RESUMO
Retinal capillary degeneration is a clinical hallmark of the early stages of diabetic retinopathy (DR). Our recent studies have revealed that diabetes-induced retinal capillary stiffening plays a crucial and previously unrecognized causal role in inflammation-mediated degeneration of retinal capillaries. The increase in retinal capillary stiffness results from the overexpression of lysyl oxidase, an enzyme that crosslinks and stiffens the subendothelial matrix. Since tackling DR at the early stage is expected to prevent or slow down DR progression and associated vision loss, subendothelial matrix, and capillary stiffness represent relevant and novel therapeutic targets for early DR management. Further, direct measurement of retinal capillary stiffness can serve as a crucial preclinical validation step for the development of new imaging techniques for non-invasive assessment of retinal capillary stiffness in animal and human subjects. With this view in mind, we here provide a detailed protocol for the isolation and stiffness measurement of mouse retinal capillaries and subendothelial matrix using atomic force microscopy.
Assuntos
Capilares , Microscopia de Força Atômica , Vasos Retinianos , Camundongos , Microscopia de Força Atômica/métodos , Animais , Vasos Retinianos/diagnóstico por imagem , Rigidez Vascular/fisiologiaRESUMO
Retinal capillary degeneration is a clinical hallmark of the early stages of diabetic retinopathy (DR). Our recent studies have revealed that diabetes-induced increase in retinal capillary stiffness plays a crucial and previously unrecognized causal role in inflammation-mediated degeneration of retinal capillaries. Retinal capillary stiffening results from overexpression of lysyl oxidase, an enzyme that crosslinks and stiffens the subendothelial matrix. Since tackling DR at the early stage is expected to prevent or slow down DR progression and associated vision loss, subendothelial matrix and capillary stiffness represent relevant and novel therapeutic targets for early DR management. Further, direct measurement of retinal capillary stiffness can serve as a crucial preclinical validation step for the development of new imaging techniques for non-invasive assessment of retinal capillary stiffness in animal and human subjects. With this view in mind, we here provide a detailed protocol for the isolation and stiffness measurement of mouse retinal capillaries and retinal subendothelial matrix using atomic force microscopy.
RESUMO
Vascular inflammation is known to cause degeneration of retinal capillaries in early diabetic retinopathy (DR), a major microvascular complication of diabetes. Past studies investigating these diabetes-induced retinal vascular abnormalities have focused primarily on the role of molecular or biochemical cues. Here we show that retinal vascular inflammation and degeneration in diabetes are also mechanically regulated by the increase in retinal vascular stiffness caused by overexpression of the collagen-cross-linking enzyme lysyl oxidase (LOX). Treatment of diabetic mice with LOX inhibitor ß-aminopropionitrile (BAPN) prevented the increase in retinal capillary stiffness, vascular intracellular adhesion molecule-1 overexpression, and leukostasis. Consistent with these anti-inflammatory effects, BAPN treatment of diabetic mice blocked the upregulation of proapoptotic caspase-3 in retinal vessels, which concomitantly reduced retinal capillary degeneration, pericyte ghost formation, and the diabetes-induced loss of contrast sensitivity in these mice. Finally, our in vitro studies indicate that retinal capillary stiffening is sufficient to increase the adhesiveness and neutrophil elastase-induced death of retinal endothelial cells. By uncovering a link between LOX-dependent capillary stiffening and the development of retinal vascular and functional defects in diabetes, these findings offer a new insight into DR pathogenesis that has important translational potential.
