RESUMEN
Inflammatory processes are activated following ischemic stroke that lead to increased tissue damage for weeks following the ischemic insult, but there are no approved therapies that target this inflammation-induced secondary injury. Here, we report that SynB1-ELP-p50i, a novel protein inhibitor of the nuclear factor kappa B (NF-κB) inflammatory cascade bound to the drug carrier elastin-like polypeptide (ELP), decreases NF-κB induced inflammatory cytokine production in cultured macrophages, crosses the plasma membrane and accumulates in the cytoplasm of both neurons and microglia in vitro, and accumulates at the infarct site where the blood-brain barrier (BBB) is compromised following middle cerebral artery occlusion (MCAO) in rats. Additionally, SynB1-ELP-p50i treatment reduces infarct volume by 11.86% compared to saline-treated controls 24 h following MCAO. Longitudinally, SynB1-ELP-p50i treatment improves survival for 14 days following stroke with no effects of toxicity or peripheral organ dysfunction. These results show high potential for ELP-delivered biologics for therapy of ischemic stroke and other central nervous system disorders and further support targeting inflammation in ischemic stroke.
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Isquemia Encefálica , Accidente Cerebrovascular Isquémico , Accidente Cerebrovascular , Ratas , Animales , FN-kappa B/metabolismo , Accidente Cerebrovascular Isquémico/metabolismo , Elastina/metabolismo , Encéfalo/metabolismo , Péptidos/farmacología , Péptidos/metabolismo , Accidente Cerebrovascular/metabolismo , Isquemia Encefálica/tratamiento farmacológico , Isquemia Encefálica/metabolismo , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Antiinflamatorios/metabolismo , Infarto de la Arteria Cerebral Media/metabolismo , Inflamación/metabolismo , Microglía/metabolismoRESUMEN
Inflammatory processes are activated following ischemic strokes and lead to increased tissue damage for weeks following the ischemic insult, but there are no approved therapies that target this inflammation-induced secondary injury. Here, we report that SynB1-ELP-p50i, a novel protein inhibitor of the nuclear factor kappa B (NF-κB) inflammatory cascade bound to drug carrier elastin-like polypeptide (ELP), is able to enter both neurons and microglia, cross the blood-brain barrier, localize exclusively in the ischemic core and penumbra in Wistar-Kyoto and spontaneously hypertensive rats (SHRs), and reduce infarct volume in male SHRs. Additionally, in male SHRs, SynB1-ELP-p50i treatment improves survival for 14 days following stroke with no effects of toxicity or peripheral organ dysfunction. These results show high potential for ELP-delivered biologics for therapy of ischemic stroke and other central nervous system disorders and further support targeting inflammation in ischemic stroke.
RESUMEN
OBJECTIVE: The aim of this paper was to identify and characterize all the segmental radiculomedullary arteries (RMAs) that supply the thoracic and lumbar spinal cord. METHODS: All RMAs from T4 to L5 were studied systematically in 25 cadaveric specimens. The RMA with the greatest diameter in each specimen was termed the artery of Adamkiewicz (AKA). Other supporting RMAs were also identified and characterized. RESULTS: A total of 27 AKAs were found in 25 specimens. Twenty-two AKAs (81%) originated from a left thoracic or a left lumbar radicular branch, and 5 (19%) arose from the right. Two specimens (8%) had two AKAs each: one specimen with two AKAs on the left side and the other specimen with one AKA on each side. Eight cadaveric specimens (32%) had 10 additional RMAs; among those, a single additional RMA was found in 6 specimens (75%), and 2 additional RMAs were found in each of the remaining 2 specimens (25%). Of those specimens with a single additional RMA, the supporting RMA was ipsilateral to the AKA in 5 specimens (83%) and contralateral in only 1 specimen (17%). The specimens containing 2 additional RMAs were all (100%) ipsilateral to their respective AKAs. CONCLUSIONS: The segmental RMAs supplying the thoracic and lumbar spinal cord can be unilateral, bilateral, or multiple. Multiple AKAs or additional RMAs supplying a single anterior spinal artery are common and should be considered when dealing with the spinal cord at the thoracolumbar level.
