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The employment of antibodies as a targeted drug delivery vehicle has proven successful which is exemplified by the emergence of antibody-drug conjugates (ADCs). However, ADCs are not without their shortcomings. Improvements may be made to the ADC platform by decoupling the cytotoxic drug from the delivery vehicle and conjugating an organometallic catalyst in its place. The resulting protein-metal catalyst conjugate was designed to uncage the masked cytotoxin administered as a separate entity. Macropinocytosis of albumin by cancerous cells suggests the potential of albumin acting as the tumor-targeting delivery vehicle. Herein reported are the first preparation and demonstration of ruthenium catalysts with cyclopentadienyl and quinoline-based ligands conjugated to albumin. The effective uncaging abilities were demonstrated on allyloxy carbamate (alloc)-protected rhodamine 110 and doxorubicin, providing a promising catalytic scaffold for the advancement of selective drug delivery methods in the future.
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Antineoplásicos , Inmunoconjugados , Rutenio , Carbamatos , Antineoplásicos/farmacología , AlbúminasRESUMEN
Intervertebral disc (IVD) degeneration is a major cause of low back pain. However, treatments directly approaching the etiology of IVD degeneration and discogenic pain are not yet established. We previously demonstrated that intradiscal implantation of cell-free bioresorbable ultra-purified alginate (UPAL) gel promotes tissue repair and reduces discogenic pain, and a combination of ultra-purified, Good Manufacturing Practice (GMP)-compliant, human bone marrow mesenchymal stem cells (rapidly expanding clones; RECs), and the UPAL gel increasingly enhanced IVD regeneration in animal models. This study investigated the therapeutic efficacy of injecting a mixture of REC and UPAL non-gelling solution for discogenic pain and IVD regeneration in a rat caudal nucleus pulposus punch model. REC and UPAL mixture and UPAL alone suppressed not only the expression of TNF-α, IL-6, and TrkA (p < 0.01, respectively), but also IVD degeneration and nociceptive behavior compared to punching alone (p < 0.01, respectively). Furthermore, REC and UPAL mixture suppressed these expression levels and nociceptive behavior compared to UPAL alone (p < 0.01, respectively). These results suggest that this minimally invasive treatment strategy with a single injection may be applied to treat discogenic pain and as a regenerative therapy.
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Degeneración del Disco Intervertebral , Células Madre Mesenquimatosas , Núcleo Pulposo , Ratas , Humanos , Animales , Alginatos/farmacología , Degeneración del Disco Intervertebral/tratamiento farmacológico , Degeneración del Disco Intervertebral/metabolismo , Células Madre Mesenquimatosas/metabolismo , Dolor/tratamiento farmacológico , Dolor/metabolismoRESUMEN
BACKGROUND: Rapidly expanding clones (RECs) are one of the single-cell-derived mesenchymal stem cell clones sorted from human bone marrow mononuclear cells (BMMCs), which possess advantageous features. The RECs exhibit long-lasting proliferation potency that allows more than 10 repeated serial passages in vitro, considerably benefiting the manufacturing process of allogenic MSC-based therapeutic products. Although RECs aid the preparation of large-variation clone libraries for a greedy selection of better-quality clones, such a selection is only possible by establishing multiple-candidate cell banks for quality comparisons. Thus, there is a high demand for a novel method that can predict "low-risk and high-potency clones" early and in a feasible manner given the excessive cost and effort required to maintain such an establishment. METHODS: LNGFR and Thy-1 co-positive cells from BMMCs were single-cell-sorted into 96-well plates, and only fast-growing clones that reached confluency in 2 weeks were picked up and passaged as RECs. Fifteen RECs were prepared as passage 3 (P3) cryostock as the primary cell bank. From this cryostock, RECs were passaged until their proliferation limitation; their serial-passage limitation numbers were labeled as serial-passage potencies. At the P1 stage, phase-contrast microscopic images were obtained over 6-90 h to identify time-course changes of 24 morphological descriptors describing cell population information. Machine learning models were constructed using the morphological descriptors for predicting serial-passage potencies. The time window and field-of-view-number effects were evaluated to identify the most efficient image data usage condition for realizing high-performance serial-passage potency models. RESULTS: Serial-passage test results indicated variations of 7-13-repeated serial-passage potencies within RECs. Such potency values were predicted quantitatively with high performance (RMSE < 1.0) from P1 morphological profiles using a LASSO model. The earliest and minimum effort predictions require 6-30 h with 40 FOVs and 6-90 h with 15 FOVs, respectively. CONCLUSION: We successfully developed a noninvasive morphology-based machine learning model to enhance the efficiency of establishing cell banks with single-cell-derived RECs for quantitatively predicting the future serial-passage potencies of clones. Conventional methods that can make noninvasive and quantitative predictions without wasting precious cells in the early stage are lacking; the proposed method will provide a more efficient and robust cell bank establishment process for allogenic therapeutic product manufacturing.
