-576-3p, IFIT1, STAT1, and EIF4A3 was validated using bioinformatics, luciferase assay, RNA pull-down assay, and RNA immunoprecipitation. The atherosclerosis mouse model was used to evaluate the function of circ_0086296 in vivo. circ_0086296 expression was significantly upregulated in human carotid artery plaques, oxidized low-density lipoprotein (ox-LDL)-treated HUVECs, and the aorta of atherosclerotic mice. Functional analysis indicated that circ_0086296 promotes ECs injury in vitro and atherosclerosis progression in vivo. The mechanism analysis indicated that circ_0086296 sponged miR-576-3p to promote IFIT1-STAT1 expression. Moreover, STAT1 upregulated circ_0086296 expression, forming the circ_0086296/miR-576-3p/IFIT1/STAT1 feedback loop. Notably, inhibition of the circ_0086296/miR-576-3p/IFIT1 axis could block atherosclerotic lesion formation both in vivo and in vitro. Finally, circ_0086296 was overexpressed in exosomes of patients with atherosclerosis and exosomes of ox-LDL-treated ECs. Therefore, the circ_0086296/miR-576-3p/IFIT1/STAT1 feedback loop participates in atherosclerosis progression and contributes to the high circ_0086296 expression observed in the exosomes of serum of patients with atherosclerosis. This study sought to provide a deep understanding of the mechanisms underlying the aberrant EC phenotype in AS.
Subject(s)
Atherosclerosis , Carotid Stenosis , MicroRNAs , RNA, Circular , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Animals , Apoptosis , Atherosclerosis/metabolism , Cell Proliferation/genetics , Feedback , Human Umbilical Vein Endothelial Cells/metabolism , Humans , In Situ Hybridization, Fluorescence , Inflammation , Lipoproteins, LDL , Mice , MicroRNAs/genetics , MicroRNAs/metabolism , RNA, Circular/genetics , RNA-Binding Proteins/genetics , STAT1 Transcription FactorABSTRACT
OBJECTIVES: This review aims to see the progress of several clinically-used monoclonal antibodies in treating ALL patients and how they improved patients' outcomes. METHODS: We searched Web of Science, Elsevier and PubMed for relevant published studies, and summarized eligible evidence on the management of newly-diagnosed and relapsed or refractory ALL with monoclonal antibodies. Ongoing trials were identified from ClinicalTrials.gov. RESULTS: Rituximab, an anti-CD20 monoclonal antibody, prolonged patients' complete remission duration and overall survival when combined with hyper-CVAD regimen. Another anti-CD20 monoclonal antibody, Ofatumumab, was reported to have similar benefits. Blinatumomab allows endogenous CD3-positive cytotoxic T cells to target and eliminate CD19-positive blasts. FDA has approved its efficacy in patients with R/R B-ALL and eliminating minimal residual disease (MRD). It serves as a bridge to eradicate MRD before transplantation, and may also be a new choice for patients unable to undergo transplantation. An anti-CD22 monoclonal antibody named Inotuzumab Ozogamicin showed great improvement in patients' outcome, but its toxicity to liver is also worthy of our attention. CONCLUSION: Monoclonal antibodies are proven to be a promising immunotherapeutic strategy to improve ALL patients' outcome in the long term. There's still a need for individualized treatment with effective and well-tolerated medicines.Trial registration: ClinicalTrials.gov identifier: NCT01363128.Trial registration: ClinicalTrials.gov identifier: NCT01466179.Trial registration: ClinicalTrials.gov identifier: NCT02013167.Trial registration: ClinicalTrials.gov identifier: NCT02000427.Trial registration: ClinicalTrials.gov identifier: NCT01564784.Trial registration: ClinicalTrials.gov identifier: NCT03677596.Trial registration: ClinicalTrials.gov identifier: NCT01363297.Trial registration: ClinicalTrials.gov identifier: NCT02981628.Trial registration: ClinicalTrials.gov identifier: NCT03094611.Trial registration: ClinicalTrials.gov identifier: NCT01371630.Trial registration: ClinicalTrials.gov identifier: NCT04224571.Trial registration: ClinicalTrials.gov identifier: NCT02458014.Trial registration: ClinicalTrials.gov identifier: NCT04546399.Trial registration: ClinicalTrials.gov identifier: NCT02879695.Trial registration: ClinicalTrials.gov identifier: NCT03913559.Trial registration: ClinicalTrials.gov identifier: NCT03441061.Trial registration: ClinicalTrials.gov identifier: NCT03739814.Trial registration: ClinicalTrials.gov identifier: NCT02877303.Trial registration: ClinicalTrials.gov identifier: NCT03698552.Trial registration: ClinicalTrials.gov identifier: NCT04601584.Trial registration: ClinicalTrials.gov identifier: NCT04684147.Trial registration: ClinicalTrials.gov identifier: NCT04681105.