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Médula Espinal , Arteria Vertebral , Humanos , Médula Espinal/cirugía , Médula Espinal/irrigación sanguínea , Región Lumbosacra , CadáverRESUMEN
Cadaveric dissection offers an important opportunity for students to develop their ideas about death and dying. However, it remains largely unknown how this experience impacts medical students' fear of death. The current study aimed to address this gap by describing how fear of death changed during a medical gross anatomy dissection course and how fear of death was associated with examination performance. Fear of death was surveyed at the beginning of the course and at each of the four block examinations using three of the eight subscales from the Multidimensional Fear of Death Scale: Fear of the Dead, Fear of Being Destroyed, and Fear for the Body After Death. One hundred forty-three of 165 medical students (86.7%) completed the initial survey. Repeated measures ANOVA showed no significant changes in Fear of the Dead (F (4, 108) = 1.45, P = 0.222) or Fear for the Body After Death (F (4, 108) = 1.83, P = 0.129). There was a significant increase in students' Fear of Being Destroyed (F (4, 108) = 6.86, P < 0.0005) after beginning dissection. This increase was primarily related to students' decreased willingness to donate their body. Concerning performance, there was one significant correlation between Fear for the Body After Death and the laboratory examination score at examination 1. Students with higher fears may be able to structure their experience in a way that does not negatively impact their performance, but educators should still seek ways to support these students and encourage body donation.
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Anatomía , Educación de Pregrado en Medicina , Estudiantes de Medicina , Anatomía/educación , Cadáver , Curriculum , Disección , Miedo , Humanos , Trastornos Fóbicos , Encuestas y CuestionariosRESUMEN
A projection by the superior colliculus to the supraoculomotor area (SOA) located dorsal to the oculomotor complex was first described in 1978. This projection's targets have yet to be identified, although the initial study suggested that vertical gaze motoneuron dendrites might receive this input. Defining the tectal targets is complicated by the fact the SOA contains a number of different cell populations. In the present study, we used anterograde tracers to characterize collicular axonal arbors and retrograde tracers to label prospective SOA target populations in macaque monkeys. Close associations were not found with either superior or medial rectus motoneurons whose axons supply singly innervated muscle fibers. S-group motoneurons, which supply superior rectus multiply innervated muscle fibers, appeared to receive a very minor input, but C-group motoneurons, which supply medial rectus multiply innervated muscle fibers, received no input. A number of labeled boutons were observed in close association with SOA neurons projecting to the spinal cord, or the reticular formation in the pons and medulla. These descending output neurons are presumed to be peptidergic cells within the centrally projecting Edinger-Westphal population. It is possible the collicular input provides a signaling function for neurons in this population that serve roles in either stress responses, or in eating and drinking behavior. Finally, a number of close associations were observed between tectal terminals and levator palpebrae superioris motoneurons, suggesting the possibility that the superior colliculus provides a modest direct input for raising the eyelids during upward saccades.
RESUMEN
The central mesencephalic reticular formation (cMRF) occupies much of the core of the midbrain tegmentum. Physiological studies indicate that it is involved in controlling gaze changes, particularly horizontal saccades. Anatomically, it receives input from the ipsilateral superior colliculus (SC) and it has downstream projections to the brainstem, including the horizontal gaze center located in the paramedian pontine reticular formation (PPRF). Consequently, it has been hypothesized that the cMRF plays a role in the spatiotemporal transformation needed to convert spatially coded collicular saccade signals into the temporally coded signals utilized by the premotor neurons of the horizontal gaze center. In this study, we used neuroanatomical tracers to examine the patterns of connectivity of the cMRF in macaque monkeys in order to determine whether the circuit organization supports this hypothesis. Since stimulation of the cMRF produces contraversive horizontal saccades and stimulation of the horizontal gaze center produces ipsiversive saccades, this would require an excitatory cMRF projection to the contralateral PPRF. Injections of anterograde tracers into the cMRF did produce labeled terminals within the PPRF. However, the terminations were denser ipsilaterally. Since the PPRF located contralateral to the movement direction is generally considered to be silent during a horizontal saccade, we then tested the hypothesis that this ipsilateral reticuloreticular pathway might be inhibitory. The ultrastructure of ipsilateral terminals was heterogeneous, with some displaying more extensive postsynaptic densities than others. Postembedding immunohistochemistry for gamma-aminobutyric acid (GABA) indicated that only a portion (35%) of these cMRF terminals are GABAergic. Dual tracer experiments were undertaken to determine whether the SC provides input to cMRF reticuloreticular neurons projecting to the ipsilateral pons. Retrogradely labeled reticuloreticular neurons were predominantly distributed in the ipsilateral cMRF. Anterogradely labeled tectal terminals were observed in close association with a portion of these retrogradely labeled reticuloreticular neurons. Taken together, these results suggest that the SC does have connections with reticuloreticular neurons in the cMRF. However, the predominantly excitatory nature of the ipsilateral reticuloreticular projection argues against the hypothesis that this cMRF pathway is solely responsible for producing a spatiotemporal transformation of the collicular saccade signal.
