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OBJECTIVE: This study focuses on dose-response investigation using a codon-optimized and de novo -synthesized E-Selectin/AAV2 (E-Sel/AAV2) vector in preparation for Investigational New Drug (IND)-enabling of subsequent clinical studies. BACKGROUND: Gene therapy is a potential solution for patients suffering from chronic limb-threatening ischemia (CLTI). Understanding the dose for effective gene delivery is crucial for future IND-enabling studies. METHODS: Expression of the codon-optimized E-Selectin gene was assessed by flow cytometry following in vitro cell transfection assay and RT-qPCR for murine limbs injected in vivo with AAV-m-E-Selectin (E-Sel/AAV2). Dose-response studies involved three cohorts of FVB/NJ mice (n=6/group) with escalating log doses of E-Selectin/AAV2 injected intramuscularly (IM) in divided aliquots, ranging from 2×10 9 VG to 2×10 11 VG, into ischemic limbs created by left femoral artery/vein ligation/excision and administration of nitric oxide synthase inhibitor, L-NAME. Limb perfusion, extent of gangrene free limb, functional limb recovery and therapeutic angiogenesis were assessed. RESULTS: Codon-optimized E-Sel/AAV2 gene therapy exhibits superior expression level than WT E-Sel/AAV2 gene therapy both in vitro and in vivo . Mice treated with a high dose (2×10 11 VG) of E-Sel/AAV2 showed significantly improved perfusion indices, lower Faber's scores, increased running stamina and neovascularization compared with lower doses tested with control groups, indicating a distinct dose-dependent response. No toxicity was detected in any of the animal groups studied. CONCLUSION: E-Sel/AAV2 Vascular Regeneration Gene Therapy (VRGT) holds promise for enhancing the recovery of ischemic hindlimb perfusion and function, with the effective dose identified in this study as 2×10 11 VG aliquots injected IM.
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A simple and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous determination of isoquercitrin, kaempferol-3-O-rutinoside and tiliroside in rat plasma. Plasma samples were deproteinized with methanol and separated on a Hypersil Gold C18 column (2.1 × 50 mm, i.d., 3.0 µm) using gradient elution with the mobile phase of water and methanol at a flow rate of 0.4 mL/min. Mass spectrometric detection was performed with negative ion electrospray ionization in selected reaction monitoring mode. All analytes showed good linearity over their investigated concentration ranges (r2 > 0.99). The lower limit of quantification was 1.0 ng/mL for isoquercitrin and 2.0 ng/mL for kaempferol-3-O-rutinoside and tiliroside, respectively. Intra- and inter-day precisions were <8.2% and accuracy ranged from -11.5 to 9.7%. The mean extraction recoveries of analytes and IS from rat plasma were >80.4%. The assay was successfully applied to investigate the pharmacokinetic study of the three ingredients after oral administration of Rubus chingii Hu to rats.
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Medicamentos Herbarios Chinos/administración & dosificación , Flavonoles/sangre , Glicósidos/sangre , Rubus , Administración Oral , Animales , Cromatografía Liquida/métodos , Flavonoles/química , Flavonoles/farmacocinética , Glicósidos/química , Glicósidos/farmacocinética , Modelos Lineales , Masculino , Ratas , Ratas Wistar , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Espectrometría de Masas en Tándem/métodosRESUMEN
Aging has been associated with pathological vascular remodeling and increased neointimal hyperplasia. The understanding of how aging exacerbates this process is fundamental to prevent cardiovascular complications in the elderly. This study proposes a mechanism by which aging sustains leukocyte adhesion, vascular inflammation, and increased neointimal thickness after injury. The effect of aging on vascular remodeling was assessed in the rat balloon injury model using microarray analysis, immunohistochemistry, and LINCOplex assays. The injured arteries in aging rats developed thicker neointimas than those in younger animals, and this significantly correlated with a higher number of tissue macrophages and increased vascular IL-18. Indeed, IL-18 was 23-fold more abundant in the injured vasculature of aged animals compared with young rats, while circulating levels were similar in both groups of animals. The depletion of macrophages in aged rats with clodronate liposomes ameliorated vascular accumulation of IL-18 and significantly decreased neointimal formation. IL-18 was found to inhibit apoptosis of vascular smooth muscle cells (VSMC) and macrophages, thus favoring both the formation and inflammation of the neointima. In addition, injured arteries of aged rats accumulated 18-fold more fibrinogen-γ than those of young animals. Incubation of rat peritoneal macrophages with immobilized IL-18 increased leukocyte adhesion to fibrinogen and suggested a proinflammatory positive feedback loop among macrophages, VSMC, and the deposition of fibrinogen during neointimal hyperplasia. In conclusion, our data reveal that concentration changes in vascular cytokine and fibrinogen following injury in aging rats contribute to local inflammation and postinjury neointima formation.
