RESUMO
Gene therapy using siRNA has become a promising strategy to achieve targeted gene knockdown for treatment of cardiovascular pathologies. However, efficient siRNA transfection often relies on cationic delivery vectors such as synthetic cell-penetrating polymers which are susceptible to interference by negatively charged molecules. Anticoagulants such as heparin, which is negatively charged and widely used in cardiovascular applications, may pose a significant barrier to effective siRNA delivery. We therefore conducted in vitro studies utilizing human smooth muscle and endothelial cells transfected with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ß2-microglobulin (B2M) siRNA in the presence of heparin, argatroban, and bivalirudin in order to determine which anticoagulant therapy is most compatible for siRNA delivery. We observed that while heparin, at clinical doses, decreases the efficiency of siRNA targeted mRNA knockdown, mRNA knockdown is not inhibited in the presence of either argatroban or bivalirudin. Our data suggests that heparin should be avoided during siRNA therapy with cationic transfection agents, and argatroban and bivalirudin should be used in its stead.
RESUMO
BACKGROUND: Vein graft failure following cardiovascular bypass surgery results in significant patient morbidity and cost to the healthcare system. Vein graft injury can occur during autogenous vein harvest and preparation, as well as after implantation into the arterial system, leading to the development of intimal hyperplasia, vein graft stenosis, and, ultimately, bypass graft failure. Although previous studies have identified maladaptive pathways that occur shortly after implantation, the specific signaling pathways that occur during vein graft preparation are not well defined and may result in a cumulative impact on vein graft failure. We, therefore, aimed to elucidate the response of the vein conduit wall during harvest and following implantation, probing the key maladaptive pathways driving graft failure with the overarching goal of identifying therapeutic targets for biologic intervention to minimize these natural responses to surgical vein graft injury. METHODS: Employing a novel approach to investigating vascular pathologies, we harnessed both single-nuclei RNA-sequencing and spatial transcriptomics analyses to profile the genomic effects of vein grafts after harvest and distension, then compared these findings to vein grafts obtained 24 hours after carotid-carotid vein bypass implantation in a canine model (n=4). RESULTS: Spatial transcriptomic analysis of canine cephalic vein after initial conduit harvest and distention revealed significant enrichment of pathways (P<0.05) involved in the activation of endothelial cells (ECs), fibroblasts, and vascular smooth muscle cells, namely pathways responsible for cellular proliferation and migration and platelet activation across the intimal and medial layers, cytokine signaling within the adventitial layer, and ECM (extracellular matrix) remodeling throughout the vein wall. Subsequent single-nuclei RNA-sequencing analysis supported these findings and further unveiled distinct EC and fibroblast subpopulations with significant upregulation (P<0.05) of markers related to endothelial injury response and cellular activation of ECs, fibroblasts, and vascular smooth muscle cells. Similarly, in vein grafts obtained 24 hours after arterial bypass, there was an increase in myeloid cell, protomyofibroblast, injury response EC, and mesenchymal-transitioning EC subpopulations with a concomitant decrease in homeostatic ECs and fibroblasts. Among these markers were genes previously implicated in vein graft injury, including VCAN, FBN1, and VEGFC, in addition to novel genes of interest, such as GLIS3 and EPHA3. These genes were further noted to be driving the expression of genes implicated in vascular remodeling and graft failure, such as IL-6, TGFBR1, SMAD4, and ADAMTS9. By integrating the spatial transcriptomics and single-nuclei RNA-sequencing data sets, we highlighted the spatial architecture of the vein graft following distension, wherein activated and mesenchymal-transitioning ECs, myeloid cells, and fibroblasts were notably enriched in the intima and media of distended veins. Finally, intercellular communication network analysis unveiled the critical roles of activated ECs, mesenchymal-transitioning ECs, protomyofibroblasts, and vascular smooth muscle cells in upregulating signaling pathways associated with cellular proliferation (MDK [midkine], PDGF [platelet-derived growth factor], VEGF [vascular endothelial growth factor]), transdifferentiation (Notch), migration (ephrin, semaphorin), ECM remodeling (collagen, laminin, fibronectin), and inflammation (thrombospondin), following distension. CONCLUSIONS: Vein conduit harvest and distension elicit a prompt genomic response facilitated by distinct cellular subpopulations heterogeneously distributed throughout the vein wall. This response was found to be further exacerbated following vein graft implantation, resulting in a cascade of maladaptive gene regulatory networks. Together, these results suggest that distension initiates the upregulation of pathological pathways that may ultimately contribute to bypass graft failure and presents potential early targets warranting investigation for targeted therapies. This work highlights the first applications of single-nuclei and spatial transcriptomic analyses to investigate venous pathologies, underscoring the utility of these methodologies and providing a foundation for future investigations.