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BACKGROUND: The maxillary artery (MA) has gained attention in neurosurgery particularly in cerebral revascularization techniques, intracranial endonasal approaches and endovascular procedures. OBJECTIVES: To describe and illustrate the anatomy of the MA and its neurosurgical importance in a detailed manner. METHODS: Six cadaveric heads (12 MAs) were injected with latex. The arteries and surrounding structures were dissected and studied using microsurgical techniques. The dimensions, course and branching patterns of the MA were recollected. In addition, 20 three-dimensional reconstruction CT head and neck angiograms (3D CTAs) of actual patients were correlated with the cadaveric findings. RESULTS: The MA can be divided in three segments: mandibular, pterygoid and pterygopalatine. Medial and lateral trunk variants regarding its course around the lateral pterygoid muscle can be found. The different branching patterns of the MA have a direct correlation with the course of its main trunk at the base of the skull. Branching and trunk variants on one side do not predict the findings on the contralateral side. CONCLUSION: In this study the highly variable course, branching patterns and relations of the MA are illustrated and described in human cadaveric heads and 3D CTAs. MA 3D CTA with bone reconstruction can be useful preoperatively for the identification of the medial or lateral course variants of this artery, particularly its pterygoid segment, which should be taken into account when considering the MA as a donor vessel for an EC-IC bypass.
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Arteria Maxilar/cirugía , Cirugía Endoscópica por Orificios Naturales/métodos , Procedimientos Neuroquirúrgicos/métodos , Adulto , Angiografía , Femenino , Humanos , Masculino , Arteria Maxilar/anatomía & histología , Arteria Maxilar/diagnóstico por imagen , Nariz/anatomía & histología , Nariz/cirugía , Músculos Pterigoideos/anatomía & histología , Músculos Pterigoideos/diagnóstico por imagen , Músculos Pterigoideos/cirugía , Cráneo/anatomía & histología , Cráneo/diagnóstico por imagen , Cráneo/cirugía , Tomografía Computarizada por Rayos XRESUMEN
BACKGROUND: Therapeutic angiogenesis with vascular endothelial growth factor (VEGF), delivered either via recombinant protein infusion or via gene therapy, has shown promise in preclinical models of various diseases including myocardial infarction, renovascular disease, preeclampsia, and neurodegenerative disorders. However, dosing, duration of expression, and tissue specificity are challenges to VEGF gene therapy, and recombinant VEGF delivery suffers from extremely rapid plasma clearance, necessitating continuous infusion and/or direct injection at the site of interest. METHODS: Here we describe a novel growth factor purification and delivery system (PADS) generated by fusion of VEGF121 to a protein polymer based on Elastin-like Polypeptide (ELP). ELP is a thermally responsive biopolymer derived from a five amino acid repeat sequence found in human tropoelastin. VEGFPADS were constructed by fusion of the ELP coding sequence in-frame with the VEGF121 coding sequence connected by a flexible di-glycine linker. In vitro activity of VEGFPADS was determined using cell proliferation, tube formation, and migration assays with vascular endothelial cells. Pharmacokinetics and biodistribution of VEGFPADS in vivo were compared to free VEGF in mice using quantitative fluorescence techniques. RESULTS: ELP fusion allowed for recombinant expression and simple, non-chromatographic purification of the ELP-VEGF121 chimera in yields as high as 90 mg/L of culture and at very high purity. ELP fusion had no effect on the VEGF activity, as the VEGFPADS were equally potent as free VEGF121 in stimulating HUVEC proliferation, tube formation, and migration. Additionally, the VEGFPADS had a molecular weight five-fold larger than free VEGF121, which lead to slower plasma clearance and an altered biodistribution after systemic delivery in vivo. CONCLUSION: PADS represent a new method of both purification and in vivo stabilization of recombinant growth factors. The use of this system could permit recombinant growth factors to become viable options for therapeutic angiogenesis in a number of disease models.