Subject(s)
Antineoplastic Agents, Immunological , Antineoplastic Agents , Burkitt Lymphoma , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Antibodies, Monoclonal/therapeutic use , Antineoplastic Agents/therapeutic use , Antineoplastic Agents, Immunological/therapeutic use , Burkitt Lymphoma/drug therapy , Humans , Inotuzumab Ozogamicin , Neoplasm, Residual , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapyABSTRACT
The neural crest stem cells derived from human induced pluripotent stem cells (iPSC-NCSCs) are a valuable autologous cell source for tissue engineering and regenerative medicine. In this study, we investigated how iPSC-NCSCs could be regulated to regenerate arteries by microenvironmental factors, including the physical factor of matrix stiffness, and the chemical factor of transforming growth factor beta-1 (TGF-ß1). We found that, compared to soft substrate, stiff substrate drove iPSC-NCSCs differentiation into smooth muscle cells, which was further enhanced by TGF-ß1. To investigate the regulatory role of TGF-ß1 in vivo, we fabricated vascular grafts composed of electrospun nanofibrous scaffolds, collagen gel, iPSC-NCSCs, and TGF-ß1, and implanted them into athymic rats. The results showed that TGF-ß1 significantly promoted extracellular matrix synthesis and increased mechanical strength of vascular grafts. This study presents a proof of concept that iPSC-NCSCs can be used as a promising autologous cell source for vascular regeneration when combined with physical and chemical engineering.
Subject(s)
Blood Vessel Prosthesis , Carotid Arteries/drug effects , Induced Pluripotent Stem Cells/drug effects , Neural Stem Cells/drug effects , Tissue Scaffolds , Transforming Growth Factor beta1/pharmacology , Animals , Biomechanical Phenomena , Carotid Arteries/cytology , Carotid Arteries/physiology , Cell Differentiation/drug effects , Collagen/chemistry , Collagen/pharmacology , Gels , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/physiology , Myocytes, Smooth Muscle/cytology , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/physiology , Nanofibers/chemistry , Nanofibers/ultrastructure , Neural Crest/cytology , Neural Crest/drug effects , Neural Crest/physiology , Neural Stem Cells/cytology , Neural Stem Cells/physiology , Polyesters/chemistry , Rats , Rats, Nude , Regeneration/drug effects , Regeneration/physiology , Tissue Engineering/methodsABSTRACT
The definition of consciousness remains a difficult issue that requires urgent understanding and resolution. Currently, consciousness research is an intensely focused area of neuroscience. However, to establish a greater understanding of the concept of consciousness, more detailed, intrinsic neurobiological research is needed. Additionally, an accurate assessment of the level of consciousness may strengthen our awareness of this concept and provide new ideas for patients undergoing clinical treatment of consciousness disorders. In addition, research efforts that help elucidate the concept of consciousness have important scientific and clinical significance. This review presents the latest progress in consciousness research and proposes our assumptions with regard to the network of consciousness.
ABSTRACT
Stem cells are often transplanted with scaffolds for tissue regeneration; however, how the mechanical property of a scaffold modulates stem cell fate in vivo is not well understood. Here we investigated how matrix stiffness modulates stem cell differentiation in a model of vascular graft transplantation. Multipotent neural crest stem cells (NCSCs) were differentiated from induced pluripotent stem cells, embedded in the hydrogel on the outer surface of nanofibrous polymer grafts, and implanted into rat carotid arteries by anastomosis. After 3 months, NCSCs differentiated into smooth muscle cells (SMCs) near the outer surface of the polymer grafts; in contrast, NCSCs differentiated into glial cells in the most part of the hydrogel. Atomic force microscopy demonstrated a stiffer matrix near the polymer surface but much lower stiffness away from the polymer graft. Consistently, in vitro studies confirmed that stiff surface induced SMC genes whereas soft surface induced glial genes. These results suggest that the scaffold's mechanical properties play an important role in directing stem cell differentiation in vivo, which has important implications in biomaterials design for stem cell delivery and tissue engineering.