Assuntos
Análise de Célula Única , Transcriptoma , Animais , Cães , Masculino , Coleta de Tecidos e Órgãos/efeitos adversos , Coleta de Tecidos e Órgãos/métodos , Feminino , Transdução de Sinais , Perfilação da Expressão Gênica/métodosRESUMO
Background: Vein graft failure (VGF) following cardiovascular bypass surgery results in significant patient morbidity and cost to the healthcare system. Vein graft injury can occur during autogenous vein harvest and preparation, as well as after implantation into the arterial system, leading to the development of intimal hyperplasia, vein graft stenosis, and, ultimately, bypass graft failure. While previous studies have identified maladaptive pathways that occur shortly after implantation, the specific signaling pathways that occur during vein graft preparation are not well defined and may result in a cumulative impact on VGF. We, therefore, aimed to elucidate the response of the vein conduit wall during harvest and following implantation, probing the key maladaptive pathways driving graft failure with the overarching goal of identifying therapeutic targets for biologic intervention to minimize these natural responses to surgical vein graft injury. Methods: Employing a novel approach to investigating vascular pathologies, we harnessed both single-nuclei RNA-sequencing (snRNA-seq) and spatial transcriptomics (ST) analyses to profile the genomic effects of vein grafts after harvest and distension, then compared these findings to vein grafts obtained 24 hours after carotid-cartoid vein bypass implantation in a canine model (n=4). Results: Spatial transcriptomic analysis of canine cephalic vein after initial conduit harvest and distention revealed significant enrichment of pathways (P < 0.05) involved in the activation of endothelial cells (ECs), fibroblasts (FBs), and vascular smooth muscle cells (VSMCs), namely pathways responsible for cellular proliferation and migration and platelet activation across the intimal and medial layers, cytokine signaling within the adventitial layer, and extracellular matrix (ECM) remodeling throughout the vein wall. Subsequent snRNA-seq analysis supported these findings and further unveiled distinct EC and FB subpopulations with significant upregulation (P < 0.00001) of markers related to endothelial injury response and cellular activation of ECs, FBs, and VSMCs. Similarly, in vein grafts obtained 24 hours after arterial bypass, there was an increase in myeloid cell, protomyofibroblast, injury-response EC, and mesenchymal-transitioning EC subpopulations with a concomitant decrease in homeostatic ECs and fibroblasts. Among these markers were genes previously implicated in vein graft injury, including VCAN (versican), FBN1 (fibrillin-1), and VEGFC (vascular endothelial growth factor C), in addition to novel genes of interest such as GLIS3 (GLIS family zinc finger 3) and EPHA3 (ephrin-A3). These genes were further noted to be driving the expression of genes implicated in vascular remodeling and graft failure, such as IL-6, TGFBR1, SMAD4, and ADAMTS9. By integrating the ST and snRNA-seq datasets, we highlighted the spatial architecture of the vein graft following distension, wherein activated and mesenchymal-transitioning ECs, myeloid cells, and FBs were notably enriched in the intima and media of distended veins. Lastly, intercellular communication network analysis unveiled the critical roles of activated ECs, mesenchymal transitioning ECs, protomyofibroblasts, and VSMCs in upregulating signaling pathways associated with cellular proliferation (MDK, PDGF, VEGF), transdifferentiation (Notch), migration (ephrin, semaphorin), ECM remodeling (collagen, laminin, fibronectin), and inflammation (thrombospondin), following distension. Conclusions: Vein conduit harvest and distension elicit a prompt genomic response facilitated by distinct cellular subpopulations heterogeneously distributed throughout the vein wall. This response was found to be further exacerbated following vein graft implantation, resulting in a cascade of maladaptive gene regulatory networks. Together, these results suggest that distension initiates the upregulation of pathological pathways that may ultimately contribute to bypass graft failure and presents potential early targets warranting investigation for targeted therapies. This work highlights the first applications of single-nuclei and spatial transcriptomic analyses to investigate venous pathologies, underscoring the utility of these methodologies and providing a foundation for future investigations.
