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Metastasis occurs frequently after resection of pancreatic cancer (PaC). In this study, we hypothesized that multi-parametric analysis of pre-metastatic liver biopsies would classify patients according to their metastatic risk, timing and organ site. Liver biopsies obtained during pancreatectomy from 49 patients with localized PaC and 19 control patients with non-cancerous pancreatic lesions were analyzed, combining metabolomic, tissue and single-cell transcriptomics and multiplex imaging approaches. Patients were followed prospectively (median 3 years) and classified into four recurrence groups; early (<6 months after resection) or late (>6 months after resection) liver metastasis (LiM); extrahepatic metastasis (EHM); and disease-free survivors (no evidence of disease (NED)). Overall, PaC livers exhibited signs of augmented inflammation compared to controls. Enrichment of neutrophil extracellular traps (NETs), Ki-67 upregulation and decreased liver creatine significantly distinguished those with future metastasis from NED. Patients with future LiM were characterized by scant T cell lobular infiltration, less steatosis and higher levels of citrullinated H3 compared to patients who developed EHM, who had overexpression of interferon target genes (MX1 and NR1D1) and an increase of CD11B+ natural killer (NK) cells. Upregulation of sortilin-1 and prominent NETs, together with the lack of T cells and a reduction in CD11B+ NK cells, differentiated patients with early-onset LiM from those with late-onset LiM. Liver profiles of NED closely resembled those of controls. Using the above parameters, a machine-learning-based model was developed that successfully predicted the metastatic outcome at the time of surgery with 78% accuracy. Therefore, multi-parametric profiling of liver biopsies at the time of PaC diagnosis may determine metastatic risk and organotropism and guide clinical stratification for optimal treatment selection.
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Neoplasias Hepáticas , Fígado , Neoplasias Pancreáticas , Humanos , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/cirurgia , Neoplasias Hepáticas/secundário , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/genética , Masculino , Feminino , Pessoa de Meia-Idade , Idoso , Fígado/patologia , Fígado/metabolismo , Biópsia , Estadiamento de Neoplasias , Pancreatectomia , Armadilhas Extracelulares/metabolismo , PrognósticoRESUMO
Extracellular vesicles and particles (EVPs) are pivotal mediators of pre-metastatic niche formation and cancer progression, including induction of vascular permeability, which facilitates tumor cell extravasation and metastasis. However, the mechanisms through which EVPs exert this effect remain poorly understood. Here, we elucidate a novel mechanism by which tumor EVPs enhance endothelial cell permeability, tumor extravasation, and lung metastasis to different degrees, depending on tumor type. Strikingly, vascular leakiness is observed within 48h following tumor implantation and as early as one hour following intravenous injection of tumour-derived EVPs in naïve mice. Surprisingly, rather than acting directly on endothelial cells, EVPs first activate interstitial macrophages (IMs) leading to activation of JAK/STAT signaling and IL-6 secretion in IMs which subsequently promote endothelial permeability. Depletion of IMs significantly reduces tumour-derived EVP-dependent vascular leakiness and metastatic potential. Tumour EVPs that strongly induce vascular leakiness express high levels of ITGα5, and ITGα5 ablation impairs IM activation, cytokine secretion, and subsequently vascular permeability and metastasis. Importantly, IL-6 expression is elevated in IMs from non-involved tumor-adjacent lung tissue compared to distal lung tissue in lung cancer patients, highlight the clinical relevance of our discovery. Our findings identify a key role for IM activation as an initiating step in tumor type-specific EVP-driven vascular permeability and metastasis, offering promising targets for therapeutic intervention.
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PRIMARY OBJECTIVE: We evaluated whether photobiomodulation with red/near infrared light applied transcranially via light emitting diodes (LED) was associated with reduced symptoms and improved cognitive functioning in patients with chronic symptoms following mild traumatic brain injury. RESEARCH DESIGN: Participants (3 men, 6 women; 22-61 years-old) underwent a 6-week intervention involving 18 40-minute transcranial LED treatment sessions. METHODS AND PROCEDURES: Reliable change indices were calculated for 10 neuropsychological test scores and 3 self-report questionnaires of subjective cognition, post-concussion symptoms, and depression at baseline and following treatment. Questionnaires were also administered after 2-week sham and at 1-month and 2-month follow-ups. MAIN OUTCOME AND RESULTS: Only 2 participants improved on neuropsychological testing. On questionnaires, 4 reported improved cognition, 5 reported improved post-concussion symptoms, and 3 reported improved depression. Significant improvement in 2 or more domains was reported by 4 participants and mostly maintained at both follow-ups. CONCLUSIONS: Most participants did not improve on neuropsychological testing. A minority self-reported improvement in symptoms, potentially explained by the intervention, psychiatric medication changes, placebo effects, or other factors. Selecting participants with different clinical characteristics, and dosing and delivery system changes, may produce different results. A study design accounting for placebo effects appears warranted in future trials.