RESUMEN
Current therapies for the treatment of pancreatic cancer are limited. The limitations of this type of treatment are abundant. The majority of chemotherapeutic agents used in clinics are highly toxic to both tumor cells and normal tissues due to the lack of specificity. Resistance can develop due to overexposure of these agents. To address these issues, these agents must be made more exclusive toward the tumor site. We have developed a macromolecular carrier based on the sequence of the biopolymer elastin-like polypeptide (ELP) that is able to aggregate upon reaching the externally heated tumor environment. This carrier is specific to the tumor as it only aggregates at the heated tumor site. ELP is soluble below its transition temperature but will aggregate when the temperature is raised above its transition temperature. ELP was modified by p21, a cell cycle inhibitory peptide, and the addition of Bac, a cell-penetrating peptide with nuclear localization capabilities. In this study, p21-ELP-Bac and its control, ELP-p21, were used in cell proliferation studies using the pancreatic cancer cell lines Panc-1, MiaPaca-2, and S2013. ELP-p21 had little effect on proliferation, while the half maximal inhibitory concentration of p21-ELP-Bac was â¼30 µM. As translocation across the plasma membrane is a limiting step for delivery of macromolecules, these polypeptides were utilized in a pancreatic xenograft model to study the plasma clearance, biodistribution, tumor accumulation, and tumor reduction capabilities of the polypeptide with and without a cell-penetrating peptide.
Asunto(s)
Biopolímeros/química , Péptidos de Penetración Celular/química , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Modelos Animales de Enfermedad , Neoplasias Experimentales/metabolismo , Neoplasias Pancreáticas/metabolismo , Temperatura , Animales , Biopolímeros/administración & dosificación , Proliferación Celular/efectos de los fármacos , Péptidos de Penetración Celular/administración & dosificación , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/química , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Ratones , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/patología , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/patología , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
Gaze changes involving the eyes and head are orchestrated by brainstem gaze centers found within the superior colliculus (SC), paramedian pontine reticular formation (PPRF), and medullary reticular formation (MdRF). The mesencephalic reticular formation (MRF) also plays a role in gaze. It receives a major input from the ipsilateral SC and contains cells that fire in relation to gaze changes. Moreover, it provides a feedback projection to the SC and feed-forward projections to the PPRF and MdRF. We sought to determine whether these MRF feedback and feed-forward projections originate from the same or different neuronal populations by utilizing paired fluorescent retrograde tracers in cats. Specifically, we tested: 1. whether MRF neurons that control eye movements form a single population by injecting the SC and PPRF with different tracers, and 2. whether MRF neurons that control head movements form a single population by injecting the SC and MdRF with different tracers. In neither case were double labeled neurons observed, indicating that feedback and feed-forward projections originate from separate MRF populations. In both cases, the labeled reticulotectal and reticuloreticular neurons were distributed bilaterally in the MRF. However, neurons projecting to the MdRF were generally constrained to the medial half of the MRF, while those projecting to the PPRF, like MRF reticulotectal neurons, were spread throughout the mediolateral axis. Thus, the medial MRF may be specialized for control of head movements, with control of eye movements being more widespread in this structure.
RESUMEN
Omnipause neurons (OPNs) within the nucleus raphe interpositus (RIP) help gate the transition between fixation and saccadic eye movements by monosynaptically suppressing activity in premotor burst neurons during fixation, and releasing them during saccades. Premotor neuron activity is initiated by excitatory input from the superior colliculus (SC), but how the tectum's saccade-related activity turns off OPNs is not known. Since the central mesencephalic reticular formation (cMRF) is a major SC target, we explored whether this nucleus has the appropriate connections to support tectal gating of OPN activity. In dual-tracer experiments undertaken in macaque monkeys (Macaca fascicularis), cMRF neurons labeled retrogradely from injections into RIP had numerous anterogradely labeled terminals closely associated with them following SC injections. This suggested the presence of an SC-cMRF-RIP pathway. Furthermore, anterograde tracers injected into the cMRF of other macaques labeled axonal terminals in RIP, confirming this cMRF projection. To determine whether the cMRF projections gate OPN activity, postembedding electron microscopic immunochemistry was performed on anterogradely labeled cMRF terminals with antibody to GABA or glycine. Of the terminals analyzed, 51.4% were GABA positive, 35.5% were GABA negative, and most contacted glycinergic cells. In summary, a trans-cMRF pathway connecting the SC to the RIP is present. This pathway contains inhibitory elements that could help gate omnipause activity and allow other tectal drives to induce the bursts of firing in premotor neurons that are necessary for saccades. The non-GABAergic cMRF terminals may derive from fixation units in the cMRF.