RESUMO
Bypass graft failure occurs in 20%-50% of coronary and lower extremity bypasses within the first-year due to intimal hyperplasia (IH). TSP-2 is a key regulatory protein that has been implicated in the development of IH following vessel injury. In this study, we developed a biodegradable CLICK-chemistry gelatin-based hydrogel to achieve sustained perivascular delivery of TSP-2 siRNA to rat carotid arteries following endothelial denudation injury. At 21 days, perivascular application of TSP-2 siRNA embedded hydrogels significantly downregulated TSP-2 gene expression, cellular proliferation, as well as other associated mediators of IH including MMP-9 and VEGF-R2, ultimately resulting in a significant decrease in IH. Our data illustrates the ability of perivascular CLICK-gelatin delivery of TSP-2 siRNA to mitigate IH following arterial injury.
Assuntos
Gelatina , Lesões do Sistema Vascular , Ratos , Animais , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Hiperplasia , Trombospondinas/genética , Proliferação de CélulasRESUMO
BACKGROUND: Cardiovascular disease remains a major health care challenge. The knowledge about the underlying mechanisms of the respective vascular disease etiologies has greatly expanded over the last decades. This includes the contribution of microRNAs, endogenous non-coding RNA molecules, known to vastly influence gene expression. In addition, short interference RNA has been established as a mechanism to temporarily affect gene expression. This review discusses challenges relating to the design of a RNA interference therapy strategy for the modulation of vascular disease. Despite advances in medical and surgical therapies, atherosclerosis (ATH), aortic aneurysms (AA) are still associated with high morbidity and mortality. In addition, intimal hyperplasia (IH) remains a leading cause of late vein and prosthetic bypass graft failure. Pathomechanisms of all three entities include activation of endothelial cells (EC) and dedifferentiation of vascular smooth muscle cells (VSMC). RNA interference represents a promising technology that may be utilized to silence genes contributing to ATH, AA or IH. Successful RNAi delivery to the vessel wall faces multiple obstacles. These include the challenge of cell specific, targeted delivery of RNAi, anatomical barriers such as basal membrane, elastic laminae in arterial walls, multiple layers of VSMC, as well as adventitial tissues. Another major decision point is the route of delivery and potential methods of transfection. A plethora of transfection reagents and adjuncts have been described with varying efficacies and side effects. Timing and duration of RNAi therapy as well as target gene choice are further relevant aspects that need to be addressed in a temporo-spatial fashion. CONCLUSIONS: While multiple preclinical studies reported encouraging results of RNAi delivery to the vascular wall, it remains to be seen if a single target can be sufficient to the achieve clinically desirable changes in the injured vascular wall in humans. It might be necessary to achieve simultaneous and/or sequential silencing of multiple, synergistically acting target genes. Some advances in cell specific RNAi delivery have been made, but a reliable vascular cell specific transfection strategy is still missing. Also, off-target effects of RNAi and unwanted effects of transfection agents on gene expression are challenges to be addressed. Close collaborative efforts between clinicians, geneticists, biologists, and chemical and medical engineers will be needed to provide tailored therapeutics for the various types of vascular diseases.
Assuntos
Artérias/anatomia & histologia , Artérias/fisiologia , Terapêutica com RNAi , Veias/anatomia & histologia , Veias/fisiologia , Animais , Humanos , MicroRNAs/metabolismo , RNA Interferente Pequeno/administração & dosagem , Pesquisa Translacional BiomédicaRESUMO
BACKGROUND: Life expectancy is short for patients with critical limb ischemia (CLI), many of whom may fear amputation more than death. In light of the reduced life expectancy of these patients, the traditional 5-year freedom from amputation (FFA) statistic may not accurately address their concern. We developed a more relevant patient-centered calculation of major amputation risk during a patient's remaining lifetime to better answer the question, Will I ever lose my leg? METHODS: We identified all limbs undergoing first-time intervention for CLI in a large institutional database from 2005 to 2013. We calculated the traditional metrics of amputation-free survival (AFS, for which failure is death or amputation) and FFA (for which failure is amputation but deaths are censored and removed from further analysis). In addition, we propose a new term, lifelong limb preservation (LLP). LLP defines amputation as failure, but deaths are not censored and therefore reflect that LLP has been achieved. All deaths before 30 days were considered a failure in all three metrics, reflecting the risk of surgery. RESULTS: There were 1006 limbs identified as having first-time intervention for CLI (22% rest pain, 45% ulcer, 27% gangrene; 46% treated by angioplasty with or without stenting, 54% bypass). Using life-table analysis, 7-year AFS was 14% (561 events), FFA was 78% (123 events), and LLP was 86% (123 events). LLP was similar between patients undergoing angioplasty with or without stenting and bypass (7-year rates, 86% and 85%, respectively). For patients undergoing intervention for rest pain, 7-year rates were 14% for AFS, 84% for FFA, and 92% for LLP. For those undergoing treatment for ulcer, 7-year rates were 14% for AFS, 77% for FFA, and 86% for LLP. Finally, in those with gangrene, rates were 10% for AFS, 67% for FFA, and 79% for LLP. Using LLP, patients presenting with an ulcer can be told that although we cannot guarantee how long they will live, with revascularization there is approximately an 86% chance they will not lose the leg. CONCLUSIONS: These results show that the durability of our limb preservation efforts often exceeds the life expectancy of our patients. Using LLP as an outcomes assessment provides a more accurate and patient-centered answer to the question, If I have this procedure, will I ever lose my leg?