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Concussão Encefálica , Terapia com Luz de Baixa Intensidade , Síndrome Pós-Concussão , Masculino , Humanos , Feminino , Adulto Jovem , Adulto , Pessoa de Meia-Idade , Concussão Encefálica/complicações , Concussão Encefálica/radioterapia , Concussão Encefálica/diagnóstico , Síndrome Pós-Concussão/radioterapia , Síndrome Pós-Concussão/psicologia , Projetos Piloto , CogniçãoRESUMO
Scanning electron microscopy (SEM) offers an unparalleled view of the membrane topography of mammalian cells by using a conventional osmium (OsO4) and ethanol-based tissue preparation. However, conventional SEM methods limit optimal resolution due to ethanol and lipid interactions and interfere with visualization of fluorescent reporter proteins. Therefore, SEM correlative light and electron microscopy (CLEM) has been hindered by the adverse effects of ethanol and OsO4 on retention of fluorescence signals. To overcome this technological gap in achieving high-resolution SEM and retain fluorescent reporter signals, we developed a freeze-drying method with gaseous nitrogen (FDGN). We demonstrate that FDGN preserves cyto-architecture to allow visualization of detailed membrane topography while retaining fluorescent signals and that FDGN processing can be used in conjunction with a variety of high-resolution imaging systems to enable collection and validation of unique, high-quality data from these approaches. In particular, we show that FDGN coupled with high resolution microscopy provided detailed insight into viral or tumor-derived extracellular vesicle (TEV)-host cell interactions and may aid in designing new approaches to intervene during viral infection or to harness TEVs as therapeutic agents.
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Primary tumors secrete a variety of factors to turn distant microenvironments into favorable and fertile 'soil' for subsequent metastases. Among these 'seeding' factors that initiate pre-metastatic niche (PMN) formation, tumor-derived extracellular vesicles (EVs) are of particular interest as tumor EVs can direct organotropism depending on their surface integrin profiles. In addition, EVs also contain versatile, bioactive cargo, which include proteins, metabolites, lipids, RNA, and DNA fragments. The cargo incorporated into EVs is collectively shed from cancer cells and cancer-associated stromal cells. Increased understanding of how tumor EVs promote PMN establishment and detection of EVs in bodily fluids highlight how tumor EVs could serve as potential diagnostic and prognostic biomarkers, as well as provide a therapeutic target for metastasis prevention. This review focuses on tumor-derived EVs and how they direct organotropism and subsequently modulate stromal and immune microenvironments at distal sites to facilitate PMN formation. We also outline the progress made thus far towards clinical applications of tumor EVs.
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Vesículas Extracelulares , Neoplasias , Humanos , Neoplasias/metabolismo , Vesículas Extracelulares/metabolismo , Comunicação Celular , Microambiente TumoralRESUMO
Cancer alters the function of multiple organs beyond those targeted by metastasis1,2. Here we show that inflammation, fatty liver and dysregulated metabolism are hallmarks of systemically affected livers in mouse models and in patients with extrahepatic metastasis. We identified tumour-derived extracellular vesicles and particles (EVPs) as crucial mediators of cancer-induced hepatic reprogramming, which could be reversed by reducing tumour EVP secretion via depletion of Rab27a. All EVP subpopulations, exosomes and principally exomeres, could dysregulate hepatic function. The fatty acid cargo of tumour EVPs-particularly palmitic acid-induced secretion of tumour necrosis factor (TNF) by Kupffer cells, generating a pro-inflammatory microenvironment, suppressing fatty acid metabolism and oxidative phosphorylation, and promoting fatty liver formation. Notably, Kupffer cell ablation or TNF blockade markedly decreased tumour-induced fatty liver generation. Tumour implantation or pre-treatment with tumour EVPs diminished cytochrome P450 gene expression and attenuated drug metabolism in a TNF-dependent manner. We also observed fatty liver and decreased cytochrome P450 expression at diagnosis in tumour-free livers of patients with pancreatic cancer who later developed extrahepatic metastasis, highlighting the clinical relevance of our findings. Notably, tumour EVP education enhanced side effects of chemotherapy, including bone marrow suppression and cardiotoxicity, suggesting that metabolic reprogramming of the liver by tumour-derived EVPs may limit chemotherapy tolerance in patients with cancer. Our results reveal how tumour-derived EVPs dysregulate hepatic function and their targetable potential, alongside TNF inhibition, for preventing fatty liver formation and enhancing the efficacy of chemotherapy.