Assuntos
Diabetes Mellitus Experimental , Retinopatia Diabética , Degeneração Retiniana , Camundongos , Animais , Células Endoteliais , Diabetes Mellitus Experimental/complicações , Aminopropionitrilo/farmacologia , Retina/patologia , Retinopatia Diabética/patologia , Inflamação/patologia , Vasos Retinianos/patologia , Camundongos Endogâmicos C57BLRESUMO
Endothelial cell (EC) activation is a crucial determinant of retinal vascular inflammation associated with diabetic retinopathy (DR), a major microvascular complication of diabetes. We previously showed that, similar to abnormal biochemical factors, aberrant mechanical cues in the form of lysyl oxidase (LOX)-dependent subendothelial matrix stiffening also contribute significantly to retinal EC activation in diabetes. Yet, how LOX is itself regulated and precisely how it mechanically controls retinal EC activation in diabetes is poorly understood. Here, we show that high-glucose-induced LOX upregulation in human retinal ECs (HRECs) is mediated by proinflammatory receptor for advanced glycation end products (RAGE). HRECs treated with methylglyoxal (MGO), an active precursor to the advanced glycation end product (AGE) MG-H1, exhibited LOX upregulation that was blocked by a RAGE inhibitor, thus confirming the ability of RAGE to promote LOX expression. Crucially, as a downstream effector of RAGE, LOX was found to mediate both the proinflammatory and matrix remodeling effects of AGE/RAGE, primarily through its ability to crosslink or stiffen matrix. Finally, using decellularized HREC-derived matrices and a mouse model of diabetes, we demonstrate that LOX-dependent matrix stiffening feeds back to enhance RAGE, thereby achieving its autoregulation and proinflammatory effects. Collectively, these findings provide fresh mechanistic insights into the regulation and proinflammatory role of LOX-dependent mechanical cues in diabetes while simultaneously implicating LOX as an alternative (downstream) target to block AGE/RAGE signaling in DR. ARTICLE HIGHLIGHTS: We investigated the regulation and proinflammatory role of retinal endothelial lysyl oxidase (LOX) in diabetes. Findings reveal that LOX is upregulated by advanced glycation end products (AGE) and receptor for AGE (RAGE) and mediates AGE/RAGE-induced retinal endothelial cell activation and subendothelial matrix remodeling. We also show that LOX-dependent subendothelial matrix stiffening feeds back to enhance retinal endothelial RAGE. These findings implicate LOX as a key proinflammatory factor and an alternative (downstream) target to block AGE/RAGE signaling in diabetic retinopathy.
Assuntos
Diabetes Mellitus , Retinopatia Diabética , Camundongos , Animais , Humanos , Retinopatia Diabética/metabolismo , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Proteína-Lisina 6-Oxidase/metabolismo , Retina/metabolismo , Endotélio/metabolismo , Produtos Finais de Glicação Avançada/metabolismo , Diabetes Mellitus/metabolismoRESUMO
Age-related macular degeneration (AMD) is the leading cause of blindness in the aging population. Yet no therapies exist for ~85% of all AMD patients who have the dry form that is marked by degeneration of the retinal pigmented epithelium (RPE) and underlying choroidal vasculature. As the choroidal vessels are crucial for RPE development and maintenance, understanding how they degenerate may lead to effective therapies for dry AMD. One likely causative factor for choroidal vascular loss is the cytolytic membrane attack complex (MAC) of the complement pathway that is abundant on choroidal vessels of humans with early dry AMD. To examine this possibility, we studied the effect of complement activation on choroidal endothelial cells (ECs) isolated from a rhesus monkey model of early AMD that, we report, exhibits MAC deposition and choriocapillaris endothelial loss similar to that seen in human early AMD. Treatment of choroidal ECs from AMD eyes with complement-competent normal human serum caused extensive actin cytoskeletal injury that was significantly less pronounced in choroidal ECs from young normal monkey eyes. We further show that ECs from AMD eyes are significantly stiffer than their younger counterparts and exhibit peripheral actin organization that is distinct from the longitudinal stress fibers in young ECs. Finally, these differences in complement susceptibility and mechanostructural properties were found to be regulated by the differential activity of the small GTPases Rac and Rho, because Rac inhibition in AMD cells led to simultaneous reduction in stiffness and complement susceptibility, while Rho inhibition in young cells exacerbated complement injury. Thus, by identifying cell stiffness and cytoskeletal regulators Rac and Rho as important determinants of complement susceptibility, the current findings offer a new mechanistic insight into choroidal vascular loss in early AMD that warrants further investigation for assessment of translational potential. © 2022 The Pathological Society of Great Britain and Ireland.