Subject(s)
Cell Differentiation/physiology , Neural Crest/cytology , Neural Stem Cells/cytology , Animals , Cell Differentiation/drug effects , Cells, Cultured , Humans , Hydrogels/pharmacology , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/drug effects , Multipotent Stem Cells/cytology , Multipotent Stem Cells/drug effects , Myocytes, Smooth Muscle/cytology , Myocytes, Smooth Muscle/drug effects , Nanofibers/chemistry , Neural Crest/drug effects , Neural Stem Cells/drug effects , Neuroglia/cytology , Neuroglia/drug effects , Polymers/chemistry , Rats , Tissue Engineering/methods , Tissue ScaffoldsABSTRACT
Due to high incidence of vascular bypass procedures, an unmet need for suitable vessel replacements exists, especially for small-diameter (<6 mm) vascular grafts. Here, we developed a novel, bilayered, synthetic vascular graft of 1-mm diameter that consisted of a microfibrous luminal layer and a nanofibrous outer layer, which was tailored to possess the same mechanical property as native arteries. We then chemically modified the scaffold with mucin, a glycoprotein lubricant on the surface of epithelial tissues, by either passive adsorption or covalent bonding using the di-amino-poly(ethylene glycol) linker to microfibers. Under static and physiological flow conditions, conjugated mucin was more stable than adsorbed mucin on the surfaces. Mucin could slightly inhibit blood clotting, and mucin coating suppressed platelet adhesion on microfibrous scaffolds. In the rat common carotid artery anastomosis model, grafts with conjugated mucin, but not adsorbed mucin, exhibited excellent patency and higher cell infiltration into the graft walls. Mucin, which can be easily obtained from autologous sources, offers a novel method for improving the hemocompatibility and surface lubrication of vascular grafts and many other implants.
Subject(s)
Blood Vessel Prosthesis , Mucins/pharmacology , Nanofibers/chemistry , Vascular Patency/drug effects , Animals , Anticoagulants/pharmacology , Biomechanical Phenomena/drug effects , Cattle , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Endothelium/drug effects , Endothelium/metabolism , Extracellular Matrix/drug effects , Extracellular Matrix/metabolism , Female , Nanofibers/ultrastructure , Platelet Adhesiveness/drug effects , Prosthesis Implantation , Rats , Rats, Nude , Rheology/drug effects , Tissue ScaffoldsABSTRACT
Small-diameter synthetic vascular grafts have high failure rate and tissue-engineered blood vessels are limited by the scalability. Here we engineered bioactive materials for in situ vascular tissue engineering, which recruits two types of endogenous progenitor cells for the regeneration of blood vessels. Heparin was conjugated to microfibrous vascular grafts to suppress thrombogenic responses, and stromal cell-derived factor-1α (SDF-1α) was immobilized onto heparin to recruit endogenous progenitor cells. Heparin-bound SDF-1α was more stable than adsorbed SDF-1α under both static and flow conditions. Microfibrous grafts were implanted in rats by anastomosis to test the functional performance. Heparin coating improved the short-term patency, and immobilized SDF-1α further improved the long-term patency. SDF-1α effectively recruited endothelial progenitor cells (EPCs) to the luminal surface of the grafts, which differentiated into endothelial cells (ECs) and accelerated endothelialization. More interestingly, SDF-1α increased the recruitment of smooth muscle progenitor cells (SMPCs) to the grafts, and SMPCs differentiated into smooth muscle cells (SMCs) in vivo and in vitro. Consistently, SDF-1α-immobilized grafts had significantly higher elastic modulus. This work demonstrates the feasibility of simultaneously recruiting progenitor cells of ECs and SMCs for in situ blood vessel regeneration. This in situ tissue engineering approach will have broad applications in regenerative medicine.