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Vesículas Extracelulares , Ácidos Graxos , Fígado Gorduroso , Fígado , Neoplasias Pancreáticas , Animais , Camundongos , Sistema Enzimático do Citocromo P-450/genética , Vesículas Extracelulares/metabolismo , Ácidos Graxos/metabolismo , Fígado Gorduroso/tratamento farmacológico , Fígado Gorduroso/etiologia , Fígado Gorduroso/metabolismo , Fígado Gorduroso/prevenção & controle , Fígado/metabolismo , Fígado/patologia , Fígado/fisiopatologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Microambiente Tumoral , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/metabolismo , Neoplasias Hepáticas/secundário , Humanos , Inflamação/metabolismo , Ácido Palmítico/metabolismo , Células de Kupffer , Fosforilação Oxidativa , Proteínas rab27 de Ligação ao GTP/deficiênciaRESUMO
STUDY OBJECTIVES: To describe the development and feasibility of a cognitive behavioral therapy for insomnia (CBT-I) program delivered via personal digital devices and fully integrated with the electronic health record (EHR). METHODS: A multidisciplinary team of clinicians and members of our Center for Digital Health collaborated to develop a Chronic Insomnia Interactive Care Plan (ChI-ICP), an application that provides personalized and just in time education and promotes self-management using CBT-I concepts, and is activated from and fully integrated into the EHR. Following development, we evaluated patient engagement and workflows, assessed changes to provider workload, and examined outcomes on measures of insomnia during a pilot deployment of the application. RESULTS: A total of 222 patients were enrolled and 179 engaged with the plan during the 3-month pilot program. Enrolled patients generated an average of 3.9 ± 2.3 In Basket messages, most being automated notifications related to noncompletion of assigned tasks, while only a few were related to patients requesting additional training or help with insomnia. Sleep efficiency improved from baseline until the completion of the program from 74.5% ± 16.7% to 87.6% ± 10.8% (P = .001), and the Insomnia Severity Index improved from 14.9 ± 5.22 to 11.6 ± 4.80 (P = .006). CONCLUSIONS: In this pilot implementation of an integrated ChI-ICP, patient engagement was favorable, workflows and workload were not significantly burdensome for the care teams, and initial evaluation of efficacy was favorable. This provides evidence for an application that is a scalable method to assist patients with chronic insomnia and future work should assess its efficacy in controlled trials. CITATION: Morgenthaler TI, Kolla BP, Anderson SE, et al. Development and acceptability of a mobile health application integrated with the electronic heath record for treatment of chronic insomnia disorder. J Clin Sleep Med. 2022;18(12):2785-2792.
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Aplicativos Móveis , Distúrbios do Início e da Manutenção do Sono , Telemedicina , Humanos , Distúrbios do Início e da Manutenção do Sono/terapia , Resultado do Tratamento , Telemedicina/métodos , EletrônicaRESUMO
Objective: To investigate the effect of Huperzine A on memory and learning in individuals with moderate-severe traumatic brain injury (TBI).Design: Randomized, double-blind, placebo-controlled Phase II clinical trial.Methods: Subjects were randomly assigned to receive Huperzine A or placebo for 12 weeks and were assessed during in-person visits at screening/baseline, and 6, 12, 24, and 52 weeks post-injury. Changes in memory and learning scores on the California Verbal Learning Test - 2nd Edition (CVLT-II) from baseline to week 12 were assessed using permutation tests and regression analyses.Results: There was no difference between the Huperzine A and placebo groups in memory performance after 12 weeks of treatment. In the placebo group, significant improvements were noted in learning and memory scores. Both groups showed clinically important improvements in depression on the Beck Depression Index.Conclusions: The clinically important improvements in cognitive and emotional outcomes observed in both the placebo and active treatment arms of this clinical trial of Huperzine A are best understood in the context of a placebo effect. Future trials involving patients with moderate-severe TBI in the subacute to chronic phases of recovery should be designed to account for placebo effects as failure to do so may lead to spurious conclusions.