Assuntos
Células Endoteliais , Degeneração Macular , Actinas/metabolismo , Idoso , Corioide/metabolismo , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Células Endoteliais/metabolismo , Humanos , Degeneração Macular/patologiaRESUMO
PURPOSE: Age-related macular degeneration (AMD) commonly causes blindness in the elderly. Yet, it is untreatable in the large fraction of all AMD patients that develop the early dry form. Dry AMD is marked by the deposition of membrane attack complex (MAC) on choriocapillaris (CC), which is implicated in CC degeneration and subsequent atrophy of overlying retinal pigment epithelium. Since MAC is also found on the CC of young eyes, here we investigated whether and how aging increases choroidal endothelial susceptibility to MAC injury. METHODS: Monkey chorioretinal endothelial cells (ECs, RF/6A) were cultured to high passages (>P60) to achieve replicative senescence. We treated ECs with complement-competent human serum to promote MAC deposition and injury, which were assessed by flow cytometry and trypan blue exclusion assay, respectively. Stiffness of EC was measured by atomic force microscopy indentation while Rho GTPase activity was quantified by Rho G-LISA assay. RESULTS: Our findings reveal that senescent ECs are significantly stiffer than their normal counterparts, which correlates with higher cytoskeletal Rho activity in these cells and their greater susceptibility to complement (MAC) injury. Importantly, inhibition of Rho activity in senescent ECs significantly reduced cell stiffness and MAC-induced lysis. CONCLUSIONS: By revealing an important role of senescence-associated choroidal EC stiffening in complement injury, these findings implicate CC stiffening as an important determinant of age-related CC atrophy seen in dry AMD. Future studies are needed to validate these findings in appropriate animal models so new therapeutic targets can be identified for treatment of dry AMD.
Assuntos
Senescência Celular/fisiologia , Corioide/efeitos dos fármacos , Complexo de Ataque à Membrana do Sistema Complemento/fisiologia , Proteínas do Sistema Complemento/farmacologia , Células Endoteliais/efeitos dos fármacos , Degeneração Macular/patologia , Degeneração Macular/fisiopatologia , Idoso , Animais , Células Cultivadas , Corioide/citologia , Corioide/fisiologia , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Células Endoteliais/fisiologia , Haplorrinos , Humanos , Masculino , Microscopia de Força Atômica , Retina/citologiaRESUMO
PURPOSE: To redesign a complement-inhibiting peptide with the potential to become a therapeutic for dry and wet age-related macular degeneration (AMD). METHODS: We present a new potent peptide (Peptide 2) of the compstatin family. The peptide is developed by rational design, based on a mechanistic binding hypothesis, and structural and physicochemical properties derived from molecular dynamics (MD) simulation. The inhibitory activity, efficacy, and solubility of Peptide 2 are evaluated using a hemolytic assay, a human RPE cell-based assay, and ultraviolet (UV) absorption properties, respectively, and compared to the respective properties of its parent peptide (Peptide 1). RESULTS: The sequence of Peptide 2 contains an arginine-serine N-terminal extension (a characteristic of parent Peptide 1) and a novel 8-polyethylene glycol (PEG) block C-terminal extension. Peptide 2 has significantly improved aqueous solubility compared to Peptide 1 and comparable complement inhibitory activity. In addition, Peptide 2 is more efficacious in inhibiting complement activation in a cell-based model that mimics the pathobiology of dry AMD. CONCLUSIONS: We have designed a new peptide analog of compstatin that combines N-terminal polar amino acid extensions and C-terminal PEGylation extensions. This peptide demonstrates significantly improved aqueous solubility and complement inhibitory efficacy, compared to the parent peptide. The new peptide overcomes the aggregation limitation for clinical translation of previous compstatin analogs and is a candidate to become a therapeutic for the treatment of AMD.