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Neuronas/fisiología , Formación Reticular/fisiología , Movimientos Sacádicos/fisiología , Colículos Superiores/fisiología , Vías Visuales/fisiología , Animales , Femenino , Inmunohistoquímica , Macaca fascicularis , Masculino , Mesencéfalo/citología , Mesencéfalo/fisiología , Microscopía Electrónica de Transmisión , Neuronas/citología , Formación Reticular/anatomía & histología , Colículos Superiores/anatomía & histología , Vías Visuales/citologíaRESUMEN
20-Hydroxyeicosatetraenoic acid (20-HETE) contributes to the migration and proliferation of vascular smooth muscle cells (VSMC) in vitro, but there are few studies that address its effects on vascular remodeling in vivo. The present study determined whether inhibition of 20-HETE production attenuates intimal hyperplasia (IH) and vascular remodeling after balloon injury (BI). Sprague Dawley rats underwent BI of the common carotid artery and were treated with vehicle, 1-aminobenzotriazole (ABT, 50 mg/kg i.p. once daily), or HET0016 (N-hydroxy-N'-(4-butyl-2-methylphenyl)-formamidine) (2 mg/kg s.c. twice daily) for 14 days. Fourteen days after BI and treatment, the animals underwent carotid angiography, and the arteries were harvested for morphometric, enzymatic and immunohistochemical analysis. There was a 96% reduction of angiographic stenosis in the rats treated with 1-ABT. There was a 61 and 66% reduction of the intima/media area ratios in the 1-ABT and HET0016 treated rats compared with the vehicle-treated group. 20-HETE levels were elevated in BI carotid arteries, and the levels were markedly suppressed in the groups treated with 1-ABT and HET0016 (P < 0.001). Immunostaining revealed that the expression of CYP4A enzyme was markedly increased in the neointima of BI arteries, and it colocalized with the expression of smooth muscle-specific actin, indicating increased proliferation of VSMC. An increase in the expression of CYP4A and the production of 20-HETE contributes to neointimal growth in BI rat carotid arteries. Systemic administration 1-ABT or HET0016 prevents the increase in 20-HETE levels and attenuates VSMC migration and proliferation, resulting in a marked reduction in IH and vascular remodeling after endothelial injury.
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Estenosis Carotídea/prevención & control , Citocromo P-450 CYP4A/antagonistas & inhibidores , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/uso terapéutico , Ácidos Hidroxieicosatetraenoicos/antagonistas & inhibidores , Neointima/prevención & control , Túnica Íntima/lesiones , Amidinas/uso terapéutico , Angioplastia Coronaria con Balón/efectos adversos , Animales , Traumatismos de las Arterias Carótidas/tratamiento farmacológico , Traumatismos de las Arterias Carótidas/etiología , Traumatismos de las Arterias Carótidas/patología , Traumatismos de las Arterias Carótidas/fisiopatología , Arteria Carótida Común/efectos de los fármacos , Arteria Carótida Común/metabolismo , Arteria Carótida Común/patología , Estenosis Carotídea/etiología , Estenosis Carotídea/metabolismo , Estenosis Carotídea/patología , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Citocromo P-450 CYP4A/metabolismo , Inhibidores Enzimáticos del Citocromo P-450 , Sistema Enzimático del Citocromo P-450/metabolismo , Familia 4 del Citocromo P450 , Ácidos Hidroxieicosatetraenoicos/metabolismo , Hiperplasia , Masculino , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Neointima/etiología , Ratas , Ratas Sprague-Dawley , Triazoles/uso terapéutico , Túnica Íntima/efectos de los fármacos , Túnica Íntima/metabolismo , Túnica Íntima/patologíaRESUMEN
Treatment of glioblastoma is complicated by the tumors' high resistance to chemotherapy, poor penetration of drugs across the blood brain barrier, and damaging effects of chemotherapy and radiation to normal neural tissue. To overcome these limitations, a thermally responsive polypeptide was developed for targeted delivery of therapeutic peptides to brain tumors using focused hyperthermia. The peptide carrier is based on elastin-like polypeptide (ELP), which is a thermally responsive biopolymer that forms aggregates above a characteristic transition temperature. ELP was modified with cell penetrating peptides (CPPs) to enhance delivery to brain tumors and mediate uptake across the tumor cells' plasma membranes and with a peptide inhibitor of c-Myc (H1). In rats with intracerebral gliomas, brain tumor targeting of ELP following systemic administration was enhanced up to 5-fold by the use of CPPs. When the lead CPP-ELP-fused c-Myc inhibitor was combined with focused hyperthermia of the tumors, an additional 3 fold increase in tumor polypeptide levels was observed, and 80% reduction in tumor volume, delayed onset of tumor-associated neurological deficits, and at least doubled median survival time including complete regression in 80% of animals was achieved. This work demonstrates that a c-Myc inhibitory peptide can be effectively delivered to brain tumors.