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Lesões Encefálicas Traumáticas , Efeito Placebo , Alcaloides , Lesões Encefálicas Traumáticas/complicações , Lesões Encefálicas Traumáticas/tratamento farmacológico , Cognição , Método Duplo-Cego , Humanos , Projetos Piloto , SesquiterpenosRESUMO
There is a clinical need for new therapeutics to improve healing of chronic impaired wounds. Thus, we investigated how biopolymer conjugation could be used to improve the wound healing performance of a key growth factor for tissue regeneration: Sonic hedgehog (Shh). We generated two multivalent Shh conjugates (mvShh) using hyaluronic acid with two different MWs, which exhibited equivalent potency and proteolytic protection in vitro. Using db/db diabetic mice, we showed that mvShh made with smaller HyA MW resulted in more rapid and robust neovascularization compared to mvShh made with larger MW HyA. Further, smaller mvShh conjugates resulted in faster wound resolution compared to the unconjugated Shh. This study is the first to show how the wound healing efficacy of multivalent protein-polymer conjugates is sensitive to the polymer MW, and our findings suggest that this parameter could be used to enhance the efficacy of growth factor conjugates.
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Fibroblastos/metabolismo , Proteínas Hedgehog , Ácido Hialurônico , Cicatrização/efeitos dos fármacos , Animais , Linhagem Celular , Proteínas Hedgehog/química , Proteínas Hedgehog/farmacologia , Ácido Hialurônico/química , Ácido Hialurônico/farmacologia , Camundongos , Camundongos Endogâmicos NOD , Peso MolecularRESUMO
Different protein kinase C (PKC) isoforms have distinct roles in regulating cell functions. The conventional (α, ß, γ) and novel (δ, É, η, θ) classes are targets of phorbol ester tumor promoters, which are surrogates of endogenous second messenger, diacylglycerol. The promoter-stimulated disappearance of filopodia was investigated by use of blocking peptides (BPs) that inhibit PKC maturation and/or docking. Filopodia were partially rescued by a peptide representing PKC É hydrophobic sequence, but also by a myristoylated PKC α/ß pseudosubstrate sequence, and an inhibitor of T-cell protein tyrosine phosphatase (TC-PTP). The ability to turn over filopodia was widely distributed among PKC isoforms. PKC α and η hydrophobic sequences enhanced filopodia in cells in the absence of tumor promoter treatment. With transcriptional knockdown of PKC α, the content of PKC É predominated over other isoforms. PKC É could decrease filopodia significantly in promoter-treated cells, and this was attributed to ruffling. The presence of PKC α counteracted the PKC É-mediated enhancement of ruffling. The results showed that there were two mechanisms of filopodia downregulation. One operated in the steady-state and relied on PKC α and η. The other was stimulated by tumor promoters and relied on PKC É. Cycles of protrusion and retraction are characteristic of filopodia and are essential for the cell to orient itself during chemotaxis and haptotaxis. By suppressing filopodia, PKC É can create a long-term "memory" of an environmental signal that may act in nature as a mnemonic device to mark the direction of a repulsive signal.