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Tyrosinase, an important oxidase involved in the primary immune response in humans, can sometimes become problematic as it can catalyze undesirable oxidation reactions. Therefore, for decades there has been a strong pharmaceutical interest in the discovery of novel inhibitors of this enzyme. Recent studies have also indicated that tyrosinase inhibitors can potentially be used in the treatment of melanoma cancer. Over the years, many new tyrosinase inhibitors have been discovered from various natural sources; however, marine natural products (MNPs) have contributed only a small number of promising candidates. Therefore, in this study we focused on the discovery of new MNP tyrosinase inhibitors of marine cyanobacterial and algal origins. A colorimetric tyrosinase inhibitory assay was used to screen over 4,500 marine extracts against mushroom tyrosinase (A. bisporus). Our results revealed that scytonemin monomer (ScyM), a pure compound from our compound library and also the monomeric last-step precursor in the biosynthesis of the well-known cyanobacterial sunscreen pigment "scytonemin," consistently showed the highest tyrosinase inhibitory score. Determination of the half maximal inhibitory concentration (IC50) further indicated that ScyM is more potent than the commonly used commercial inhibitor standard "kojic acid" (KA; IC50 of ScyM: 4.90 µM vs. IC50 of KA: 11.31 µM). After a scaled-up chemical synthesis of ScyM as well as its O-methyl analog (ScyM-OMe), we conducted a series of follow-up studies on their structures, inhibitory properties, and mode of inhibition. Our results supported ScyM as the second case ever of a novel tyrosinase inhibitory compound based on a marine cyanobacterial natural product. The excellent in vitro performance of ScyM makes it a promising candidate for applications such as a skin-whitening agent or an adjuvant therapy for melanoma cancer treatment.
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BACKGROUND: Lumbar intervertebral disc (IVD) herniations are associated with significant disability. Discectomy is the conventional treatment option for IVD herniations but causes a defect in the IVD, which has low self-repair ability, thereby representing a risk of further IVD degeneration. An acellular, bioresorbable, and good manufacturing practice (GMP)-compliant in situ-forming gel, which corrects discectomy-associated IVD defects and prevents further IVD degeneration had been developed. However, this acellular matrix-based strategy has certain limitations, particularly in elderly patients, whose tissues have low self-repair ability. The aim of this study was to investigate the therapeutic efficacy of using a combination of newly-developed, ultra-purified, GMP-compliant, human bone marrow mesenchymal stem cells (rapidly expanding clones; RECs) and the gel for IVD regeneration after discectomy in a sheep model of severe IVD degeneration. METHODS: RECs and nucleus pulposus cells (NPCs) were co-cultured in the gel. In addition, RECs combined with the gel were implanted into IVDs following discectomy in sheep with degenerated IVDs. FINDINGS: Gene expression of NPC markers, growth factors, and extracellular matrix increased significantly in the co-culture compared to that in each mono-culture. The REC and gel combination enhanced IVD regeneration after discectomy (up to 24 weeks) in the severe IVD degeneration sheep model. INTERPRETATION: These findings demonstrate the translational potential of the combination of RECs with an in situ-forming gel for the treatment of herniations in degenerative human IVDs. FUNDING: Ministry of Education, Culture, Sports, Science, and Technology of Japan, Japan Agency for Medical Research and Development, and the Mochida Pharmaceutical Co., Ltd.