Assuntos
Angioplastia , Técnicas de Apoio para a Decisão , Isquemia/terapia , Salvamento de Membro , Extremidade Inferior/irrigação sanguínea , Doença Arterial Periférica/terapia , Idoso , Amputação Cirúrgica , Angioplastia/efeitos adversos , Angioplastia/instrumentação , Angioplastia/mortalidade , Comunicação , Estado Terminal , Intervalo Livre de Doença , Feminino , Humanos , Isquemia/diagnóstico , Isquemia/mortalidade , Isquemia/psicologia , Estimativa de Kaplan-Meier , Expectativa de Vida , Tábuas de Vida , Masculino , Doença Arterial Periférica/diagnóstico , Doença Arterial Periférica/mortalidade , Doença Arterial Periférica/psicologia , Relações Médico-Paciente , Estudos Retrospectivos , Medição de Risco , Fatores de Risco , Stents , Fatores de Tempo , Resultado do TratamentoRESUMO
In an effort to inhibit the response to vascular injury that leads to intimal hyperplasia, this study investigated the in vivo efficacy of intraluminal delivery of thrombospondin-2 (TSP-2) small interfering RNA (siRNA). Common carotid artery (CCA) balloon angioplasty injury was performed in rats. Immediately after denudation, CCA was transfected intraluminally (15 min) with one of the following: polyethylenimine (PEI)+TSP-2 siRNA, saline, PEI only, or PEI+control siRNA. CCA was analyzed at 24 h or 21 d by using quantitative real-time PCR and immunohistochemistry. TSP-2 gene and protein expression were significantly up-regulated after endothelial denudation at 24 h and 21 d compared with contralateral untreated, nondenuded CCA. Treatment with PEI+TSP-2 siRNA significantly suppressed TSP-2 gene expression (3.1-fold) at 24 h and TSP-2 protein expression, cell proliferation, and collagen deposition up to 21 d. These changes could be attributed to changes in TGF-ß and matrix metalloproteinase-9, the downstream effectors of TSP-2. TSP-2 knockdown induced anti-inflammatory M2 macrophage polarization at 21 d; however, it did not significantly affect intima/media ratios. In summary, these data demonstrate effective siRNA transfection of the injured arterial wall and provide a clinically effective and translationally applicable therapeutic strategy that involves nonviral siRNA delivery to ameliorate the response to vascular injury.-Bodewes, T. C. F., Johnson, J. M., Auster, M., Huynh, C., Muralidharan, S., Contreras, M., LoGerfo, F. W., Pradhan-Nabzdyk, L. Intraluminal delivery of thrombospondin-2 small interfering RNA inhibits the vascular response to injury in a rat carotid balloon angioplasty model.
Assuntos
Angioplastia com Balão , Lesões das Artérias Carótidas/metabolismo , Regulação da Expressão Gênica/fisiologia , RNA Interferente Pequeno/administração & dosagem , Trombospondinas/metabolismo , Animais , Proliferação de Células , Colágeno , Macrófagos , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Ratos , Trombospondinas/genética , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
BACKGROUND: RNA interference (RNAi) is a powerful platform utilized to target transcription of specific genes and downregulate the protein product. To achieve effective silencing, RNAi is usually applied to cells or tissue with a transfection reagent to enhance entry into cells. A commonly used control is the same transfection reagent plus a "noncoding RNAi". However, this does not control for the genomic response to the transfection reagent alone or in combination with the noncoding RNAi. These control effects while not directly targeting the gene in question may influence expression of other genes that in turn alter expression of the target. The current study was prompted by our work focused on prevention of vascular bypass graft failure and our experience with gene silencing in human aortic smooth muscle cells (HAoSMCs) where we suspected that off target effects through this mechanism might be substantial. We have used Next Generation Sequencing (NGS) technology and bioinformatics analysis to examine the genomic response of HAoSMCs to the transfection reagent alone (polyethyleneimine (PEI)) or in combination with commercially obtained control small interfering RNA (siRNAs) (Dharmacon and Invitrogen). RESULTS: Compared to untreated cells, global gene expression of HAoSMcs after transfection either with PEI or in combination with control siRNAs displayed significant alterations in gene transcriptome after 24 h. HAoSMCs transfected by PEI alone revealed alterations of 213 genes mainly involved in inflammatory and immune responses. HAoSMCs transfected by PEI complexed with siRNA from either Dharmacon or Invitrogen showed substantial gene variation of 113 and 85 genes respectively. Transfection of cells with only PEI or with PEI and control siRNAs resulted in identification of 20 set of overlapping altered genes. Further, systems biology analysis revealed key master regulators in cells transfected with control siRNAs including the cytokine, Interleukin (IL)-1, transcription factor GATA Binding Protein (GATA)-4 and the methylation enzyme, Enhancer of zeste homolog 2 (EZH-2) a cytokine with an apical role in initiating the inflammatory response. CONCLUSIONS: Significant off-target effects in HAoSMCs transfected with PEI alone or in combination with control siRNAs may lead to misleading conclusions concerning the effectiveness of a targeted siRNA strategy. The lack of structural information about transfection reagents and "non coding" siRNA is a hindrance in the development of siRNA based therapeutics.