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Carcinógenos/toxicidade , Proteína Quinase C/metabolismo , Pseudópodes/metabolismo , Actinas/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Ativação Enzimática/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Isoenzimas/metabolismo , Peptídeos/química , Fosfoproteínas Fosfatases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Pseudópodes/efeitos dos fármacos , Ratos , Bibliotecas de Moléculas Pequenas/farmacologia , Acetato de Tetradecanoilforbol/toxicidadeRESUMO
Limited transendothelial permeability across tumor microvessels represents a significant bottleneck in the development of tumor-specific diagnostic agents and theranostic drugs. Here, we show an approach to increase transendothelial permeability of macromolecular and nanoparticle-based contrast agents via inhibition of the type I TGF-ß receptor, activin-like kinase 5 (Alk5), in tumors. Alk5 inhibition significantly increased tumor contrast agent delivery and enhancement on imaging studies, while healthy organs remained relatively unaffected. Imaging data correlated with significantly decreased tumor interstitial fluid pressure, while tumor vascular density remained unchanged. This immediately clinically translatable concept involving Alk5 inhibitor pretreatment prior to an imaging study could be leveraged for improved tumor delivery of macromolecular and nanoparticle-based imaging probes and, thereby, facilitate development of more sensitive imaging tests for cancer diagnosis, enhanced tumor characterization, and personalized, image-guided therapies.
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Despite their preclinical promise, few recombinant growth factors have been fully developed into effective therapies, in part, due to the short interval of therapeutic activity after administration. To address this problem, we developed nanoscale polymer conjugates for multivalent presentation of therapeutic proteins that enhance the activation of targeted cellular responses. As an example of this technology, we conjugated multiple Sonic hedgehog (Shh) proteins onto individual hyaluronic acid biopolymers to generate multivalent protein clusters at defined ratios (i.e., valencies) that yield enhanced Shh pathway activation at equivalent concentrations relative to unconjugated Shh. In this study, we investigated whether these multivalent conjugates (mvShh) could be used to improve the therapeutic function of Shh. We found that a single treatment with mvShh significantly accelerated the closure of full-thickness wounds in diabetic (db/db) mice compared to either an equivalent dose of unconjugated Shh or the vehicle control. Furthermore, we identified specific indicators of wound healing in fibroblasts and endothelial cells (i.e., transcriptional activation and cell migration) that were activated by mvShh in vitro and at concentrations approximately an order of magnitude lower than the unconjugated Shh. Taken together, our findings suggest that mvShh conjugates exhibit greater potency to activate the Shh pathway, and this multivalency advantage improves its therapeutic effect to accelerate wound closure in a diabetic animal model. Our strategy of multivalent protein presentation using nanoscale polymer conjugates has the potential to make a significant impact on the development of protein-based therapies by improving their in vivo performance.
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Diabetes Mellitus/patologia , Proteínas Hedgehog/farmacologia , Cicatrização/efeitos dos fármacos , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Neovascularização Fisiológica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fatores de TempoRESUMO
HoxA5 is expressed in quiescent endothelial cells (EC), but absent in activated angiogenic EC. To examine the efficacy of targeting HoxA5 therapeutically to quell pathologic or tumor angiogenesis, we generated an inducible, transgenic mouse model of sustained HoxA5 expression in ECs. During pathologic angiogenesis, sustained HoxA5 regulates expression several angiogenic effector molecules, notably increased expression of TSP-2 and reduced expression of VEGF, thus leading to inhibition of pathological angiogenesis in tissues. To evaluate if this impressive reduction of vascularization could also impact tumor angiogenesis, HoxA5 mice were bred with a mouse model of de novo squamous carcinogenesis, e.g., K14-HPV16 mice. Activation of EC-HoxA5 significantly reduced infiltration by mast cells into neoplastic skin, an early hallmark of progression to dysplasia, reduced angiogenic vasculature, and blunted characteristics of tumor progression. To evaluate HoxA5 as a therapeutic, topical application of a HoxA5 transgene onto early neoplastic skin of K14-HPV16 mice similarly resulted in a significant impairment of angiogenic vasculature and progression to dysplasia to a similar extent as observed with genetic delivery of HoxA5. Together these data indicate that HoxA5 represents a novel molecule for restricting pathological and tumorigenic angiogenesis.