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Degeneración del Disco Intervertebral , Disco Intervertebral , Implantes Absorbibles , Anciano , Animales , Discectomía , Humanos , Disco Intervertebral/metabolismo , Degeneración del Disco Intervertebral/metabolismo , Ovinos , Células Madre/metabolismoRESUMEN
Acute liver injury (ALI) induced by chemicals or viruses can progress rapidly to acute liver failure (ALF), often resulting in death of patients without liver transplantation. Since liver transplantation is limited due to a paucity of donors, expensive surgical costs, and severe immune rejection, novel therapies are required to treat liver injury. Extracellular vesicles (EVs) are used for cellular communication, carrying RNAs, proteins, and lipids and delivering them intercellularly after being endocytosed by target cells. Recently, it was reported that EVs secreted from human hepatocytes have an ability to modulate the immune responses; however, these roles of EVs secreted from human hepatocytes were studied only with in vitro experiments. In the present study, we evidenced that EVs secreted from human hepatocytes attenuated the CCL4-induced ALI by inhibiting the recruitment of monocytes through downregulation of chemokine receptor in the bone marrow and recruitment of neutrophils through the reduction of C-X-C motif chemokine ligand 1 (CXCL1) and CXCL2 expression levels in the liver.
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Tetracloruro de Carbono/efectos adversos , Vesículas Extracelulares/metabolismo , Hepatocitos/metabolismo , Fallo Hepático Agudo/inducido químicamente , Animales , Femenino , Humanos , RatonesRESUMEN
Laucysteinamide A (4) is a marine natural product isolated from the cyanobacterium Caldora penicillata and contains structural motifs found in promising cancer drug leads. The first total synthesis of 4 and its analogues was achieved, which also enabled a concise formal synthesis of somocystinamide A (3), a dimeric congener of 4 that previously showed extremely potent antiproliferative activities. This work provides further insights on structure-activity relationships in this class of natural products.
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Antineoplásicos/síntesis química , Disulfuros/química , Tiazoles/síntesis química , Antineoplásicos/farmacología , Productos Biológicos/química , Productos Biológicos/farmacología , Línea Celular Tumoral , Cianobacterias/química , Humanos , Estructura Molecular , Relación Estructura-Actividad , Tiazoles/farmacologíaRESUMEN
This study was performed to examine the applicability of the newly developed nano-biocomposite, ß-tricalcium phosphate (ß-TCP)/u-HA/poly-d/l-lactide (PDLLA), to bone defects in the oral and maxillofacial area. This novel nano-biocomposite showed several advantages, including biocompatibility, biodegradability, and osteoconductivity. In addition, its optimal plasticity also allowed its utilization in irregular critical bone defect reconstructive surgery. Here, three different nano-biomaterials, i.e., ß-TCP/PDLLA, ß-TCP, and PDLLA, were implanted into critical bone defects in the right lateral mandible of 10-week-old Sprague-Dawley (SD) rats as bone graft substitutes. Micro-computed tomography (Micro-CT) and immunohistochemical staining for the osteogenesis biomarkers, Runx2, osteocalcin, and the leptin receptor, were performed to investigate and compare bone regeneration between the groups. Although the micro-CT results showed the highest bone mineral density (BMD) and bone volume to total volume (BV/TV) with ß-TCP, immunohistochemical analysis indicated better osteogenesis-promoting ability of ß-TCP/PDLLA, especially at an early stage of the bone healing process. These results confirmed that the novel nano-biocomposite, ß-TCP/PDLLA, which has excellent biocompatibility, bioresorbability and bioactive/osteoconductivity, has the potential to become a next-generation biomaterial for use as a bone graft substitute in maxillofacial reconstructive surgery.