Assuntos
Proteínas do Sistema Complemento/metabolismo , Degeneração Macular/tratamento farmacológico , Peptídeos/uso terapêutico , Sequência de Aminoácidos , Animais , Linhagem Celular , Hemólise/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Peptídeos/síntese química , Peptídeos/química , Peptídeos/farmacologia , Peptídeos Cíclicos/farmacologia , Peptídeos Cíclicos/uso terapêutico , Coelhos , SolubilidadeRESUMO
Leukocyte-endothelial adhesion is a critical early step in chronic vascular inflammation associated with diabetes, emphysema, and aging. Importantly, these conditions are also marked by abnormal subendothelial matrix crosslinking (stiffness). Yet, whether and how abnormal matrix stiffness contributes to leukocyte-endothelial adhesion remains poorly understood. Using a co-culture of human monocytic cells and human microvascular endothelial cells (ECs) grown on matrices of tunable stiffness, we demonstrate that matrix stiffness exerts biphasic control over monocyte-EC adhesion, with both matrix softening and stiffening eliciting a two-fold increase in this adhesive interaction. This preferential endothelial adhesivity on softer and stiffer matrices was consistent with a significant increase in α-actinin-4-associated endothelial ICAM-1 clustering, a key determinant of monocyte-EC adhesion. Further, the enhanced ICAM-1 clustering on soft and stiff matrices correlated strongly with an increase in Rho activity and ROCK2 expression. Importantly, inhibition of Rho/ROCK activity blocked the effects of abnormal matrix stiffness on ICAM-1 clustering and monocyte-EC adhesion. Thus, these findings implicate matrix stiffness-dependent ICAM-1 clustering as an important regulator of vascular inflammation and provide the rationale for closely examining mechanotransduction pathways as new molecular targets for anti-inflammatory therapy.
Assuntos
Células Endoteliais/citologia , Molécula 1 de Adesão Intercelular/metabolismo , Monócitos/citologia , Quinases Associadas a rho/metabolismo , Resinas Acrílicas/química , Actinina/metabolismo , Adesão Celular , Análise por Conglomerados , Técnicas de Cocultura , Citoesqueleto/metabolismo , Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Inflamação , Leucócitos/citologia , Mecanotransdução Celular , Microcirculação , Pressão , Transdução de Sinais , Células U937RESUMO
Inflammation plays an important role in the pathogenesis of diabetic retinopathy (DR). We have previously reported increased monocyte (Mono) trafficking into the retinas of diabetic animals. In this study, we have examined the effect of activated Monos on retinal endothelial cells (ECs). The U937 MÏ-conditioned medium (CM) significantly decreased the transendothelial resistance of EC monolayers as measured by electric cell-substrate impedance sensing (P= 0.007). The CM was fractioned, and the effective fraction (30-100 kDa) was analyzed by liquid chromatography-mass spectrometry, and cathepsin D (CD) was identified as a major secreted product. Immunoprecipitated CD resulted in decreased resistance in ECs (P= 0.006). The specificity of CD in mediating alterations of the EC barrier was confirmed using small interfering RNA. The decreased resistance correlated with a significantly increased gap between ECs. CD altered the Ras homolog gene family, member A/Rho-associated kinase pathway with increased stress actin filament formation in the EC layer. Increased CD levels were found in the retinas of diabetic mice (3-fold) and serum samples of patients with diabetic macular edema (1.6-fold) measured by Western blot and ELISA. These findings suggest an important role for MÏ-derived CD in altering the blood-retinal barrier and reveal a potential therapeutic target in the treatment of DR.-Monickaraj, F., McGuire, P. G., Nitta, C. F., Ghosh, K., Das, A. Cathepsin D: an MÏ-derived factor mediating increased endothelial cell permeability with implications for alteration of the blood-retinal barrier in diabetic retinopathy.