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Células Endoteliais/metabolismo , Células Endoteliais/patologia , Proteínas de Homeodomínio/genética , Neovascularização Patológica/genética , Fosfoproteínas/genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Animais , Carcinogênese/genética , Carcinogênese/patologia , Progressão da Doença , Células Endoteliais/virologia , Papillomavirus Humano 16 , Camundongos , Camundongos Transgênicos/genética , Processos Neoplásicos , Neovascularização Patológica/patologia , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/patologia , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/patologia , Lesões Pré-Cancerosas/virologia , Neoplasias Cutâneas/virologia , Trombospondinas/genética , Fatores de Transcrição , Transgenes/genética , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Filopodia are sensors which, along with microtubules, regulate the persistence of locomotion. To determine whether protrusions were involved in sensing adhesion, epithelial cells were cultured on platinum and tantalum gradients. Protrusions were defined by an unbiased statistical method of classification as factors 4 (filopodia), 5 (mass distribution), and 7 (nascent neurites). When the prevalence of protrusions was measured in zones of high (H), middle (M), and low (L) adhesiveness, the main differences were in factor 4. Its values were highest at H and declined at M and L regardless of the gradient composition. The significance of the differences was enhanced when T (top/adhesive end) and B (bottom/nonadhesive end) sides of cells were analyzed separately. Since information about sidedness increased the statistical power of the test, this result suggested that cells pointed more filopodia toward the adhesive end. Trends occurred in factors 5 and 7 only when conditions allowed for a marked trend in factor 4. The data showed that gradient sensing is proportional to the prevalence of filopodia, and filopodia are the only protrusions engaged in comparing adhesiveness across a cell. The probability (P) of the significance of a trend was then used to determine how cells sense the gradient. Binding peptides (BPs) were introduced representing sequences critical for Cdc42 docking on a specific partner. BPs for IQGAP (IQ(calmodulin-binding domain)-containing GTPase-activating protein) and ACK (Cdc42-associated kinase) reduced factor 4 values and prevented cell orientation on the gradient. Micrographs showed attenuated or stubby filopodia. These effectors may be implicated in gradient sensing. Another IQGAP BP increased filopodia prevalence and enhanced orientation on the gradient (P<0.00015). A Wiskott-Aldrich syndrome protein (WASP) BP had no effect. When sensing and orientation were abolished, they both failed at the level of filopodia, indicating that filopodia are both sensors and implementers of signals transduced by adhesion.
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Pseudópodes/fisiologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/metabolismo , Linhagem Celular , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/metabolismo , Simulação de Dinâmica Molecular , Ligação Proteica , Proteína Quinase C-épsilon/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Pseudópodes/efeitos dos fármacos , Ratos , Tantálio/farmacologia , Proteína da Síndrome de Wiskott-Aldrich/antagonistas & inibidores , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Proteínas Ativadoras de ras GTPase/antagonistas & inibidores , Proteínas Ativadoras de ras GTPase/metabolismoRESUMO
Objective: Homeobox (HOX) transcription factors coordinate gene expression in wound repair and angiogenesis. Previous studies have shown that gene transfer of HoxA3 to wounds of diabetic mice accelerates wound healing, increasing angiogenesis and keratinocyte migration. In this study, we examined whether HoxA3 can also improve angiogenesis, epidermal integrity, and viability of composite skin grafts. Approach: To determine the effects of HoxA3 on composite skin grafts, we constructed bilayered composite grafts incorporating fibroblasts engineered to constitutively secrete HoxA3. We then transplanted these composite grafts in vivo. Results: The composite grafts produced a stratified epidermal layer after seventeen days in culture and following transplantation in vivo, these grafts exhibit normal epidermal differentiation and reduced contraction compared to controls. In addition, HoxA3 grafts showed increased angiogenesis. Quantitative polymerase chain reaction (PCR) analyses of HoxA3 graft tissue reveal an increase in the downstream HoxA3 target genes MMP-14 and uPAR expression, as well as a reduction in CCL-2 and CxCl-12. Innovation: Expression of secreted HoxA3 in composite grafts represents a comprehensive approach that targets both keratinocytes and endothelial cells to promote epidermal proliferation and angiogenesis. Conclusion: Secreted HoxA3 improves angiogenesis, reduces expression of inflammatory mediators, and prolongs composite skin graft integrity.
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Homeobox genes represent a family of highly conserved transcription factors originally discovered to regulate organ patterning during development. More recently, several homeobox genes were shown to affect processes in adult tissue, including angiogenesis and wound healing. Whereas a subset of members of the Hox-family of homeobox genes activate growth and migration to promote angiogenesis or wound healing, other Hox genes function to restore or maintain quiescent, differentiated tissue function. Pathological tissue remodeling is linked to differential expression of activating or stabilizing Hox genes and dysregulation of Hox expression can contribute to disease progression. Studies aimed at understanding the role and regulation of Hox genes have provided insight into how these potent morphoregulatory genes can be applied to enhance tissue engineering or limit cancer progression.