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Antineoplásicos/farmacología , Péptidos de Penetración Celular/farmacología , Glioma/terapia , Hipertermia Inducida , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Péptidos de Penetración Celular/administración & dosificación , Péptidos de Penetración Celular/química , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Portadores de Fármacos , Glioma/mortalidad , Glioma/patología , Calor , Humanos , Rayos Infrarrojos/uso terapéutico , Masculino , Trasplante de Neoplasias , Ratas , Distribución TisularRESUMEN
The small diameter of the carotid artery is not compatible with the evaluation of clinically available endovascular devices in the carotid balloon-injury (BI) model. We developed an endovascular BI model in the rat descending aorta, whose size is compatible with available endovascular instruments. We also tested the hypothesis that neointima formation is enhanced in the aorta of obese Zucker rats (OZR) compared with lean Zucker rats (LZR). Left external carotid arteriotomies and BI of the thoracic and abdominal aorta were performed by using a balloon catheter. Aortograms and aortic pathology were examined at 2, 4, and 10 wk after BI. At 10 wk after BI, the abdominal aorta in OZR had narrowed 8.3% ± 1.1% relative to baseline compared with an expansion of 2.4% ± 2.2% in LZR. Simultaneously, the thoracic aorta had expanded 9.5% ± 4.3% in LZR compared with stenosis of 2.8% ± 1.6% in OZR. Calculation of the intimal:medial thickness ratio revealed significantly increased neointimal formation in the OZR descending aorta compared with that in LNR. In conclusion, this minimally invasive BI model involving the rat descending aorta is compatible with available endovascular instruments. The descending aorta of OZR demonstrates enhanced neointimal formation and constrictive vascular remodeling after BI.
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Angioplastia de Balón/efectos adversos , Aorta/lesiones , Aorta/fisiopatología , Procedimientos Endovasculares/instrumentación , Modelos Animales , Neointima/patología , Obesidad/fisiopatología , Análisis de Varianza , Angiografía , Animales , Masculino , Ratas , Ratas Zucker , Factores de TiempoRESUMEN
Elastin-like polypeptide (ELP) is a macromolecular carrier with thermally responsive properties that can passively accumulate in solid tumors and additionally aggregate in tumor tissue when exposed to hyperthermia. In this study, ELP was conjugated to the anticancer drug doxorubicin (DOXO) and three different cell penetrating peptides (CPP) in order to inhibit tumor growth in mice compared to free doxorubicin. Fluorescence microscopy studies in MCF-7 breast carcinoma cells demonstrated that the three different CPP-ELP-DOXO conjugates delivered doxorubicin to the cell nucleus. All CPP-ELP-DOXO conjugates showed cytotoxicity with IC(50) values in the range of 12-30 µM at 42 °C, but the ELP carrier with SynB1 as the cell penetrating peptide had the lowest intrinsic cytotoxicity. Therefore, the antitumor efficacy of SynB1-ELP-DOXO was compared to doxorubicin under hyperthermic conditions. C57BL/6 female mice bearing syngeneic E0771 murine breast tumors were treated with either free doxorubicin or the SynB1-ELP-DOXO conjugate with or without focused hyperthermia on the tumor. Under hyperthermic conditions, tumor inhibition with SynB1-ELP-DOXO was 2-fold higher than under therapy with free doxorubicin at the equivalent dose, and is thus a promising lead candidate for optimizing thermally responsive drug polymer conjugates.