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Envejecimiento/metabolismo , Fibrinógeno/metabolismo , Mediadores de Inflamación/metabolismo , Interleucina-18/metabolismo , Macrófagos/metabolismo , Músculo Liso Vascular/metabolismo , Neointima , Comunicación Paracrina , Lesiones del Sistema Vascular/metabolismo , Factores de Edad , Envejecimiento/inmunología , Envejecimiento/patología , Animales , Apoptosis , Adhesión Celular , Células Cultivadas , Quimiotaxis , Ácido Clodrónico/farmacología , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Hiperplasia , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/patología , Masculino , Monocitos/inmunología , Monocitos/metabolismo , Músculo Liso Vascular/inmunología , Músculo Liso Vascular/patología , Comunicación Paracrina/efectos de los fármacos , Ratas , Ratas Endogámicas F344 , Transducción de Señal , Factores de Tiempo , Lesiones del Sistema Vascular/genética , Lesiones del Sistema Vascular/inmunología , Lesiones del Sistema Vascular/patología , Lesiones del Sistema Vascular/prevención & controlRESUMEN
Endocrine resistance is a crucial challenge in estrogen receptor alpha (ERα)-positive breast cancer (BCa). Aberrant alteration in modulation of E2/ERα signaling pathway has emerged as the putative contributor for endocrine resistance in BCa. Herein, we demonstrate that MYSM1 as a deubiquitinase participates in modulating ERα action via histone and non-histone deubiquitination. MYSM1 is involved in maintenance of ERα stability via ERα deubiquitination. MYSM1 regulates relevant histone modifications on cis regulatory elements of ERα-regulated genes, facilitating chromatin decondensation. MYSM1 is highly expressed in clinical BCa samples. MYSM1 depletion attenuates BCa-derived cell growth in xenograft models and increases the sensitivity of antiestrogen agents in BCa cells. A virtual screen shows that the small molecule Imatinib could potentially interact with catalytic MPN domain of MYSM1 to inhibit BCa cell growth via MYSM1-ERα axis. These findings clarify the molecular mechanism of MYSM1 as an epigenetic modifier in regulation of ERα action and provide a potential therapeutic target for endocrine resistance in BCa.
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Neoplasias de la Mama , Humanos , Femenino , Neoplasias de la Mama/genética , Receptor alfa de Estrógeno/genética , Moduladores de los Receptores de Estrógeno/farmacología , Moduladores de los Receptores de Estrógeno/uso terapéutico , Histonas/metabolismo , Proliferación Celular , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica , Línea Celular Tumoral , Transactivadores/metabolismo , Proteasas Ubiquitina-Específicas/genética , Proteasas Ubiquitina-Específicas/metabolismoRESUMEN
Background: Arteriovenous fistula (AVF) postoperative stenosis is a persistent healthcare problem for hemodialysis patients. We have previously demonstrated that fibrotic remodeling contributes to AVF non-maturation and lysyl oxidase (LOX) is upregulated in failed AVFs compared to matured. Herein, we developed a nanofiber scaffold for the periadventitial delivery of ß-aminopropionitrile (BAPN) to determine whether unidirectional periadventitial LOX inhibition is a suitable strategy to promote adaptive AVF remodeling in a rat model of AVF remodeling. Methods: Bilayer poly (lactic acid) ([PLA)-]- poly (lactic-co-glycolic acid) ([PLGA)] scaffolds were fabricated with using a two-step electrospinning process to confer directionality. BAPN-loaded and vehicle control scaffolds were wrapped around the venous limb of a rat femoral-epigastric AVF during surgery. AVF patency and lumen diameter were followed monitored using Doppler ultrasound surveillance and flow was measured before euthanasia. AVFs were harvested after 21 days for histomorphometry and immunohistochemistry. AVF compliance was measured using pressure myography. RNA from AVF veins was sequenced to analyze changes in gene expression due to LOX inhibition. Results: Bilayer periadventitial nanofiber scaffolds extended BAPN release compared to the monolayer design (p < 0.005) and only released BAPN in one direction. Periadventitial LOX inhibition led to significant increases in AVF dilation and flow after 21 days. Histologically, BAPN trended toward increased lumen and significantly reduced fibrosis compared to control scaffolds (p < 0.01). Periadventitial BAPN reduced downregulated markers associated with myofibroblast differentiation including SMA, FSP-1, LOX, and TGF-ß while increasing the contractile marker MYH11. RNA sequencing revealed differential expression of matrisome genes. Conclusion: Periadventitial BAPN treatment reduces fibrosis and promotes AVF compliance. Interestingly, the inhibition of LOX leads to increased accumulation of contractile VSMC while reducing myofibroblast-like cells. Periadventitial LOX inhibition alters the matrisome to improve AVF vascular remodeling.