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Regulação Neoplásica da Expressão Gênica , Genes Homeobox , Neovascularização Patológica/genética , Cicatrização , Animais , Diferenciação Celular , Sobrevivência Celular , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Progressão da Doença , Células Endoteliais/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Metástase Neoplásica , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Ativação TranscricionalRESUMO
The unlimited differentiation and proliferation capacity of embryonic stem cells represents a great resource for regenerative medicine. Here, we describe a method for differentiating, isolating, and expanding endothelial cells (ECs) from mouse embryonic stem cells (mESCs). First, mESCs are expanded on a mouse embryonic fibroblast (mEF) feeder layer and partially differentiated into embryoid bodies (EBs) by growing the cells in an ultra-low attachment plate for up to 5 days. The EBs are then differentiated along the endothelial lineage using endothelial growth medium supplemented with 40 ng/mL vascular endothelial growth factor (VEGF). The differentiated endothelial population expresses both Fetal Liver Kinase 1 (Flk-1) and VE-Cadherin on the cell surface which can be further purified using a fluorescence-activated cell sorting (FACS) system and subsequently expanded on 0.1 % gelatin-coated plates. The differentiated cells can be analyzed by real-time PCR and flow cytometry to confirm enrichment of EC-specific genes and proteins.
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Diferenciação Celular , Células-Tronco Embrionárias/citologia , Células Endoteliais/citologia , Citometria de Fluxo/métodos , Animais , Antígenos CD/metabolismo , Biomarcadores/metabolismo , Caderinas/metabolismo , Proliferação de Células , Corpos Embrioides/citologia , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
A COMPUTERIZED MORPHOMETRIC CLASSIFICATION TECHNIQUE BASED ON LATENT FACTORS REVEALS MAJOR PROTRUSION CLASSES: factors 4, 5, and 7. Previous work showed that factor 4 represented filopodia, 5 the distribution of lamellar cytoplasm, and 7 a blunt protrusion. We explore the relationship of focal contact (FC) characteristics and their integrated actin cables to factors values. The results show that FC maturation/cytoskeletal integration affects factor 5, because FC elongation/integration was correlated with its values. On the contrary, 7 values decreased with maturation, so cable or FC size or their integration must be restricted to form these protrusions. Where integration did occur, the cables showed distinctive size and orientation, as indicated by correlation of 7 values with FC shape. Results obtained with myosin inhibitors support the interpretation that a central, isometric, contractile network puts constraints on both factor 5 and 7 protrusions. We conclude that cells establish functional domains by rearranging the cytoskeleton.
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Although growth factor-initiated cascades in cells are networked with mechanisms such as "inside-out signaling", it is not known how these pathways are integrated. Earlier studies reported that ruffling was enhanced and filopodia reduced in transformed cells. Since dissecting relationships among features was impossible if subjective recognition was relied upon, features in two epithelial cell lines were recognized by latent factor analysis. Factor-based classification revealed four protrusion classes, but none of them corresponded to ruffles. Loss of filopodia, defined by factor 4 (F4) values, accounted for the greatest change in features of oncogenically transformed cells. Factor 5 (F5, lamella) was unchanged during transformation of an airway epithelium cell line. The tumor promoter, phorbol 12-myristate 13-acetate (PMA), increased ruffling but decreased filopodia. F4 retained this relationship to ruffling in untreated cells and at multiple times after treatment. F5 values decreased but were positively correlated with measures of ruffling. Because factors are created as mutually orthogonal variables, this suggested that ruffles were not flagged in factor analysis because they originate from other features. Actin filament capping with sub-micromolar cytochalasin D (Cyto D) suppressed ruffling without affecting F4 or F5. Cyto D increased factor 7 (F7) values, thus showing specificity for this feature. However, cytochalasin treatment of PMA-treated cells that had developed stress fibers increased F4 and decreased F5. The results suggest that PMA changes the state of the cytoskeleton, causing protrusions to show novel responses to Cyto D compared to untreated cells. Results suggest that the factors identify physiologically distinct features.