Subject(s)
Anticoagulants/pharmacology , Blood Vessel Prosthesis , Chemokine CXCL12/pharmacology , Endothelial Cells/drug effects , Heparin/pharmacology , Myocytes, Smooth Muscle/drug effects , Stem Cells/drug effects , Animals , Anticoagulants/administration & dosage , Cell Differentiation/drug effects , Cells, Cultured , Chemokine CXCL12/administration & dosage , Coated Materials, Biocompatible/chemistry , Elastic Modulus , Endothelial Cells/cytology , Heparin/administration & dosage , Male , Myocytes, Smooth Muscle/cytology , Rats , Rats, Sprague-Dawley , Stem Cells/cytology , Vascular Grafting , Vascular Patency/drug effectsABSTRACT
Neural crest stem cells (NCSCs) play an important role in the development and represent a valuable cell source for tissue engineering. However, how mechanical factors in vivo regulate NCSC differentiation is not understood. Here NCSCs were derived from induced pluripotent stem cells and used as a model to determine whether vascular mechanical strain modulates the differentiation of NCSCs into smooth muscle (SM) lineage. NCSCs were cultured on micropatterned membranes to mimic the organization of smooth muscle cells (SMCs), and subjected to cyclic uniaxial strain. Mechanical strain enhanced NCSC proliferation and ERK2 phosphorylation. In addition, mechanical strain induced contractile marker calponin-1 within 2 days and slightly induced SM myosin within 5 days. On the other hand, mechanical strain suppressed the differentiation of NCSCs into Schwann cells. The induction of calponin-1 by mechanical strain was inhibited by neural induction medium but further enhanced by TGF-ß. For NCSCs pre-treated with TGF-ß, mechanical strain induced the gene expression of both calponin-1 and SM myosin. Our results demonstrated that mechanical strain regulates the differentiation of NCSCs in a manner dependent on biochemical factors and the differentiation stage of NCSCs. Understanding the mechanical regulation of NCSC differentiation will shed light on the development and remodeling of vascular tissues, and how transplanted NCSCs respond to mechanical factors.
Subject(s)
Cell Differentiation , Embryonic Stem Cells/cytology , Microtechnology , Myocytes, Smooth Muscle/cytology , Neural Crest/cytology , Stress, Mechanical , Biomarkers/metabolism , Calcium-Binding Proteins/metabolism , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Embryonic Stem Cells/drug effects , Embryonic Stem Cells/metabolism , Gene Expression Regulation/drug effects , Humans , Membranes, Artificial , Microfilament Proteins/metabolism , Myocytes, Smooth Muscle/drug effects , Myosin Heavy Chains/metabolism , Schwann Cells/cytology , Schwann Cells/drug effects , Surface Properties , Time Factors , Transforming Growth Factor beta/pharmacology , CalponinsABSTRACT
Induced pluripotent stem cells (iPSCs) hold great promise for cell therapies and tissue engineering. Neural crest stem cells (NCSCs) are multipotent and represent a valuable system to investigate iPSC differentiation and therapeutic potential. Here we derived NCSCs from human iPSCs and embryonic stem cells (ESCs), and investigated the potential of NCSCs for neural tissue engineering. The differentiation of iPSCs and the expansion of derived NCSCs varied in different cell lines, but all NCSC lines were capable of differentiating into mesodermal and ectodermal lineages, including neural cells. Tissue-engineered nerve conduits were fabricated by seeding NCSCs into nanofibrous tubular scaffolds, and used as a bridge for transected sciatic nerves in a rat model. Electrophysiological analysis showed that only NCSC-engrafted nerve conduits resulted in an accelerated regeneration of sciatic nerves at 1 month. Histological analysis demonstrated that NCSC transplantation promoted axonal myelination. Furthermore, NCSCs differentiated into Schwann cells and were integrated into the myelin sheath around axons. No teratoma formation was observed for up to 1 year after NCSC transplantation in vivo. This study demonstrates that iPSC-derived multipotent NCSCs can be directly used for tissue engineering and that the approach that combines stem cells and scaffolds has tremendous potential for regenerative medicine applications.