Assuntos
Barreira Hematorretiniana/metabolismo , Catepsina D/metabolismo , Retinopatia Diabética/metabolismo , Células Endoteliais/metabolismo , Adulto , Idoso , Animais , Western Blotting , Permeabilidade Capilar , Catepsina D/sangue , Catepsina D/genética , Permeabilidade da Membrana Celular , Células Cultivadas , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Retinopatia Diabética/sangue , Ensaio de Imunoadsorção Enzimática , Humanos , Macrófagos/enzimologia , Edema Macular/sangue , Edema Macular/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Microscopia Confocal , Pessoa de Meia-Idade , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células U937 , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismoRESUMO
Endothelial activation is a hallmark of the high-glucose (HG)-induced retinal inflammation associated with diabetic retinopathy (DR). However, precisely how HG induces retinal endothelial activation is not fully understood. We hypothesized that HG-induced up-regulation of lysyl oxidase (LOX), a collagen-cross-linking enzyme, in retinal capillary endothelial cells (ECs) enhances subendothelial basement membrane (BM) stiffness, which, in turn, promotes retinal EC activation. Diabetic C57BL/6 mice exhibiting a 70 and 50% increase in retinal intercellular adhesion molecule (ICAM)-1 expression and leukocyte accumulation, respectively, demonstrated a 2-fold increase in the levels of BM collagen IV and LOX, key determinants of capillary BM stiffness. Using atomic force microscopy, we confirmed that HG significantly enhances LOX-dependent subendothelial matrix stiffness in vitro, which correlated with an â¼2.5-fold increase in endothelial ICAM-1 expression, a 4-fold greater monocyte-EC adhesion, and an â¼2-fold alteration in endothelial NO (decrease) and NF-κB activation (increase). Inhibition of LOX-dependent subendothelial matrix stiffening alone suppressed HG-induced retinal EC activation. Finally, using synthetic matrices of tunable stiffness, we demonstrated that subendothelial matrix stiffening is necessary and sufficient to promote EC activation. These findings implicate BM stiffening as a critical determinant of HG-induced retinal EC activation and provide a rationale for examining BM stiffness and underlying mechanotransduction pathways as therapeutic targets for diabetic retinopathy.
Assuntos
Membrana Basal/patologia , Diabetes Mellitus Experimental/complicações , Retinopatia Diabética/induzido quimicamente , Endotélio/patologia , Retina/patologia , Animais , Linhagem Celular , Retinopatia Diabética/metabolismo , Retinopatia Diabética/patologia , Células Endoteliais/metabolismo , Regulação da Expressão Gênica/fisiologia , Haplorrinos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Monócitos , Proteína-Lisina 6-Oxidase/antagonistas & inibidores , Proteína-Lisina 6-Oxidase/genética , Proteína-Lisina 6-Oxidase/metabolismoRESUMO
Nitroglycerin (NTG) markedly enhances nitric oxide (NO) bioavailability. However, its ability to mimic the anti-inflammatory properties of NO remains unknown. Here, we examined whether NTG can suppress endothelial cell (EC) activation during inflammation and developed NTG nanoformulation to simultaneously amplify its anti-inflammatory effects and ameliorate adverse effects associated with high-dose NTG administration. Our findings reveal that NTG significantly inhibits human U937 cell adhesion to NO-deficient human microvascular ECs in vitro through an increase in endothelial NO and decrease in endothelial ICAM-1 clustering, as determined by NO analyzer, microfluorimetry, and immunofluorescence staining. Nanoliposomal NTG (NTG-NL) was formulated by encapsulating NTG within unilamellar lipid vesicles (DPhPC, POPC, Cholesterol, DHPE-Texas Red at molar ratio of 6:2:2:0.2) that were ~155 nm in diameter and readily uptaken by ECs, as determined by dynamic light scattering and quantitative fluorescence microscopy, respectively. More importantly, NTG-NL produced a 70-fold increase in NTG therapeutic efficacy when compared with free NTG while preventing excessive mitochondrial superoxide production associated with high NTG doses. Thus, these findings, which are the first to reveal the superior therapeutic effects of an NTG nanoformulation, provide the rationale for their detailed investigation for potentially superior vascular normalization therapies.