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The estrogen receptor alpha (ERα) signaling pathway is a crucial target for ERα-positive breast cancer therapeutic strategies. Co-regulators and other transcription factors cooperate for effective ERα-related enhancer activation. Recent studies demonstrate that the transcription factor CTCF is essential to participate in ERα/E2-induced enhancer transactivation. However, the mechanism of how CTCF is achieved remains unknown. Here, we provided evidence that BAP18 is required for CTCF recruitment on ERα-enriched enhancers, facilitating CTCF-mediated chromatin accessibility to promote enhancer RNAs transcription. Consistently, GRO-seq demonstrates that the enhancer activity is positively correlated with BAP18 enrichment. Furthermore, BAP18 interacts with SMARCA1/BPTF to accelerate the recruitment of CTCF to ERα-related enhancers. Interestingly, BAP18 is involved in chromatin accessibility within enhancer regions, thereby increasing enhancer transactivation and enhancer-promoter looping. BAP18 depletion increases the sensitivity of anti-estrogen and anti-enhancer treatment in MCF7 cells. Collectively, our study indicates that BAP18 coordinates with CTCF to enlarge the transactivation of ERα-related enhancers, providing a better understanding of BAP18/CTCF coupling chromatin remodeling and E-P looping in the regulation of enhancer transcription.
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Neoplasias de la Mama , Cromatina , Femenino , Humanos , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Elementos de Facilitación Genéticos/genética , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , EstrógenosRESUMEN
To address the problem of high failure rate and low accuracy in computed tomography (CT) image edge segmentation, we proposed a CT sequence image edge segmentation optimization algorithm using improved convolution neural network. Firstly, the pattern clustering algorithm is applied to cluster the pixels with relationship in the CT sequence image space to extract the edge information of the real CT image; secondly, Euclidean distance is used to calculate similarity and measure similarity, according to the measurement results, convolution neural network (CNN) hierarchical optimization is carried out to improve the convergence ability of CNN; finally, the pixel classification of CT sequence images is carried out, and the edge segmentation of CT sequence images is optimized according to the classification results. The results show that the overall recognition rate of this method is at a high level. The training time is obviously reduced when the training times exceed 12 times, the recall rate is always about 90%, and the accuracy of image segmentation is high, which solves the problem of large failure rate and low accuracy.
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Procesamiento de Imagen Asistido por Computador , Redes Neurales de la Computación , Algoritmos , Análisis por Conglomerados , Procesamiento de Imagen Asistido por Computador/métodos , Tomografía Computarizada por Rayos X/métodosRESUMEN
Objective: The aim of this study was to measure the expression of PD-L1, CD1a (a marker for immature dendritic cells), and CD83 (a marker for mature dendritic cells) and further examine the associations of PD-L1, CD83, and CD1a with overall survival (OS) in triple-negative breast carcinoma patients. Methods: PD-L1, CD1a, and CD83 expression in breast carcinoma tissues and CD83 expression in lymph node tissues were examined by immunohistochemistry and tissue microarray in 159 patients. Patients were classified into the low, medium, and high PD-L1, CD1a, and CD83 levels. Pearson χ2 test was used to analyze the correlations between PD-L1, CD1a, and CD83. The Kaplan-Meier method was used to calculate the OS. Multivariate analysis was used to identify determinants of 3- and 5-year OS. Results: 25.1, 25.8, and 49.1% of the patients had low, medium, and high PD-L1 levels, respectively. PD-L1 levels significantly correlated with CD1a (r = 0.30409, p < 0.001) and CD83 levels (r = 0.6146, p < 0.001) in breast carcinoma tissue, as well as CD83 levels (r = 0.17508, p = 0.027) in lymph node. The median OS was 83 months (range 12-106), and the 3- and 5-year OS rates were 94.97% (95% CI 91.57-98.37) and 86.79% (95% CI 81.53-92.06), respectively. Moreover, patients with high median CD1a levels had a significantly lower 5-year OS rate (75.6%) than those with low median CD1a levels (93.5%, p = 0.038). Conclusion: PD-L1, CD1a, and CD83 are variably expressed in triple-negative breast carcinoma tissues, and PD-L1 expression correlates with CD1a and CD83. Higher CD1a levels correlate with PD-L1 expression and predict worse OS in triple-negative breast carcinoma.