Assuntos
Aorta/efeitos dos fármacos , Biologia Computacional , Regulação da Expressão Gênica/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Aorta/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste , Fator de Transcrição GATA4/biossíntese , Regulação da Expressão Gênica/genética , Inativação Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Interleucina-1/biossíntese , Músculo Liso Vascular/metabolismo , Complexo Repressor Polycomb 2/biossíntese , Polietilenoimina/administração & dosagem , RNA Interferente Pequeno/genética , Transfecção/métodosRESUMO
A principal challenge in wound healing is a lack of cell recruitment, cell infiltration, and vascularization, which occurs in the absence of temporal and spatial cues. We hypothesized that a scaffold that expands due to local changes in pH may alter oxygen and nutrient transport and the local cell density, leading to enhanced cell deposition and survival. In this study, we present a pH-responsive scaffold that increases oxygen transport, as confirmed by our finite element model analysis, and cell proliferation relative to a non-responsive scaffold. In vivo, responsive scaffolds induce a pro-healing gene expression profile indicative of enhanced angiogenesis, granulation tissue formation, and tissue remodeling. Scaffolds that stretch in response to their environment may be a hallmark for tissue regeneration.
Assuntos
Metacrilatos/química , Oxigênio/metabolismo , Alicerces Teciduais/química , Cicatrização , Animais , Proliferação de Células , Sobrevivência Celular , Análise de Elementos Finitos , Concentração de Íons de Hidrogênio , Camundongos , Células NIH 3T3 , Porosidade , Ratos Wistar , Engenharia TecidualRESUMO
Diabetic foot ulceration is a major complication of diabetes. Substance P (SP) is involved in wound healing, but its effect in diabetic skin wounds is unclear. We examined the effect of exogenous SP delivery on diabetic mouse and rabbit wounds. We also studied the impact of deficiency in SP or its receptor, neurokinin-1 receptor, on wound healing in mouse models. SP treatment improved wound healing in mice and rabbits, whereas the absence of SP or its receptor impaired wound progression in mice. Moreover, SP bioavailability in diabetic skin was reduced as SP gene expression was decreased, whereas the gene expression and protein levels of the enzyme that degrades SP, neutral endopeptidase, were increased. Diabetes and SP deficiency were associated with absence of an acute inflammatory response important for wound healing progression and instead revealed a persistent inflammation throughout the healing process. SP treatment induced an acute inflammatory response, which enabled the progression to the proliferative phase and modulated macrophage activation toward the M2 phenotype that promotes wound healing. In conclusion, SP treatment reverses the chronic proinflammatory state in diabetic skin and promotes healing of diabetic wounds.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Neuropatias Diabéticas/metabolismo , Inflamação/metabolismo , Macrófagos/metabolismo , Substância P/metabolismo , Substância P/farmacologia , Cicatrização/efeitos dos fármacos , Animais , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Neuropatias Diabéticas/genética , Neuropatias Diabéticas/patologia , Inflamação/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Camundongos , Coelhos , Receptores da Neurocinina-1/genética , Receptores da Neurocinina-1/metabolismo , Pele/efeitos dos fármacos , Pele/metabolismo , Pele/patologia , Substância P/genética , Cicatrização/fisiologiaRESUMO
Intimal hyperplasia (IH) is the leading cause of late vein and prosthetic bypass graft failure. Injury at the time of graft implantation leading to the activation of endothelial cells and dedifferentiation of vascular smooth muscle cells to a synthetic phenotype are known causes of IH. Prior attempts to develop therapy to mitigate these cellular changes to prevent IH and graft failure have failed. Small interfering RNA (siRNA) mediated targeted gene silencing is a promising tool to prevent IH. Several studies have been performed in this direction to target genes that are involved in IH. In this review we discuss siRNA targets that are being investigated for prevention and treatment of IH.