Assuntos
Anti-Inflamatórios/farmacologia , Células Endoteliais/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Nitroglicerina/farmacologia , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/química , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Relação Dose-Resposta a Droga , Células Endoteliais/metabolismo , Feminino , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Lipossomos , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Nanopartículas/química , Nanopartículas/ultraestrutura , Óxido Nítrico/metabolismo , Nitroglicerina/administração & dosagem , Nitroglicerina/química , Gravidez , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/fisiologia , Ovinos , Superóxidos/metabolismo , Células U937 , Vasodilatação/efeitos dos fármacosRESUMO
Angiogenesis is the formation of new blood vessels from a pre-existing vascular bed. It is a multi-step process beginning with enzymatic degradation of the capillary basement membrane, followed by endothelial cell (EC) proliferation, migration, tube formation, assembly of a new basement membrane, and pericyte stabilization. Aberrant angiogenesis plays a major role in the pathogenesis of many diseases. The regulation of this complex process is an important therapeutic target. Success in this pursuit, however, requires the development of in vivo angiogenesis models that provide a reliable and facile platform for mechanistic studies of angiogenic regulation as well as drug development and testing (Carmeliet and Jain, 2011). Postnatal development of mouse retinal vasculature offers a unique and powerful in vivo angiogenesis model because, unlike other species, mice undergo extensive angiogenesis-dependent maturation of their retinal vessels after birth. As such, this model is also very useful for the mechanistic study of embryonic vascularization (Stahl et al., 2010; Adini et al., 2003). This protocol describes the steps involved in the whole mount processing of mouse eyes for visualization of the retinal vasculature.
RESUMO
PURPOSE: Environmental tobacco smoke (ETS) is widely regarded as a major modifiable risk factor for age-related macular degeneration (AMD). Yet, precisely how it exerts its pathologic effects is poorly understood. Since early-stage AMD is characterized by choroidal capillary loss, this study examined the effect of sidestream smoke (SS), the major component of ETS, on the viability of choroidal endothelial cells (EC), with an emphasis on the role of aberrant cell and basement membrane (BM) architecture in mediating SS-induced response. METHODS: Chorioretinal ECs (RF/6A) were treated with SS, and cell viability and architecture were analyzed by colorimetric assay and actin cytoskeletal organization, respectively. The structure of RF/6A EC-secreted BM was examined by immunofluorescence for collagen IV and immunoblotting for lysyl oxidase (LOX), a collagen-crosslinking enzyme. Finally, fresh RF/6A ECs were cultured on decellularized SS-treated BM to evaluate its active role in EC dysfunction. RESULTS: The RF/6A EC viability decreased progressively with increasing SS dose, which correlated strongly with a significant decline in actin cytoskeleton-dependent EC spreading. Sidestream smoke also caused marked disruption of the RF/6A EC-secreted BM that was accompanied by suppression of LOX expression. Further, fresh, non-SS-treated RF/6A ECs exhibited a significant loss in viability and actin cytoskeletal organization when cultured on SS-treated corrupt BM. CONCLUSIONS: These findings indicate that aberrant physical cues in the form of EC and BM architecture likely have an important role in choriocapillaris dysfunction seen in SS-associated early AMD and implicate choroidal BM as a potential target for AMD management strategies.