Subject(s)
Induced Pluripotent Stem Cells/physiology , Nerve Regeneration/physiology , Nerve Tissue/physiology , Neural Crest/cytology , Tissue Engineering/methods , Animals , Cell Differentiation/physiology , Electrophysiology , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/transplantation , Nerve Tissue/cytology , Rats , Sciatic Nerve/cytology , Sciatic Nerve/transplantation , Tissue ScaffoldsABSTRACT
Trauma injuries often cause peripheral nerve damage and disability. A goal in neural tissue engineering is to develop synthetic nerve conduits for peripheral nerve regeneration having therapeutic efficacy comparable to that of autografts. Nanofibrous conduits with aligned nanofibers have been shown to promote nerve regeneration, but current fabrication methods rely on rolling a fibrous sheet into the shape of a conduit, which results in a graft with inconsistent size and a discontinuous joint or seam. In addition, the long-term effects of nanofibrous nerve conduits, in comparison with autografts, are still unknown. Here we developed a novel one-step electrospinning process and, for the first time, fabricated a seamless bi-layer nanofibrous nerve conduit: the luminal layer having longitudinally aligned nanofibers to promote nerve regeneration, and the outer layer having randomly organized nanofibers for mechanical support. Long-term in vivo studies demonstrated that bi-layer aligned nanofibrous nerve conduits were superior to random nanofibrous conduits and had comparable therapeutic effects to autografts for nerve regeneration. In summary, we showed that the engineered nanostructure had a significant impact on neural tissue regeneration in situ. The results from this study will also lead to the scalable fabrication of engineered nanofibrous nerve conduits with designed nanostructure. This technology platform can be combined with drug delivery and cell therapies for tissue engineering.
Subject(s)
Guided Tissue Regeneration/methods , Nanofibers/chemistry , Nerve Regeneration/physiology , Peripheral Nerves/physiology , Tissue Engineering/methods , Tissue Scaffolds/chemistry , Animals , Axons/pathology , Biomechanical Phenomena , Electrophysiological Phenomena/physiology , Female , Materials Testing , Myelin Sheath/pathology , Peripheral Nerves/pathology , Rats , Rats, Inbred Lew , Recovery of Function/physiologyABSTRACT
Brain arteriovenous malformations (BAVMs) are an important cause of intracranial hemorrhage (ICH) in young adults. A small percent of BAVMs is due to hereditary hemorrhagic telangiectasia 1 and 2 (HHT1 and 2), which are caused by mutations in two genes involved in TGF-ß signaling: endoglin (ENG) and activin-like kinase 1 (ALK1). The BAVM phenotype is an incomplete penetrant in HHT patients, and the mechanism is unknown. We tested the hypothesis that a "response-to-injury" triggers abnormal vascular (dysplasia) development, using Eng and Alk1 haploinsufficient mice. Adeno-associated virus (AAV) expressing vascular endothelial growth factor (VEGF) was used to mimic the injury conditions. VEGF overexpression caused a similar degree of angiogenesis in the brain of all groups, except that the cortex of Alk1(+/-) mice had a 33% higher capillary density than other groups. There were different levels of cerebrovascular dysplasia in haploinsufficient mice (Eng(+/)>Alk1(+/-)), which simulates the relative penetrance of BAVM in HHT patients (HHT1>HHT2). Few dysplastic capillaries were observed in AAV-LacZ-injected mice. Our data indicate that both angiogenic stimulation and genetic alteration are necessary for the development of dysplasia, suggesting that anti-angiogenic therapies might be adapted to slow the progression of the disease and decrease the risk of spontaneous ICH.
ABSTRACT
Endothelial progenitor cell-based therapy offers great potential to reduce morbidity and mortality in patients with vascular diseases. In animal models of ischemia, circulating endothelial progenitor cells (EPCs) have been shown to home to sites of active angiogenesis and differentiate into endothelial cells in response to tissue ischemia, vascular trauma, or tumor growth. These studies indicate a thorough understanding of EPCs function and role in angiogenesis as a potential therapeutic target for vascular diseases. In this chapter, we describe the procedures for: (1) isolation of EPCs from circulating blood and bone marrow; (2) culturing these cells; (3) characterization of EPCs by FCAS, DiI-ac-LDL up-taking, and FITC-UEA-1 binding assays; and (4) determination of EPCs' function by examining proliferation, migration, and tube formation in vitro.