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To elucidate the source of neointimal cells, experimental fistulas were created in Lewis wild-type (WT) and transgenic rats that constitutively expressed the green fluorescent protein (GFP) in all tissues. Arteriovenous fistula (AVFs) were created by anastomosing the left renal vein to the abdominal aorta. The contribution of bone marrow (BM)-derived cells to the AVF neointima was examined in lethally irradiated WT rats that had been rescued with GFP BM cells. Neointimal cells in these chimeric rats were mostly GFP negative indicating the non-BM origin of those cells. Then, the contribution of arterial cells to the AVF neointima was assessed in a fistula made with a GFP aorta that had been implanted orthotopically into a WT rat. Most of the neointimal cells were also GFP negative demonstrating that AVF neointimal cells are not derived from the feeding artery. Finally to study local resident cells contribution to the formation of neointimal lesions, a composite fistula was created by interposing a GFP vein between the renal vein and the aorta in a WT recipient rat. GFP neointimal cells were only found in the transplanted vein. This study suggests that neointimal cells originate from the local resident cells in the venous limb of the fistula.
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Arterias/citología , Derivación Arteriovenosa Quirúrgica , Células de la Médula Ósea/patología , Neointima/patología , Venas/citología , Animales , Arterias/patología , Derivación Arteriovenosa Quirúrgica/efectos adversos , Proteínas Fluorescentes Verdes/metabolismo , Microscopía Confocal , Ratas , Ratas Endogámicas Lew/genética , Ratas Transgénicas , Células Madre , Venas/patologíaRESUMEN
Aberrant leptin signaling and overexpression of fibroblast growth factor receptor 1 (FGFR1) are both implicated in the pathogenesis of letrozole resistance in breast cancer (BCa), but it remains unknown whether these two pathways are involved in letrozole resistance in a coordinated manner. Here, we demonstrate that expression levels of the pre-B-cell leukemia homeobox transcription factor 3 (PBX3), a pioneer factor that governs divergent biological processes, were significantly upregulated in letrozole-resistant BCa cells and tissues, and this upregulation correlated to a poorer progression-free survival in patients. By leveraging a patient-derived xenograft model with pharmacological approaches, we demonstrated that leptin activated PBX3 expression in a STAT3 (signal transducer and activator of transcription 3)-dependent manner. Our loss- and gain-of-function study further showed that PBX3 attenuated response to letrozole by potentiating BCa cell survival and anchorage-independent growth in BCa cells. By profiling BCa cells with ectopic PBX3 expression, we revealed that PBX3 conferred letrozole resistance via transactivation of the FGFR1 signaling, and this molecular event must coordinate a synergistic transcription activation programs through interacting with MTA1-HDAC2 (metastasis associated 1-histone deacetylase 2) complex. Overall, the available data reveal a novel role of leptin/PBX3 cascade linking energy homeostasis (i.e. hyperleptinemia) and endocrine therapy failure (i.e. letrozole resistance) in BCa.
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BACKGROUND: The arteriovenous fistula (AVF) is the preferred hemodialysis access for end-stage renal disease (ESRD) patients. Yet, establishment of a functional AVF presents a challenge, even for the most experienced surgeons, since postoperative stenosis frequently occludes the AVF. Stenosis results from the loss of compliance in fibrotic areas of the fistula which turns intimal hyperplasia into an occlusive feature. Fibrotic remodeling depends on deposition and crosslinking of collagen by lysyl oxidase (LOX), an enzyme that catalyzes the deamination of lysine and hydroxylysine residues, facilitating intra/intermolecular covalent bonds. We postulate that pharmacological inhibition of lysyl oxidase (LOX) increases postoperative venous compliance and prevents stenosis in a rat AVF model. METHODS: LOX gene expression and vascular localization were assayed in rat AVFs and human pre-access veins, respectively. Collagen crosslinking was measured in humans AVFs that matured or failed, and in rat AVFs treated with ß-aminopropionitrile (BAPN), an irreversible LOX inhibitor. BAPN was either injected systemically or delivered locally around rat AVFs using nanofiber scaffolds. The major endpoints were AVF blood flow, wall fibrosis, collagen crosslinking, and vascular distensibility. RESULTS: Non-maturation of human AVFs was associated with higher LOX deposition in pre-access veins (N=20, P=0.029), and increased trivalent crosslinks (N=18, P=0.027) in human AVF tissues. Systemic and local inhibition of LOX increased AVF distensibility, while reducing wall fibrosis and collagen crosslinking in rat fistulas. CONCLUSIONS: Our results demonstrate that BAPN-mediated inhibition of LOX significantly improves vascular remodeling in experimental fistulas.