Assuntos
Membrana Basal/efeitos dos fármacos , Corioide/citologia , Células Endoteliais/efeitos dos fármacos , Retina/citologia , Fumaça/efeitos adversos , Actinas/ultraestrutura , Análise de Variância , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Corioide/efeitos dos fármacos , Doenças da Coroide/induzido quimicamente , Doenças da Coroide/patologia , Citoesqueleto/ultraestrutura , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Humanos , Proteína-Lisina 6-Oxidase/metabolismo , Poluição por Fumaça de Tabaco/efeitos adversosRESUMO
Studies have established that pigmentation can provide strong, protective effects against certain human diseases. For example, angiogenesis-dependent diseases such as wet age-related macular degeneration and infantile hemangioma are more common in light-skinned individuals of mixed European descent than in African-Americans. Here we found that melanocytes from light-skinned humans and albino mice secrete high levels of fibromodulin (FMOD), which we determined to be a potent angiogenic factor. FMOD treatment stimulated angiogenesis in numerous in vivo systems, including laser-induced choroidal neovascularization, growth factor-induced corneal neovascularization, wound healing, and Matrigel plug assays. Additionally, FMOD enhanced vascular sprouting during normal retinal development. Deletion of Fmod in albino mice resulted in a marked reduction in the amount of neovascularization induced by retinal vein occlusion, corneal growth factor pellets, and Matrigel plugs. Our data implicate the melanocyte-secreted factor FMOD as a key regulator of angiogenesis and suggest an underlying mechanism for epidemiological differences between light-skinned individuals of mixed European descent and African-Americans. Furthermore, inhibition of FMOD in humans has potential as a therapeutic strategy for treating angiogenesis-dependent diseases.
Assuntos
Proteínas da Matriz Extracelular/metabolismo , Melanócitos/metabolismo , Neovascularização Fisiológica , Proteoglicanas/metabolismo , Animais , Movimento Celular , Proliferação de Células , Células Cultivadas , Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Fibromodulina , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Pigmentação da Pele , Fator de Crescimento Transformador beta1/metabolismoRESUMO
En masse cell migration is more relevant compared with single-cell migration in physiological processes of tissue formation, such as embryogenesis, morphogenesis, and wound healing. In these situations, cells are influenced by the proximity of other cells including interactions facilitated by substrate mechanics. Here, we found that when fibroblasts migrated en masse over a hydrogel, they established a well-defined deformation field by traction forces and migrated along a trajectory defined by field gradients. The mechanics of the hydrogel determined the magnitude of the gradient. For materials stiff enough to withstand deformation related to cellular traction forces, such patterns did not form. Furthermore, migration patterns functioned poorly on very soft matrices where only a minimal traction gradient could be established. The largest degree of alignment and migration velocity occurred on the gels with the largest gradients. Granulation tissue formation in punch wounds of juvenile pigs was correlated strongly with the modulus of the implanted gel, in agreement with in vitro en masse cell migration studies. These findings provide basic insight into the biomechanical influences on fibroblast movement in early wounds and relevant design criteria for the development of tissue-engineered constructs that aim to stimulate en masse cell recruitment for rapid wound healing.
Assuntos
Movimento Celular/fisiologia , Fibroblastos/citologia , Fibroblastos/fisiologia , Cicatrização/fisiologia , Adulto , Contagem de Células , Matriz Extracelular/fisiologia , Feminino , Tecido de Granulação/fisiologia , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato , Cultura Primária de Células , Sefarose , Engenharia Tecidual/métodosRESUMO
Prominin-1, a pentaspan transmembrane protein, is a unique cell surface marker commonly used to identify stem cells, including endothelial progenitor cells and cancer stem cells. However, recent studies have shown that prominin-1 expression is not restricted to stem cells but also occurs in modified forms in many mature adult human cells. Although prominin-1 has been studied extensively as a stem cell marker, its physiological function of the protein has not been elucidated. We investigated prominin-1 function in two cell lines, primary human endothelial cells and B16-F10 melanoma cells, both of which express high levels of prominin-1. We found that prominin-1 directly interacts with the angiogenic and tumor survival factor vascular endothelial growth factor (VEGF) in both the primary endothelial cells and the melanoma cells. Knocking down prominin-1 in the endothelial cells disrupted capillary formation in vitro and decreased angiogenesis in vivo. Similarly, tumors derived from prominin-1 knockdown melanoma cells had a reduced growth rate in vivo. Further, melanoma cells with knocked down prominin-1 had diminished ability to interact with VEGF, which was associated with decreased bcl-2 protein levels and increased apoptosis. In vitro studies with soluble prominin-1 showed that it stabilized dimer formation of VEGF164, but not VEGF121. Taken together, our findings support the notion that prominin-1 plays an active role in cell growth through its ability to interact and potentiate the anti-apoptotic and pro-angiogenic activities of VEGF. Additionally, prominin-1 promotes tumor growth by supporting angiogenesis and inhibiting tumor cell apoptosis.