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Human mesenchymal stem/stromal cells (hMSCs) have garnered enormous interest as a potential resource for cell-based therapies. However, the molecular mechanisms regulating senescence in hMSCs remain unclear. To elucidate these mechanisms, we performed gene expression profiling to compare clonal immature MSCs exhibiting multipotency with less potent MSCs. We found that the transcription factor Frizzled 5 (FZD5) is expressed specifically in immature hMSCs. The FZD5 cell surface antigen was also highly expressed in the primary MSC fraction (LNGFR+ THY-1+ ) and cultured MSCs. Treatment of cells with the FZD5 ligand WNT5A promoted their proliferation. Upon FZD5 knockdown, hMSCs exhibited markedly attenuated proliferation and differentiation ability. The observed increase in the levels of senescence markers suggested that FZD5 knockdown promotes cellular senescence by regulating the noncanonical Wnt pathway. Conversely, FZD5 overexpression delayed cell cycle arrest during the continued culture of hMSCs. These results indicated that the intrinsic activation of FZD5 plays an essential role in negatively regulating senescence in hMSCs and suggested that controlling FZD5 signaling offers the potential to regulate hMSC quality and improve the efficacy of cell-replacement therapies using hMSCs.
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Diferenciación Celular/fisiología , Senescencia Celular/fisiología , Receptores Frizzled/metabolismo , Células Madre Mesenquimatosas/metabolismo , Proliferación Celular/fisiología , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Células Cultivadas , Humanos , Trasplante de Células Madre Mesenquimatosas/métodosRESUMEN
Coibamide A is a highly potent antiproliferative cyclic depsipeptide, which was originally isolated from a Panamanian marine cyanobacterium. In this study, the synthesis of coibamide A has been investigated using Fmoc-based solid-phase peptide synthesis followed by the cleavage of the resulting linear peptide from the resin and its subsequent macrolactonization. The peptide sequence of the linear coibamide A precursor was constructed on a solid-support following the optimization of the coupling conditions, where numerous coupling agents were evaluated. The macrocyclization of the resulting linear peptide provided the [d-MeAla(11)]-epimer of coibamide A, which exhibited nanomolar cytotoxic activity towards a number of human cancer cell lines.
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Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Depsipéptidos/síntesis química , Depsipéptidos/farmacología , Neoplasias/tratamiento farmacológico , Humanos , Estructura Molecular , Neoplasias/patología , Técnicas de Síntesis en Fase Sólida , Estereoisomerismo , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
Credneramides A (1) and B (2), two vinyl chloride-containing metabolites, were isolated from a Papua New Guinea collection of cf. Trichodesmium sp. nov. and expand a recently described class of vinyl chloride-containing natural products. The precursor fatty acid, credneric acid (3), was isolated from both the aqueous and organic fractions of the parent fraction as well as from another geographically and phylogenetically distinct cyanobacterial collection (Panama). Credneramides A and B inhibited spontaneous calcium oscillations in murine cerebrocortical neurons at low micromolar concentrations (1, IC(50) 4.0 µM; 2, IC(50) 3.8 µM).