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Fístula Arteriovenosa , Derivación Arteriovenosa Quirúrgica , Aminopropionitrilo/farmacología , Animales , Fístula Arteriovenosa/tratamiento farmacológico , Derivación Arteriovenosa Quirúrgica/métodos , Humanos , Proteína-Lisina 6-Oxidasa , Ratas , VenasRESUMEN
OBJECTIVE: To observe the expression change of mitophagy-related proteins in skeletal muscle in rats with spleen deficiency syndrome and to explain the partial action mechanism of acupuncture at Zusanli (ST 36) for spleen deficiency syndrome. METHODS: Forty male SD rats, after normal feeding, were randomly divided into a normal group, a spleen deficiency group, a Zusanli group and a non-acupoint group, ten rats in each group. Except the normal group, the three factors modeling method was used for 14 days to establish the model of spleen deficiency syndrome on the other 3 groups. The rats in the Zusanli group were treated with EA at bilateral "Zusanli" (ST 36), while the rats in the non-acupoint group were treated with EA at bilateral non acupoint (dense-sparse wave, frequency of 2 Hz/100 Hz, 20 min per treatment, once a day for 10 days). The rats in the normal group and spleen deficiency group were treated with immobilization for 20 min per day, and no EA was given. The HPLC method was applied to measure the content of adenosine triphosphate (ATP) and adenosine monophosphate (AMP) in skeletal muscle. The Western blotting method was applied to measure the expression of adenosine monophosphate activated protein kinase (AMPK), p-AMPK, ULK1, p-ULK1ï¼LC3-â and LC3-â ¡ in skeletal muscle. RESULTS: The ATP content in the spleen deficiency group was significantly lower than that in the normal group (P<0.01); the ATP content in the Zusanli group was significantly higher than that in the spleen deficiency group (P<0.05) but lower than that in the normal group (P<0.05), there was no significant difference between the non-acupoint group and the spleen deficiency group (P>0.05). Compared with the normal group, the AMP/ATP in the spleen deficiency group and the Zusanli group were significantly up-regulated (P<0.01, P<0.05). The differences of p-AMPK/AMPK between the spleen deficiency group and the normal group was not significant (P>0.05). Compared with the normal group and spleen deficiency group, the p-AMPK/AMPK in the Zusanli group was significantly up-regulated (both P<0.05). The p-ULK1/ULK1 and LC3-â ¡/LC3-â in the Zusanli group was higher than those in the normal group and spleen deficiency group (all P<0.01). CONCLUSION: EA at "Zusanli" (ST 36) might activate AMPK and produce stable ULK1/AMPK compound and increase the mitochondrial autophagy, which could regulate spleen-stomach and treat spleen deficiency.