Subject(s)
Endothelial Cells/cytology , Neovascularization, Physiologic , Stem Cells/cytology , Animals , Blood Circulation , Bone Marrow Cells/cytology , Cell Culture Techniques , Cell Movement , Cell Proliferation , Cell Separation , Endothelial Cells/metabolism , Flow Cytometry , Humans , RatsABSTRACT
The difficulty in spinal cord regeneration is related to the inhibitory factors for axon growth and the lack of appropriate axon guidance in the lesion region. Here we developed scaffolds with aligned nanofibers for nerve guidance and drug delivery in spinal cord. Blended polymers including Poly (l-lactic acid) (PLLA) and Poly (lactide-co-glycolide) (PLGA) were used to electrospin nanofibrous scaffolds with two-layer structure: aligned nanofibers in the inner layer and random nanofibers in the outer layer. Rolipram, a small molecule that can enhance cAMP activity in neurons and suppress inflammatory responses, was immobilized onto nanofibers. To test the therapeutic effects of nanofibrous scaffolds, the nanofibrous scaffolds loaded with rolipram were used to bridge the hemisection lesion in 8-week old athymic rats. The scaffolds with rolipram increased axon growth through the scaffolds and in the lesion, promoted angiogenesis through the scaffold, and decreased the population of astrocytes and chondroitin sulfate proteoglycans in the lesion. Locomotor scale rating analysis showed that the scaffolds with rolipram significantly improved hindlimb function after 3 weeks. This study demonstrated that nanofibrous scaffolds offered a valuable platform for drug delivery for spinal cord regeneration.
ABSTRACT
Hemangiomas are angiogenesis-dependent benign vascular tumors that can rupture and cause intracranial hemorrhages. We previously showed that the transcription factor homeobox A5 (HoxA5), which is absent in activated angiogenic endothelial cells can block angiogenesis. Here, we investigated whether restoring expression of HoxA5 blocks hemangioma growth by transplanting mouse hemangioendothelioma endothelial cells (EOMA) or HoxA5-expressing EOMA cells into the brains of mice. The EOMA cells induced brain hemangiomas characterized by large cystlike spaces lined by thin walls of endothelial cells surrounded by scant smooth muscle cells. When HoxA5-expressing EOMA cells were injected, lesion volumes were reduced between 5- and 20-fold compared with the EOMA control group (p < 0.05). Restoration of HoxA5 was associated with increased thrombospondin-2, which inhibits angiogenesis and reduced hypoxia-inducible factor 1alpha expression. These data suggest that restoring HoxA5 can attenuate experimental brain hemangioma development.
Subject(s)
Brain Neoplasms/metabolism , Brain Neoplasms/physiopathology , Gene Expression Regulation, Neoplastic , Hemangioma/metabolism , Hemangioma/physiopathology , Homeodomain Proteins/metabolism , Phosphoproteins/metabolism , Actins/metabolism , Animals , Brain Neoplasms/etiology , Brain Neoplasms/surgery , Cell Line, Tumor , Disease Models, Animal , Endothelial Cells/metabolism , Endothelial Cells/transplantation , Gene Expression Regulation, Neoplastic/genetics , Hemangioma/etiology , Hemangioma/surgery , Homeodomain Proteins/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Lectins , Male , Mice , Phosphoproteins/genetics , RNA, Messenger/metabolism , Thrombospondins/metabolism , Time Factors , Transcription Factors , Transfection/methodsABSTRACT
Cerebral venous hypertension (VH) and angiogenesis are implicated in the pathogenesis of brain arteriovenous malformation and dural arteriovenous fistulae. We studied the association of VH and angiogenesis using a mouse brain VH model. Sixty mice underwent external jugular vein and common carotid artery (CCA) anastomosis (VH model), CCA ligation, or sham dissection (n=20). Hypoxia-inducible factor-1alpha (HIF-1alpha), vascular endothelial growth factor (VEGF) and stromal-cell-derived factor-1alpha (SDF-1alpha) expression, and matrix metalloproteinase (MMP) activity were analyzed. We found VH animals had higher (P<0.05) sagittal sinus pressure (8+/-1 mm Hg) than control groups (1+/-1 mm Hg). Surface cerebral blood flow and mean arterial pressure did not change. Hypoxia-inducible factor-1alpha, VEGF, and SDF-1alpha expression increased (P<0.05). Neutrophils and MMP-9 activity increased 10-fold 1 day after surgery, gradually decreased afterward, and returned to baseline 2 weeks after surgery. Macrophages began to increase 3 days after surgery (P<0.05), which coincided with the changes in SDF-1alpha expression. Capillary density in the parasagittal cortex increased 17% compared with the controls. Our findings suggest that mild nonischemic VH results in a pro-angiogenic stage in the brain by upregulating HIF-1 and its downstream targets, VEGF and SDF-1alpha, increasing leukocyte infiltration and MMP-9 activity.