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Aminas/aislamiento & purificación , Aminas/farmacología , Ácidos Grasos/química , Ácidos Grasos/farmacología , Neurotransmisores/aislamiento & purificación , Neurotransmisores/farmacología , Fenetilaminas/farmacología , Aminas/química , Animales , Secuencia de Bases , Cianobacterias , Ácidos Grasos/aislamiento & purificación , Ratones , Estructura Molecular , Neurotransmisores/química , Papúa Nueva Guinea , Fenetilaminas/química , Fenetilaminas/aislamiento & purificación , Percepción de Quorum/fisiologíaRESUMEN
The structural assignment of new natural product molecules supports research in a multitude of disciplines that may lead to new therapeutic agents and or new understanding of disease biology. However, reports of numerous structural revisions, even of recently elucidated natural products, inspired the present survey of techniques used in structural misassignments and subsequent revisions in the context of constitutional or configurational errors. Given the comparatively recent development of marine natural products chemistry, coincident with modern spectroscopy, it is of interest to consider the relative roles of spectroscopy and chemical synthesis in the structure elucidation and revision of those marine natural products that were initially misassigned. Thus, a tabulated review of all marine natural product structural revisions from 2005 to 2010 is organized according to structural motif revised. Misassignments of constitution are more frequent than perhaps anticipated by reliance on HMBC and other advanced NMR experiments, especially when considering the full complement of all natural products. However, these techniques also feature prominently in structural revisions, specifically of marine natural products. Nevertheless, as is the case for revision of relative and absolute configuration, total synthesis is a proven partner for marine, as well as terrestrial, natural products structure elucidation. It also becomes apparent that considerable 'detective work' remains in structure elucidation, in spite of the spectacular advances in spectroscopic techniques.
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Organismos Acuáticos/química , Productos Biológicos/química , Animales , Organismos Acuáticos/metabolismo , Productos Biológicos/síntesis química , Productos Biológicos/metabolismo , Resonancia Magnética Nuclear Biomolecular/métodos , EstereoisomerismoRESUMEN
An efficient synthetic methodology for 3-hydroxy-2,2-dimethyloctynoic acid (DHOYA) and several variants, which are increasingly common fragments encountered in bioactive marine cyanobacterial metabolites, was developed. These fragments were obtained in three steps via a tertiary aldol reaction utilizing an Evans' chiral auxiliary to afford the desired stereochemistry at the ß-hydroxy carbon. Thus far, this methodology has been successfully applied in determination of the absolute stereochemistry of eight cyanobacterial natural products, including the VGSC activator palymramide A.
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Expression of the Wnt modulator secreted frizzled related protein 4 (Sfrp4) is upregulated after heart ischemic injury. We show that intramuscular administration of recombinant Sfrp4 to rat heart ischemic injury and recanalization models prevents further deterioration of cardiac function after the ischemic injury. The effect of Sfrp4 persisted for at least 20 weeks when Sfrp4 was administered in a slow release system (Sfrp4-polyhedra) to both acute and subacute ischemic models. The histology of the dissected heart showed that the cardiac wall was thicker and the area of acellular scarring was smaller in Sfrp4-treated hearts than in controls. Increased amounts of both the inactive serine 9-phosphorylated form of glycogen synthase kinase (GSK)-3ß and the active form of ß-catenin were observed by immunohistology 3 days after lateral anterior descendant ligation in control, but not in Sfrp4-treated hearts. All together, we show that administration of Sfrp4 interferes with canonical Wnt signaling that could mediate the formation of acellular scar and consequently contributes to the prevention of aggravation of cardiac function.