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Electroacupuntura , Puntos de Acupuntura , Animales , Masculino , Mitofagia , Músculo Esquelético , Ratas , Ratas Sprague-DawleyRESUMEN
OBJECTIVE: To observe the effect of electroacupuncture (EA) of "Zusanli" (ST 36) on mitochondrial oxidative stress of skeletal muscle in rats with chronic fatigue syndrome (CFS) based on adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK)/ peroxlsome proliferator-activated receptor-γ coactivator-1 α (PGC-1 α) signaling, in order to reveal its mechanism underlying improvement of CFS. METHODS: Forty SD rats were randomly divided into normal control, CFS model, EA-Zusanli (ST 36) and EA-non-acupoint groups (n=10 rats in each group). The CFS model was established by forced exhausted load-bearing swimming (twice daily), chronic constraint (1 h) and sleep deprivation (20 h/day) for 14 days. Following modeling, EA (2 Hz/100 Hz, 2 V) was applied to bilateral Zusanli (ST 36) or non-acupoint (about 10-15 mm superior to the bilateral Iliac creast and about 20 mm lateral to the posterior median line) for 20 min, once a day for 10 days. The expression levels of ATP synthase, AMPK, phosphorylated (p)-AMPK, silent mating type information regulation 2 homolog-1 (SIRT 1) and PGC-1 α proteins, and ATP synthase, SIRT 1 and PGC-1 α mRNAs of the quadriceps femoris muscle were detected by Western blot and fluorescence quantitative PCR, respectively. The rats' grabbing force was detected by using a grabbing-force detector. RESULTS: Compared with the normal group, the grabbing force, and the expression levels of ATP synthase and PGC-1 α proteins and mRNAs were significantly decreased (P<0.05, P<0.01), while the expression of SIRT 1 protein was significantly up-regulated (P<0.05) in the CFS model group. Following EA intervention, the grabbing force and the expression levels of ATP synthase mRNA, SIRT 1 and PGC-1 α proteins and mRNAs, and p-AMPK/AMPK were significantly up-regulated in the EA-Zusanli (ST 36) group (P<0.05, P<0.01). CONCLUSION: EA of ST 36 can raise the grabbing force of CFS rats, which may be related to its effects in up-regulating the expression of ATP synthase mRNA, SIRT 1 and PGC-1 α proteins and mRNAs, and p-AMPK/AMPK to reduce mitochondrial oxidative stress reaction and in increasing ATP synthesis.
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Electroacupuntura , Síndrome de Fatiga Crónica , Puntos de Acupuntura , Adenilato Quinasa , Animales , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND: Critical limb ischemia (CLI) is the extreme manifestation of peripheral artery disease, a major unmet clinical need for which lower limb amputation is the only option for many patients. After 2 decades in development, therapeutic angiogenesis has been tested clinically via intramuscular delivery of proangiogenic proteins, genes, and stem cells. Efficacy has been modest to absent, and the largest phase 3 trial of gene therapy for CLI reported a worsening trend of plasmid fibroblast growth factor. In all clinical trials to date, gene therapy has used unregulated vectors with limited duration of expression. Only unregulated extended expression vectors such as adeno-associated virus (AAV) and lentivirus have been tested in preclinical models. METHODS AND RESULTS: We present preclinical results of ischemia (hypoxia)-regulated conditionally silenced (CS) AAV-human vascular endothelial growth factor (hVEGF) gene delivery that shows efficacy and safety in a setting where other strategies fail. In a BALB/c mouse model of CLI, we show that gene therapy with AAV-CS-hVEGF, but not unregulated AAV or plasmid, vectors conferred limb salvage, protection from necrosis, and vascular regeneration when delivered via intramuscular or intra-arterial routes. All vector treatments conferred increased capillary density, but organized longitudinal arteries were selectively generated by AAV-CS-hVEGF. AAV-CS-hVEGF therapy reversibly activated angiogenic and vasculogenic genes, including Notch, SDF1, Angiopoietin, and Ephrin-B2. Reoxygenation extinguished VEGF expression and inactivated the program with no apparent adverse side effects. CONCLUSIONS: Restriction of angiogenic growth factor expression to regions of ischemia supports the safe and stable reperfusion of hindlimbs in a clinically relevant murine model of CLI.
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Arteria Femoral/fisiología , Terapia Genética/métodos , Miembro Posterior/irrigación sanguínea , Isquemia/terapia , Regeneración/fisiología , Factor A de Crecimiento Endotelial Vascular/administración & dosificación , Adenoviridae , Animales , Silenciador del Gen/fisiología , Técnicas de Transferencia de Gen , Vectores Genéticos , Humanos , Ratones Endogámicos BALB C , Neovascularización Fisiológica/fisiología , Enfermedad Arterial Periférica/terapia , Reperfusión/métodos , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
BACKGROUND: The role of immune cells in arteriovenous fistulae (AVF) maturation is poorly understood and has received, until quite recently, little attention. This study examines the function of T lymphocytes in AVF vascular remodeling. METHODS: Experimental fistulae were created in athymic rnu nude rats lacking mature T lymphocytes and euthymic control animals by anastomosing the left superior epigastric vein to the nearby femoral artery. Blood flow rates, wall morphology, and histologic changes were assessed in AVF 21 days after creation. The effect of CD4(+) lymphocytes on AVF maturation in athymic animals was analyzed by adoptive transfer of cells after fistula creation. RESULTS: The absence of T lymphocytes compromised blood flow in experimental fistulae. Histopathologic inspection of AVF from athymic rats revealed that T-cell immunodeficiency negatively affected venous vascular remodeling, as evidenced by a reduced lumen, a thick muscular layer, and a low number of inflammatory cells compared with control animals. Adoptive transfer of CD4(+) lymphocytes from euthymic rats into athymic animals after fistula creation improved blood flow and reduced intima-media thickness. CONCLUSION: These results point at the protective role of CD4(+) lymphocytes in the remodeling of the AVF vascular wall.