Subject(s)
Cerebral Veins/pathology , Gene Expression Regulation/drug effects , Hypertension/pathology , Hypoxia-Inducible Factor 1, alpha Subunit/biosynthesis , Matrix Metalloproteinase 9/biosynthesis , Neovascularization, Pathologic/genetics , Neutrophils/enzymology , Animals , Blood Flow Velocity/physiology , Blotting, Western , Cerebral Veins/metabolism , Cerebrovascular Circulation/physiology , Chemokine CXCL12/biosynthesis , Chemokine CXCL12/genetics , Disease Models, Animal , Enzyme-Linked Immunosorbent Assay , Hypertension/genetics , Hypertension/metabolism , Immunohistochemistry , Macrophages/pathology , Male , Matrix Metalloproteinase 9/genetics , Mice , Mice, Inbred C57BL , Neovascularization, Pathologic/enzymology , Neovascularization, Pathologic/metabolism , Neovascularization, Pathologic/pathology , Neutrophil Infiltration , Vascular Endothelial Growth Factor A/biosynthesis , Vascular Endothelial Growth Factor A/geneticsABSTRACT
Developmental endothelial locus-1 (Del-1) is a novel angiomatrix protein that has been shown to stimulate a potent angiogenic response and promote functional recovery in hind-limb and cardiac ischemia in animal models; however, its impact on cerebral angiogenesis is unknown. In this study, we investigated whether Del-1 overexpression via gene transfer induces cerebral angiogenesis in a murine model, and examined Del-1 expression after ischemic stroke. Cerebral Del-1 overexpression was achieved with AAV (adeno-associated virus) transduction system via stereotactic injection. Control mice were injected with AAV-lacZ. Del-1 gene transduction led to a significant induction of cerebral angiogenesis compared to AAV-lacZ treatment at 4 weeks after gene transfer (Del-1: 97+/-7 vs lacZ: 64+/-28, vessels/field, p<0.05). Mice transduced with AAV-Del-1 showed significantly elevated vascular densities for up to 6 weeks after gene delivery. In addition, double immunofluorescent staining showed co-localization of endothelial cell marker CD31 with BrdU (specific marker for proliferating cells), indicating that Del-1 promoted endogenous endothelial cell proliferation and angiogenesis. Our immunohistochemical staining also showed that Del-1 expression was markedly up-regulated in the peri-infarct area at 3 days after permanent focal cerebral ischemia compared to the sham-operated non-ischemic control. Our data suggest that Del-1 may participate in modulating cerebral angiogenesis, and that gene transfer of Del-1 may provide a novel and potent means for stimulating cerebral angiogenesis.
Subject(s)
Brain Ischemia/therapy , Carrier Proteins/genetics , Carrier Proteins/metabolism , Neovascularization, Physiologic/genetics , Transduction, Genetic/methods , Analysis of Variance , Animals , Brain Ischemia/pathology , Bromodeoxyuridine/metabolism , Calcium-Binding Proteins , Cell Adhesion Molecules , Cell Line, Transformed , Cerebral Cortex/blood supply , Dependovirus/physiology , Disease Models, Animal , Humans , Intercellular Signaling Peptides and Proteins , Male , Mice , Platelet Endothelial Cell Adhesion Molecule-1/metabolism , Time Factors , Transfection/methodsABSTRACT
Circulating blood endothelial progenitor cells (EPCs) contribute to postnatal vasculogenesis, providing a novel therapeutic target for vascular diseases. However, the molecular mechanism of EPC-induced vasculogenesis is unknown. Interleukin-6 plays multiple functions in angiogenesis and vascular remodeling. Our previous study demonstrated that the polymorphism (174G>C) in IL-6 gene promoter was associated with brain vascular disease. In this study, we investigated if IL-6 receptor is expressed in human EPCs derived from circulating mononuclear cells, and if interleukin-6 (IL-6) stimulates EPC angiogenesis in vitro. First, we isolated and cultured mononuclear cells from adult human circulating blood. We obtained EPC clones that were further cultured and expended for the angiogenesis study. We found that the EPCs possessed human mature endothelial cell phenotypes; however, they proliferated much faster than mature endothelial cells (P<0.05). We then found that IL-6 receptor (gp-80) was expressed in the EPCs, and that administration of IL-6 could activate receptor gp80/gp130 signaling pathways including downstream extracellular signal-regulated kinase 1/2 and STAT3 phosphorylation in EPCs. Furthermore, IL-6 stimulated EPC proliferation, migration, and matrigel tube formation in a dose-dependent manner (P<0.05); anti-IL-6 antibodies or IL-6 receptor could abolish these effects (P<0.05). These results suggest that IL-6 plays a crucial role in the biologic behavior of blood-derived EPCs, which may help clarify the mechanism of IL-6 inflammatory-related diseases.