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Pruebas de Función Cardíaca , Isquemia Miocárdica/tratamiento farmacológico , Isquemia Miocárdica/fisiopatología , Proteínas Proto-Oncogénicas/uso terapéutico , Animales , Materiales Biocompatibles/farmacología , Cardiotónicos/farmacología , Cardiotónicos/uso terapéutico , Modelos Animales de Enfermedad , Sistemas de Liberación de Medicamentos , Fibrosis , Humanos , Inyecciones Intramusculares , Masculino , Isquemia Miocárdica/genética , Isquemia Miocárdica/patología , Proteínas Proto-Oncogénicas/administración & dosificación , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/farmacología , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéutico , Transducción de Señal/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Regulación hacia Arriba/genética , Proteínas Wnt/metabolismo , beta Catenina/metabolismoRESUMEN
Primary fractions from the extract of a tropical red alga mixed with filamentous cyanobacteria, collected from Papua New Guinea, were active in a neurotoxicity assay. Bioassay-guided isolation led to two natural products (1,2) with relatively potent calcium ion influx properties. The more prevalent of the neurotoxic compounds (1) was characterized by extensive NMR, mass spectrometry, and X-ray crystallography, and shown to be identical to a polybrominated diphenyl ether metabolite present in the literature, but reported with different NMR properties. To clarify this anomalous result, we synthesized a candidate isomeric polybrominated diphenyl ether (3), but this clearly had different NMR shifts than the reported compound. We conclude that the original isolate of 3,4,5-tribromo-2-(2,4-dibromophenoxy)phenol was contaminated with a minor compound, giving rise to the observed anomalous NMR shifts. The second and less abundant natural product (2) isolated in this study was a more highly brominated species. All three compounds showed a low micromolar ability to increase intracellular calcium ion concentrations in mouse neocortical neurons as well as toxicity to zebrafish. Because polybrominated diphenyl ethers have both natural as well as anthropomorphic origins, and accumulate in marine organisms at higher trophic level (mammals, fish, birds), these neurotoxic properties are of environmental significance and concern.
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Cianobacterias/química , Toxinas Marinas/toxicidad , Bifenilos Polibrominados/toxicidad , Rhodophyta/química , Animales , Bioensayo , Calcio/metabolismo , Células Cultivadas , Cristalografía por Rayos X , Citoplasma/química , Citoplasma/metabolismo , Citometría de Flujo , Indicadores y Reactivos , Espectroscopía de Resonancia Magnética , Toxinas Marinas/química , Ratones , Modelos Moleculares , Neocórtex/citología , Neocórtex/efectos de los fármacos , Neocórtex/metabolismo , Neuronas/metabolismo , Neurotoxinas/toxicidad , Papúa Nueva Guinea , Bifenilos Polibrominados/química , Espectrometría de Masa por Ionización de Electrospray , Pez CebraRESUMEN
The first total synthesis of somocystinamide A, a disulfide dimer with extremely labile enamide functional groups, was accomplished in a concise and stereospecific manner. Somocystinamide A is reported to possess exceptionally potent antiangiogenic and tumoricidal activities. The current work should enable further pharmacological investigation of this important natural product.
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Disulfuros/síntesis química , Amidas/química , Productos Biológicos/química , Disulfuros/química , Estructura Molecular , EstereoisomerismoRESUMEN
Cancer cell toxicity-guided fractionation of extracts of the Papua New Guinea marine cyanobacteria Lyngbya majuscula and Lyngbya sordida led to the isolation of apratoxin D (1). Compound 1 contains the same macrocycle as apratoxins A and C but possesses the novel 3,7-dihydroxy-2,5,8,10,10-pentamethylundecanoic acid as the polyketide moiety. The planar structures and stereostructures of compound 1 were determined by extensive 1D and 2D NMR and MS data analyses and by comparison with the spectroscopic data of apratoxins A and C. Apratoxin D (1) showed potent in vitro cytotoxicity against H-460 human lung cancer cells with an IC 50 value of 2.6 nM.