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Derivación Arteriovenosa Quirúrgica , Linfocitos T CD4-Positivos/metabolismo , Arteria Femoral/cirugía , Remodelación Vascular/inmunología , Animales , Biomarcadores/metabolismo , Velocidad del Flujo Sanguíneo , Arteria Femoral/inmunología , Arteria Femoral/patología , Inmunohistoquímica , Masculino , Ratas , Ratas DesnudasRESUMEN
AIMS: The aim of this study was to investigate the mechanisms by which nicotine increases vascular smooth muscle cell (VSMC) proliferation and post-injury neointimal formation. METHODS AND RESULTS: Vascular injury was inflicted in the right iliac artery of nicotine-treated and control rats. Nicotine increased post-injury VSMC proliferation (Ki67(+) cells) and neointimal formation (neointima/media ratio, 0.42 ± 0.23 vs. 0.14 ± 0.07, P= 0.02). To determine the mechanisms by which nicotine exacerbates VSMC proliferation, cultured cells were exposed to nicotine, and signalling pathways leading to cell proliferation were studied. Nicotine activated extracellular signal-regulated kinase (ERK) 1/2 in a dose- and time-dependent manner. The blockade of this signalling axis abolished nicotine-mediated proliferation. Functional nicotinic acetylcholine receptors and Ca(2+) influx were necessary for ERK1/2 activation and nicotine-induced mitogenesis in VSMCs. Downstream to ERK1/2, nicotine induced the phosphorylation of Ets-like gene 1 in a timely co-ordinated manner with the up-regulation of the atherogenic transcription factor, early growth response 1 (Egr-1). The treatment of balloon-injured arteries with a lentivirus vector carrying a short hairpin RNA against Egr-1 abolished the deleterious effect of nicotine on vascular remodelling. CONCLUSION: Nicotine acts through its receptors in VSMC to activate the ERK-Egr-1 signaling cascade that induces cell proliferation and exacerbates post-injury neointimal development.
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Aterosclerosis/inducido químicamente , Proliferación Celular/efectos de los fármacos , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Nicotina/toxicidad , Agonistas Nicotínicos/toxicidad , Túnica Íntima/efectos de los fármacos , Administración Oral , Animales , Aterosclerosis/metabolismo , Aterosclerosis/patología , Aterosclerosis/prevención & control , Señalización del Calcio/efectos de los fármacos , Cateterismo/efectos adversos , Células Cultivadas , Quelantes/farmacología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Activación Enzimática , Arteria Ilíaca/efectos de los fármacos , Arteria Ilíaca/metabolismo , Arteria Ilíaca/patología , Antígeno Ki-67/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Músculo Liso Vascular/lesiones , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Nicotina/administración & dosificación , Agonistas Nicotínicos/administración & dosificación , Antagonistas Nicotínicos/farmacología , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Interferencia de ARN , Ratas , Ratas Sprague-Dawley , Receptores Nicotínicos/efectos de los fármacos , Receptores Nicotínicos/metabolismo , Factores de Tiempo , Transfección , Túnica Íntima/lesiones , Túnica Íntima/metabolismo , Túnica Íntima/patología , Proteína Elk-1 con Dominio ets/metabolismoRESUMEN
BACKGROUND AND AIMS: In-stent restenosis (ISR) is the major complication that occurs after percutaneous coronary interventions to facilitate coronary revascularization. Herein we described a simple and cost-effective model, which reproduces important features of ISR in the mouse. METHODS AND RESULTS: Microvascular bare metal stents were successfully implanted in the abdominal aorta of atherosclerotic ApoE-null mice. Patency of implanted stents was interrogated using ultrasound biomicroscopy. Aortas were harvested at different time points after implantation and processed for histopathological analysis. Thrombus formation was histologically detected after 1 day. Leukocyte adherence and infiltration were evident after 7 days and decreased thereafter. Neointimal formation, neointimal thickness and luminal stenosis simultaneously increased up to 28 days after stent implantation. Using multichannel fluorescence molecular tomography (FMT) for spatiotemporal resolution of MMP activities, we observed that MMP activity in the stented aorta of Apo-E null mice was 2-fold higher than that of wild-type mice. Finally, we compared neointimal formation in response to stenting in two genetically different mouse strains. In-stent neointimas in FVB/NJ mice were 2-fold thicker than in C57BL/6J mice (p=0.002). CONCLUSION: We have developed a model that can take advantage of the multiple genetic resources available for the mouse to study the mechanisms of in-stent restenosis.