Subject(s)
Endothelial Cells/metabolism , Interleukin-6/pharmacology , Neovascularization, Physiologic/physiology , Stem Cells/metabolism , Adult , Cell Movement/drug effects , Cell Movement/physiology , Cell Proliferation/drug effects , Cells, Cultured , Cerebrovascular Disorders/genetics , Cerebrovascular Disorders/metabolism , Cerebrovascular Disorders/pathology , Cytokine Receptor gp130/agonists , Cytokine Receptor gp130/metabolism , Dose-Response Relationship, Drug , Endothelial Cells/cytology , Female , Humans , Inflammation/genetics , Inflammation/metabolism , Interleukin-6/genetics , Interleukin-6/metabolism , Leukocytes, Mononuclear/cytology , Leukocytes, Mononuclear/metabolism , MAP Kinase Signaling System/drug effects , MAP Kinase Signaling System/physiology , Male , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Neovascularization, Physiologic/drug effects , Phosphorylation/drug effects , Polymorphism, Single Nucleotide , Promoter Regions, Genetic , Receptors, Interleukin-6/agonists , Receptors, Interleukin-6/metabolism , STAT3 Transcription Factor/metabolism , Stem Cells/cytologyABSTRACT
BACKGROUND AND PURPOSE: Human brain arteriovenous malformation tissue displays increased levels of vascular endothelial growth factor (VEGF) as well as matrix metalloproteinase (MMP)-9, a tissue protease associated with various intracerebral hemorrhage (ICH). We hypothesized that increased MMP-9 was associated with ICH induced by vascular endothelial growth factor hyperstimulation and that this effect could be attenuated by nonspecific MMP inhibition. METHODS: We used a mouse model with adenoviral vector-mediated vascular endothelial growth factor transduction in the brain. The association of MMP-9 expression and the brain tissue hemoglobin levels, an index of ICH, after stereotactic injection of adenoviral vector-mediated vascular endothelial growth factor into caudate putamen was assessed. A dose-response study with adenoviral vector-mediated vascular endothelial growth factor and a time course study at both 24 and 48 hours postinjection were performed. Effects of minocycline, a nonspecific MMP inhibitor, and pyrrolidine dithiocarbamate, an upstream regulator of MMPs, on MMP-9 activity and thereby the degree of ICH were also tested. RESULTS: Adenoviral vector-mediated vascular endothelial growth factor at the higher dose and at 48 hours induced MMP-9 levels 6-fold (n=6, P=0.02) and increased brain tissue hemoglobin (43.4+/-11.5 versus 30.3+/-4.1 mug/mg, n=6, P=0.003) compared with the adenoviral vector control. Immnunostaining was positive for MMP-9 around the cerebral vessels and the hemorrhagic areas. Minocycline and pyrrolidine dithiocarbamate administration suppressed vascular endothelial growth factor-induced MMP-9 activity (n=6, P=0.003 and P=0.01, respectively) and the associated increases in hemoglobin levels (n=5-6, P=0.001 and P=0.02, respectively). CONCLUSIONS: Vascular endothelial growth factor-induced ICH is associated with increased MMP-9 expression. Suppression of MMP-9 by minocycline or pyrrolidine dithiocarbamate attenuated ICH, suggesting the therapeutic potential of MMP inhibitors in cerebral vascular rupture.