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Antibióticos Antineoplásicos/administración & dosificación , Péptidos de Penetración Celular/administración & dosificación , Doxorrubicina/análogos & derivados , Portadores de Fármacos/administración & dosificación , Elastina/administración & dosificación , Hidrazonas/administración & dosificación , Péptidos/administración & dosificación , Animales , Antibióticos Antineoplásicos/química , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Péptidos de Penetración Celular/química , Doxorrubicina/administración & dosificación , Doxorrubicina/química , Portadores de Fármacos/química , Elastina/química , Femenino , Humanos , Hidrazonas/química , Concentración de Iones de Hidrógeno , Células MCF-7 , Ratones , Ratones Endogámicos C57BL , Péptidos/química , Carga Tumoral/efectos de los fármacosRESUMEN
Elastin-like polypeptides (ELP) aggregate in response to mild hyperthermia, but remain soluble under normal physiologic conditions. ELP macromolecules can accumulate in solid tumors because of the enhanced permeability and retention effect. Tumor retention of ELPs can be further enhanced through hyperthermia-induced aggregation of ELPs by local heating of the tumor. We evaluated the therapeutic potential of ELPs in delivering doxorubicin in the E0771 syngeneic mouse breast cancer model. The ELP-Dox conjugate consisted of a cell-penetrating peptide at the N-terminus and the 6-maleimidocaproyl hydrazone derivative of doxorubicin at the C-terminus of ELP. The acid-sensitive hydrazone linker ensured release of doxorubicin in the lysosomes/endosomes after cellular uptake of the drug conjugate. ELP-Dox dosed at 5 mg doxorubicin equivalent/kg, extended the plasma half-life of doxorubicin to 5.5 hours. In addition, tumor uptake of ELP-Dox increased 2-fold when hyperthermia was applied, and was also enhanced compared to free doxorubicin. Although high levels of doxorubicin were found in the heart of animals treated with free doxorubicin, no detectable levels of doxorubicin were found in ELP-Dox-treated animals, indicating a correlation between tumor targeting and reduction of potential cardiac toxicity by ELP-Dox. At an optimal dose of 12 mg doxorubicin equivalent/kg, ELP-Dox in combination with hyperthermia induced a complete tumor growth inhibition, which was distinctly superior to free drug that only moderately inhibited tumor growth. In summary, our findings show that thermal targeting of ELP increases the potency of doxorubicin underlying the potential of exploiting ELPs to enhance the therapeutic efficacy of conventional anticancer drugs.
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Péptidos de Penetración Celular/farmacología , Doxorrubicina/química , Doxorrubicina/farmacología , Elastina/química , Hipertermia Inducida , Animales , Transporte Biológico , Línea Celular Tumoral , Péptidos de Penetración Celular/administración & dosificación , Péptidos de Penetración Celular/química , Terapia Combinada , Modelos Animales de Enfermedad , Doxorrubicina/administración & dosificación , Femenino , Humanos , Dosis Máxima Tolerada , Ratones , Ratones Endogámicos C57BL , Neoplasias/terapiaRESUMEN
Although surgical resection with adjuvant chemotherapy and/or radiotherapy are used to treat breast tumors, normal tissue tolerance, development of metastases, and inherent tumor resistance to radiation or chemotherapy can hinder a successful outcome. We have developed a thermally responsive polypeptide, based on the sequence of Elastin-like polypeptide (ELP), that inhibits breast cancer cell proliferation by blocking the activity of the oncogenic protein c-Myc. Following systemic administration, the ELP - delivered c-Myc inhibitory peptide was targeted to tumors using focused hyperthermia, and significantly reduced tumor growth in an orthotopic mouse model of breast cancer. This work provides a new modality for targeted delivery of a specific oncogene inhibitory peptide, and this strategy may be expanded for delivery of other therapeutic peptides or small molecule drugs.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Hipertermia Inducida , Péptidos/administración & dosificación , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Sistemas de Liberación de Medicamentos , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Trasplante de Neoplasias , Péptidos/farmacocinéticaRESUMEN
INTRODUCTION: Calcium entry plays a critical role in the proliferation and survival of certain tumors. Ca(2+) release activated Ca(2+) (CRAC) channels constitute one of the most important pathways for calcium entry especially that of store-operated calcium entry (SOCE). ORAI1 and stromal interaction molecule1 (STIM1) are essential protein components of CRAC channels. In this study we tested the effect of inhibiting CRAC through ORAI1 and STIM1 on glioblastoma multiforme (GBM) tumor cell proliferation and survival. METHODS: Two glioblastoma cell lines, C6 (rat) and U251 (human), were used in the study. ORAI1 and STIM1 expressions were examined using Western blot and immunohistochemistry. CRAC channel activity and its components were inhibited with ion channel blockers and using siRNA knockdown. Changes in intracellular calcium concentration were recorded using Fura-2 fluorescent calcium imaging. Cell proliferation and apoptosis were examined using MTS and TUNEL assays, respectively. RESULTS: CRAC blockers, such as SKF-96365 (1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl) propoxy]ethyl-1H-imidazole), 2-aminoethoxydiphenyl borate (2-APB) and Diethylstilbestrol (DES), inhibited cell proliferations and SOCE in GBM cells. Knockdown of ORAI1 and STIM1 proteins using siRNA significantly inhibited C6 cell proliferation and SOCE compared with those in control cells, and a more significant effect was observed in cells with ORAI1 siRNA knockdown than that of STIM1-treated cells. Both CRAC blockers and siRNA treatments increased apoptosis in C-6 cells compared with control. CONCLUSION: Calcium entry via ORAI1 and CRAC channels are important for GBM proliferation and survival.