Assuntos
Angioplastia/efeitos adversos , Ponte de Artéria Coronária/efeitos adversos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/uso terapêutico , Túnica Íntima/patologia , Animais , Implante de Prótese Vascular/efeitos adversos , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Inibidores do Crescimento/uso terapêutico , Substâncias de Crescimento/genética , Humanos , Hiperplasia , Mediadores da Inflamação/antagonistas & inibidores , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Peptídeo Hidrolases/genética , Inibidores de Proteases/uso terapêutico , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/genética , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genéticaRESUMO
Atherosclerosis (ATH) and aortic aneurysms (AA) remain challenging chronic diseases that confer high morbidity and mortality despite advances in medical, interventional, and surgical care. RNA interference represents a promising technology that may be utilized to silence genes contributing to ATH and AA. Despite positive results in preclinical and some clinical feasibility studies, challenges such as target/sequence validation, tissue specificity, transfection efficiency, and mitigation of unwanted off-target effects remain to be addressed. In this review the most current targets and some novel approaches in siRNA delivery are being discussed. Due to the plethora of investigated targets, only studies published between 2010 and 2014 were included.
Assuntos
Aneurisma Aórtico/metabolismo , Aterosclerose/metabolismo , RNA Interferente Pequeno/metabolismo , Animais , Aneurisma Aórtico/genética , Aterosclerose/genética , Membrana Celular/metabolismo , Ensaios Clínicos como Assunto , Sistemas de Liberação de Medicamentos , Inativação Gênica , Técnicas Genéticas , Humanos , Fatores Imunológicos , Camundongos , Interferência de RNARESUMO
An excessive tissue response to prosthetic arterial graft material leads to intimal hyperplasia (IH), the leading cause of late graft failure. Seroma and abnormal capsule formation may also occur after prosthetic material implantation. The matricellular protein Thrombospondin-2 (TSP-2) has shown to be upregulated in response to biomaterial implantation. This study evaluates the uptake and release of small interfering RNA (siRNA) from unmodified and surface functionalized electrospun PET graft materials. ePET graft materials were synthesized using electrospinning technology. Subsets of the ePET materials were then chemically modified to create surface functional groups. Unmodified and surface-modified ePET grafts were dip-coated in siRNAs alone or siRNAs complexed with transfection reagents polyethyleneimine (PEI) or Lipofectamine RNAiMax. Further, control and TSP-2 siRNA-PEI complex treated ePET samples were placed onto a confluent layer of human aortic smooth muscle cells (AoSMCs). Complexation of all siRNAs with PEI led to a significant increase in adsorption to unmodified ePET. TSP-2 siRNA-PEI released from unmodified-ePET silenced TSP-2 in AoSMC. Regardless of the siRNA-PEI complex evaluated, AoSMC migrated into the ePET. siRNA-PEI complexes delivered to AoSMC from dip-coated ePET can result in gene knockdown. This methodology for siRNA delivery may improve the tissue response to vascular and other prosthetics.
Assuntos
Aorta/citologia , Materiais Revestidos Biocompatíveis/farmacologia , Inativação Gênica/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Polietilenoglicóis/farmacologia , Polietilenoimina/farmacologia , RNA Interferente Pequeno/metabolismo , Adsorção , Adesão Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/ultraestrutura , Polietilenotereftalatos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , TransfecçãoRESUMO
OBJECTIVE: The present study is designed to understand the contribution of peripheral vascular disease and peripheral neuropathy to the wound-healing impairment associated with diabetes. Using a rabbit model of diabetic neuroischemic wound healing, we investigated rate of healing, leukocyte infiltration, and expression of cytokines, interleukin-8 and interleukin-6, and neuropeptides, substance P, and neuropeptide Y. METHODS: Diabetes was induced in New Zealand White rabbits by administering alloxan while control rabbits received saline. Ten days later, animals in both groups underwent surgery. One ear served as a sham, and the other was made ischemic (ligation of central+rostral arteries) or neuroischemic (ischemia+ resection of central+rostral nerves). Four 6-mm punch biopsy wounds were created in both ears and wound healing was followed for 10 days using computerized planimetry. RESULTS: Nondiabetic sham and ischemic wounds healed significantly more rapidly than diabetic sham and ischemic wounds. Healing was slowest in neuroischemic wounds, irrespective of diabetic status. A high M1/M2 macrophage ratio and a high proinflammatory cytokine expression, both indicators of chronic proinflammatory state, and low neuropeptide expression were seen in preinjury diabetic skin. Postinjury, in diabetic wounds, the M1/M2 ratio remained high, the reactive increase in cytokine expression was low, and neuropeptide expression was further decreased in neuroischemic wounds. CONCLUSIONS: This rabbit model illustrates how a combination of a high M1/M2 ratio, a failure to mount postinjury cytokine response as well as a diminished neuropeptide expression, contribute to wound-healing impairment in diabetes. The addition of neuropathy to ischemia leads to equivalently severe impaired wound-healing irrespective of diabetes status, suggesting that in the presence of ischemia, loss of neuropeptide function contributes to the impaired healing associated with diabetes.