Asunto(s)
Reestenosis Coronaria/patología , Stents/efectos adversos , Animales , Aorta Abdominal/diagnóstico por imagen , Aorta Abdominal/patología , Aorta Abdominal/cirugía , Apolipoproteínas E/deficiencia , Reestenosis Coronaria/diagnóstico por imagen , Modelos Animales de Enfermedad , Masculino , Metaloproteinasas de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente , Tomografía Óptica , Túnica Íntima/patología , UltrasonografíaRESUMEN
Alteration of VSMC (vascular smooth-muscle cell) physiology is associated with the development of atherosclerosis and restenosis. We hypothesize that aging up-regulates the expression of p16 INK4a in VSMCs, which may increase the susceptibility of blood vessels to vascular occlusive diseases. Aortic VSMCs were obtained from young and aged mice. Cells from aged mice grew more slowly than those from their younger counterparts. Progression of cell cycle in response to serum stimulation was significantly inhibited in those cells with aging, as determined by FACS after propidium iodide staining. A significant up-regulation of p16 INK4a (2.5-fold, P=0.0012) was found in VSMC from aged animals using gene arrays. The up-regulation of this gene was further confirmed by quantitative RT-PCR (reverse transcription-PCR) and Western-blot experiments. Immunostaining for p16 INK4a confirmed that aortas from aged mice contained more p16 INK4a+ SMA (smooth-muscle cell actin)+ cells than aortas from young animals (26.79+/-2.45 versus 7.06+/-1.44, P=0.00027, n=4). In conclusion, we have shown that aging up-regulates the expression of p16 INK4a in VSMC in both cultures and arteries. The increase in p16 INK4a in the vasculature with aging may modify VSMC's response to post-injury stress and therefore accelerate the development of age-related cardiovascular diseases.
Asunto(s)
Envejecimiento/metabolismo , Aterosclerosis/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Músculo Liso Vascular/metabolismo , Actinas/metabolismo , Animales , Aorta/metabolismo , Aorta/fisiopatología , Aterosclerosis/fisiopatología , Proteínas Sanguíneas/farmacología , Ciclo Celular/efectos de los fármacos , Ciclo Celular/fisiología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Reestenosis Coronaria/metabolismo , Reestenosis Coronaria/fisiopatología , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Músculo Liso Vascular/citología , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia Arriba/genéticaRESUMEN
AIMS: The origin of post-injury neointimal cells is still a matter of debate. This study aims to determine the anatomic source of neointimal cells in one of the most important animal models for the study of vascular stenosis in response to injury, the rat balloon injury model. METHODS AND RESULTS: Chimeric rats were generated by rescuing lethally irradiated animals with green fluorescent protein (GFP)(+) bone marrow (BM) cells from transgenic rats. Neointimal formation was induced in the right iliac artery of these animals using a balloon angioplasty catheter. Injured and non-injured contra-lateral arteries were harvested at 7, 14, and 30 days post-surgery. BM-derived monocytes/macrophages (CD68(+) GFP(+)) were abundant in the media and adventitia of injured vessels harvested at 7 days as determined by immunofluorescence and confocal microscopy. The number of GFP(+) cells declined in the vascular wall with time. Post-injury neointimal cells were mostly GFP(-)/smooth muscle actin (SMA)(+), which indicated that those cells originated in the recipient. Only a few neointimal cells seemed to come from circulating progenitors (GFP(+) SMA(+), 2.34% +/- 1.61). The vascular origin of cells in the neointima was further confirmed by transplanting injured GFP arteries into wild-type recipients. In these grafts, 94.23 +/- 0.44% of medial and 92.95 +/- 19.34% of neointimal cells were GFP(+) SMA(+). Finally, we tested the capacity of vascular smooth muscle cells (VSMC) to migrate through the vascular wall using a novel in vivo assay. As expected, VSMC migrated and populated the neointima only in response to injury. CONCLUSION: Our results suggest that neointimal cells in the rat balloon injury model mostly derive from pre-existing vascular cells and that only a small population of those cells come from BM-derived progenitors.