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Antineoplásicos/aislamiento & purificación , Antineoplásicos/farmacología , Cianobacterias/química , Depsipéptidos/aislamiento & purificación , Depsipéptidos/farmacología , Toxinas de Lyngbya/aislamiento & purificación , Toxinas de Lyngbya/farmacología , Toxinas Marinas/aislamiento & purificación , Toxinas Marinas/farmacología , Antineoplásicos/química , Depsipéptidos/química , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Concentración 50 Inhibidora , Toxinas de Lyngbya/química , Toxinas Marinas/química , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Papúa Nueva GuineaRESUMEN
Screening for novel anticancer drugs in chemical libraries isolated from marine organisms, we identified the lipopeptide somocystinamide A (ScA) as a pluripotent inhibitor of angiogenesis and tumor cell proliferation. The antiproliferative activity was largely attributable to induction of programmed cell death. Sensitivity to ScA was significantly increased among cells expressing caspase 8, whereas siRNA knockdown of caspase 8 increased survival after exposure to ScA. ScA rapidly and efficiently partitioned into liposomes while retaining full antiproliferative activity. Consistent with the induction of apoptosis via the lipid compartment, we noted accumulation and aggregation of ceramide in treated cells and subsequent colocalization with caspase 8. Angiogenic endothelial cells were extremely sensitive to ScA. Picomolar concentrations of ScA disrupted proliferation and endothelial tubule formation in vitro. Systemic treatment of zebrafish or local treatment of the chick chorioallantoic membrane with ScA resulted in dose-dependent inhibition of angiogenesis, whereas topical treatment blocked tumor growth among caspase-8-expressing tumors. Together, the results reveal an unexpected mechanism of action for this unique lipopeptide and suggest future development of this and similar agents as antiangiogenesis and anticancer drugs.
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Apoptosis/efectos de los fármacos , Caspasa 8/metabolismo , Disulfuros/farmacología , Lipoproteínas/farmacología , Inhibidores de la Angiogénesis/farmacología , Animales , Animales Modificados Genéticamente , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Pollos , Disulfuros/química , Embrión no Mamífero/irrigación sanguínea , Embrión no Mamífero/efectos de los fármacos , Embrión no Mamífero/embriología , Humanos , Estructura Molecular , Océanos y Mares , Fosfolípidos/metabolismo , Sensibilidad y EspecificidadRESUMEN
Therapeutic angiogenesis can be induced by the implantation of bone marrow cells (BMCs). However, the mechanism of BMC-mediated neovascularization remains to be clarified. We investigated the differential activities of bone marrow subpopulations in angiogenesis and cytokine production. BMCs were separated into positive and negative fractions by surface expression of Mac-1, Gr-1, CD19, and c-kit, respectively. After 7 days of culture in the presence of vascular endothelial growth factor (VEGF), the cells produced adherent cells which incorporate acetylated low-density lipoprotein (acLDL). Mac-1(+) and Mac-1(-) cells produced almost equal numbers of acLDL(+) cells, but only Mac-1(-) cells expressed endothelial markers, including Flk-1, vWF, and CD31. Similarly, the expression of endothelial markers was detected in Gr-1(-), CD19(-), and c-kit(+) BMC fractions at 7-day cultures, but not in Gr-1(+), CD19(+), or c-kit(-) cells. In contrast, freshly isolated Mac-1(+) and Gr-1(+) BMCs expressed higher levels of mRNAs for angiogenic cytokines (including VEGF-A, FGF-2, and HGF) than Mac-1(-) and Gr-1(-) cells, respectively. Moreover, Mac-1(+)/c-kit(+) BMC subpopulation expressed higher levels of VEGF-A and SDF-1 mRNAs than other subpopulations. These data demonstrate that a relatively small proportion of VEGF-cultured adherent cells are true endothelial cells with a Flk-1(+)/vWF(+)/CD31(+) phenotype. Moreover, endothelial stem/progenitor cells (EPCs) are limited primarily to Mac-1(-), Gr-1(-), and c-kit(+) BMC populations. In contrast, angiogenic cytokine mRNAs were also produced by Mac-1(+), Gr-1(+), and c-kit(-) BMCs, suggesting the heterogeneity of effector cell types for neovasculatization therapy.
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Células de la Médula Ósea/citología , Citocinas/genética , Endotelio Vascular/citología , Células Madre/citología , Animales , Células de la Médula Ósea/fisiología , Técnicas de Cultivo de Célula , Separación Celular , Endotelio Vascular/fisiología , Fémur , Citometría de Flujo , Ratones , Ratones Endogámicos BALB C , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Short and practical syntheses of epiquinamide and its enantiomer were accomplished with high overall yields and high stereoselectivity from readily available starting materials.