Assuntos
Citocinas/metabolismo , Diabetes Mellitus Experimental/complicações , Angiopatias Diabéticas/etiologia , Neuropatias Diabéticas/etiologia , Mediadores da Inflamação/metabolismo , Isquemia/etiologia , Neuropeptídeos/metabolismo , Úlcera Cutânea/etiologia , Pele , Cicatrização , Animais , Citocinas/genética , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Experimental/metabolismo , Angiopatias Diabéticas/genética , Angiopatias Diabéticas/imunologia , Angiopatias Diabéticas/metabolismo , Angiopatias Diabéticas/patologia , Neuropatias Diabéticas/genética , Neuropatias Diabéticas/imunologia , Neuropatias Diabéticas/metabolismo , Neuropatias Diabéticas/patologia , Regulação para Baixo , Isquemia/genética , Isquemia/imunologia , Isquemia/metabolismo , Isquemia/patologia , Macrófagos/imunologia , Neuropeptídeos/genética , Coelhos , Pele/imunologia , Pele/metabolismo , Pele/patologia , Úlcera Cutânea/genética , Úlcera Cutânea/imunologia , Úlcera Cutânea/metabolismo , Úlcera Cutânea/patologia , Fatores de Tempo , Regulação para CimaRESUMO
BACKGROUND: RNAi technology is a promising tool for gene therapy of vascular disease. However, the biological heterogeneity between endothelial (EC) and vascular smooth muscle cells (SMC) and within different vascular beds make them differentially susceptible to siRNA. This is further complicated by the task of choosing the right transfection reagent that leads to consistent gene silencing across all cell types with minimal toxicity. The goal of this study was to investigate the intrinsic RNAi susceptibility of primary human aortic and coronary artery endothelial and vascular smooth muscle cells (AoEC, CoEC, AoSMC and CoSMC) using adherent cell cytometry. METHODS: Cells were seeded at a density of 5000cells/well of a 96well plate. Twenty four hours later cells were transfected with either non-targeting unlabeled control siRNA (50nM), or non-targeting red fluorescence labeled siRNA (siGLO Red, 5 or 50nM) using no transfection reagent, HiPerFect or Lipofectamine RNAiMAX. Hoechst nuclei stain was used to label cells for counting. For data analysis an adherent cell cytometer, Celigo was used. RESULTS: Red fluorescence counts were normalized to the cell count. EC displayed a higher susceptibility towards siRNA delivery than SMC from the corresponding artery. CoSMC were more susceptible than AoSMC. In all cell types RNAiMAX was more potent compared to HiPerFect or no transfection reagent. However, after 24h, RNAiMAX led to a significant cell loss in both AoEC and CoEC. None of the other transfection conditions led to a significant cell loss. CONCLUSION: This study confirms our prior observation that EC are more susceptible to siRNA than SMC based on intracellular siRNA delivery. RNAiMax treatment led to significant cell loss in AoEC and CoEC, but not in the SMC populations. Additionally, this study is the first to demonstrate that coronary SMC are more susceptible to siRNA than aortic SMC.