Asunto(s)
Apoptosis/genética , Canales de Calcio/metabolismo , Señalización del Calcio , Proliferación Celular , Glioblastoma , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Animales , Compuestos de Boro/farmacología , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Puntos de Control del Ciclo Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Dietilestilbestrol/farmacología , Retículo Endoplásmico/metabolismo , Fura-2 , Regulación Neoplásica de la Expresión Génica , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Imidazoles/farmacología , Proteína ORAI1 , ARN Interferente Pequeño , Ratas , Molécula de Interacción Estromal 1RESUMEN
RATIONALE: The rat carotid balloon-injury (BI) model is a widely used model of intimal hyperplasia (IH) and vascular remodeling. A variable degree of IH after BI has been previously reported, and we have encountered technical challenges and suboptimal results with the original method. OBJECTIVE: To evaluate the original rat carotid artery BI method with the use of micro-angiography. We tested the hypothesis that in order to obtain an optimal arterial response, BI should be limited to the common carotid artery with preservation of blood flow. METHODS AND RESULTS: The left common carotid artery (CCA) was injured by one of three different methods. Carotid angiograms and pathology were examined 14 days after BI. A 2F Fogarty balloon catheter inflated to 2 atm inside the aortic arch would not slide back into the common carotid artery until deflation to 0.5 to 0.7 atm. Four out of five (80%) vessels injured with this method developed excessive inflammation without discernible IH. Six out of nine (66%) arteries that underwent BI limited to the CCA at 2 atm developed the largest angiographic stenosis (p=0.003) and IH (0.20±0.03 mm(2), p=0.028). Ten out of eleven (91%) arteries injured with a variable pressure of 1.5 to 2.2 atm, based on the operator's feedback, developed considerable IH (0.12±0.02 mm(2)). All injured carotid arteries with preserved blood flow on angiography developed IH with intact histological boundaries. CONCLUSIONS: Optimal IH with preservation of histological boundaries is achieved by graded BI limited to the CCA that preserves carotid blood flow.
Asunto(s)
Angiografía , Arteria Carótida Común/diagnóstico por imagen , Arteria Carótida Común/patología , Cateterismo , Animales , Aorta Torácica/diagnóstico por imagen , Constricción Patológica/complicaciones , Constricción Patológica/diagnóstico por imagen , Constricción Patológica/patología , Modelos Animales de Enfermedad , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
OBJECTIVE: Minimally invasive spine surgery (MISS) is among the fastest growing technologies in general neurosurgical practice. In addition, great demand exists to teach these skills to neurosurgery residents. With newly enforced work hour restrictions, opportunities to acquire these skills are limited, necessitating development of alternative strategies of education. We describe a novel simulation model for MISS supplemented by resident self-assessment analysis and evaluation. METHODS: The simulator was constructed using a nontransparent Plexiglas frame supplemented with a modified halo frame on which to affix spine specimens. Interchangeable copper tubing was affixed to a 360-degree pivot system to replicate a working portal. Deer skulls and spines were then collected and prepared accordingly. Laboratory exercises were based on the resident's level of training with emphasis on proper drilling techniques. Eight neurosurgery residents were asked to complete the exercises and complete a self-assessment survey regarding their competence level on a scale of 0 to 5, both before and after completing the skill sets. Additionally, they were asked to complete an exit survey that was used to assess the simulation exercises. RESULTS: All exercises were completed successfully with the exception of placing 2 separate pedicle screws through the same portal, which posed difficulty on some specimens because of the of lack of lordosis of the specimens, leading to unfavorable trajectories using a free-hand technique. With regard to the resident self-assessment analysis, the mean confidence rating for performing an MISS laminectomy improved by a difference of 1.25 points (n = 8; 95% confidence interval, 0.66-1.84; P = 0.0015), from 2.50 to 3.75 before and after simulation exercises, respectively, and reached statistical significance. For the senior-level residents, the mean confidence rating for performing MISS placement of pedicle screws using a free-hand technique improved by a difference of 1.00 (n = 3; 95% confidence interval, -1.48-3.48; P = 0.225), from 3.33 to 4.33 before and after simulation exercises, respectively. Results of the exit survey were encouraging. CONCLUSION: The MISS simulator is a feasible, inexpensive, and reproducible adjunct to neurosurgery resident training and provides a new teaching method for spine surgery. Further investigation of this technology is warranted, although multicenter, randomized, controlled trials assessing its validity may not be practical because of ethical constraints with regard to patient safety.