Assuntos
Aorta/citologia , Vasos Coronários/citologia , Endotélio Vascular/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transfecção , Adesão Celular , Contagem de Células , Citometria de Fluxo , Fluorescência , Humanos , Músculo Liso Vascular/citologia , RNA Interferente Pequeno/genéticaRESUMO
Vein graft failure occurs between 1 and 6 months after implantation due to obstructive intimal hyperplasia, related in part to implantation injury. The cell-specific and temporal response of the transcriptome to vein graft implantation injury was determined by transcriptional profiling of laser capture microdissected endothelial cells (EC) and medial smooth muscle cells (SMC) from canine vein grafts, 2 hours (H) to 30 days (D) following surgery. Our results demonstrate a robust genomic response beginning at 2 H, peaking at 12-24 H, declining by 7 D, and resolving by 30 D. Gene ontology and pathway analyses of differentially expressed genes indicated that implantation injury affects inflammatory and immune responses, apoptosis, mitosis, and extracellular matrix reorganization in both cell types. Through backpropagation an integrated network was built, starting with genes differentially expressed at 30 D, followed by adding upstream interactive genes from each prior time-point. This identified significant enrichment of IL-6, IL-8, NF-κB, dendritic cell maturation, glucocorticoid receptor, and Triggering Receptor Expressed on Myeloid Cells (TREM-1) signaling, as well as PPARα activation pathways in graft EC and SMC. Interactive network-based analyses identified IL-6, IL-8, IL-1α, and Insulin Receptor (INSR) as focus hub genes within these pathways. Real-time PCR was used for the validation of two of these genes: IL-6 and IL-8, in addition to Collagen 11A1 (COL11A1), a cornerstone of the backpropagation. In conclusion, these results establish causality relationships clarifying the pathogenesis of vein graft implantation injury, and identifying novel targets for its prevention.
Assuntos
Transcriptoma , Veias/transplante , Animais , Cães , Controle de QualidadeRESUMO
OBJECTIVE: To investigate the expression of neuropeptides and their receptors that play a role in cardiac homeostasis in the right atrium of nondiabetic and diabetic patients undergoing coronary artery bypass graft surgery. BACKGROUND: The cardioactive neuropeptides and their receptors investigated in this study were Neuropeptide Y (NPY), and its receptors, NPY Receptor1 (NPY1R), NPY Receptor2 (NPY2R), NPY Receptor5 (NPY5R) and Substance P (SP) and its receptor, Neurokinin1R (NK1R). METHODS: The gene and protein expression of NPY, NPY1R, NPY2R, NPY5R, SP and NK1R from the atrial tissue of 10 nondiabetic and diabetic patients undergoing coronary artery bypass grafting (CABG) was assessed by Q-RTPCR, immunohistochemistry, Western blot, and ELISA. RESULTS: Gene expression of NPY2R, NPY5R, preproTachykinin A (SP gene), and NK1R and their respective protein expression were significantly reduced whereas that of NPY and NPY1R were unchanged in the right atrium of diabetic patients compared to nondiabetic patients. CONCLUSIONS: These results demonstrate that the expression of neuropeptides and their receptors in the diabetic heart is significantly impaired, and may be the link between neuropathy and cardiac complications. Further studies are warranted to delineate pathophysiologic mechanisms associated with dysregulation of the cardiac neuropeptide system and the relationship to cardiac complications in diabetes.
Assuntos
Diabetes Mellitus/metabolismo , Átrios do Coração/metabolismo , Neuropeptídeo Y/metabolismo , Receptores da Neurocinina-1/metabolismo , Receptores de Neuropeptídeo Y/metabolismo , Substância P/metabolismo , Idoso , Diabetes Mellitus/genética , Regulação da Expressão Gênica , Átrios do Coração/patologia , Humanos , Pessoa de Meia-Idade , Receptores da Neurocinina-1/genética , Receptores de Neuropeptídeo Y/genéticaRESUMO
Cardiovascular autonomic neuropathy (CAN), in which patients present with damage of autonomic nerve fibres, is one of the most common complications of diabetes. CAN leads to abnormalities in heart rate and vascular dynamics, which are features of diabetic heart failure. Dysregulated neurohormonal activation, an outcome of diabetic neuropathy, has a significant pathophysiological role in diabetes-associated cardiovascular disease. Key players in neurohormonal activation include cardioprotective neuropeptides and their receptors, such as substance P (SP), neuropeptide Y (NPY), calcitonin-gene-related peptide (CGRP), atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP). These neuropeptides are released from the peripheral or autonomic nervous system and have vasoactive properties. They are further implicated in cardiomyocyte hypertrophy, calcium homeostasis, ischaemia-induced angiogenesis, protein kinase C signalling and the renin-angiotensin-aldosterone system. Therefore, dysregulation of the expression of neuropeptides or activation of the neuropeptide signalling pathways can negatively affect cardiac homeostasis. Targeting neuropeptides and their signalling pathways might thus serve as new therapeutic interventions in the treatment of heart failure associated with diabetes. This review discusses how neuropeptide dysregulation in diabetes might affect cardiac functions that contribute to the development of heart failure.