Assuntos
Antígenos CD/metabolismo , Glicoproteínas/metabolismo , Peptídeos/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Antígeno AC133 , Apoptose , Western Blotting , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Humanos , Imuno-Histoquímica , Melanoma/patologia , Ligação Proteica , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Obstruction of critical blood vessels due to thrombosis or embolism is a leading cause of death worldwide. Here, we describe a biomimetic strategy that uses high shear stress caused by vascular narrowing as a targeting mechanism--in the same way platelets do--to deliver drugs to obstructed blood vessels. Microscale aggregates of nanoparticles were fabricated to break up into nanoscale components when exposed to abnormally high fluid shear stress. When coated with tissue plasminogen activator and administered intravenously in mice, these shear-activated nanotherapeutics induce rapid clot dissolution in a mesenteric injury model, restore normal flow dynamics, and increase survival in an otherwise fatal mouse pulmonary embolism model. This biophysical strategy for drug targeting, which lowers required doses and minimizes side effects while maximizing drug efficacy, offers a potential new approach for treatment of life-threatening diseases that result from acute vascular occlusion.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Fibrinolíticos/administração & dosagem , Oclusão Vascular Mesentérica/tratamento farmacológico , Nanopartículas , Embolia Pulmonar/tratamento farmacológico , Trombose/tratamento farmacológico , Ativador de Plasminogênio Tecidual/administração & dosagem , Animais , Materiais Biomiméticos , Circulação Sanguínea , Hemodinâmica , Hemorreologia , Ácido Láctico , Masculino , Artérias Mesentéricas , Camundongos , Camundongos Endogâmicos C57BL , Técnicas Analíticas Microfluídicas , Modelos Anatômicos , Ácido Poliglicólico , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Estresse Mecânico , Trombose/prevenção & controleRESUMO
A cancer nanotherapeutic has been developed that targets the extracellular matrix (ECM)-modifying enzyme lysyl oxidase (LOX) and alters the ECM structure. Poly(d,l-lactide-co-glycolide) nanoparticles (â¼220 nm) coated with a LOX inhibitory antibody bind to ECM and suppress mammary cancer cell growth and invasion in vitro as well as tumor expansion in vivo, with greater efficiency than soluble anti-LOX antibody. This nanomaterials approach opens a new path for treating cancer with higher efficacy and decreased side effects.
Assuntos
Proliferação de Células/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/patologia , Nanocápsulas/administração & dosagem , Proteína-Lisina 6-Oxidase/administração & dosagem , Animais , Linhagem Celular Tumoral , CamundongosRESUMO
Here we report a proof-of-concept for development of pancreatic islet-targeting nanoparticles for immunomodulatory therapy of autoimmune type 1 diabetes. Modified with a unique islet-homing peptide, these polymeric nanomaterials exhibit 3-fold greater binding to islet endothelial cells and a 200-fold greater anti-inflammatory effect through targeted islet endothelial cell delivery of an immunosuppressant drug. Our findings also underscore the need to carefully tailor drug loading and nanoparticle dosage to achieve maximal vascular targeting and immunosuppression.
Assuntos
Imunossupressores/administração & dosagem , Imunossupressores/farmacocinética , Imunoterapia/métodos , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Nanocápsulas/química , Polímeros/química , Animais , Células Cultivadas , CamundongosRESUMO
Cellular traction forces, resulting in cell-substrate physical interactions, are generated by actin-myosin complexes and transmitted to the extracellular matrix through focal adhesions. These processes are highly dynamic under physiological conditions and modulate cell migration. To better understand the precise dynamics of cell migration, we measured the spatiotemporal redistribution of cellular traction stresses (force per area) during fibroblast migration at a submicron level and correlated it with nuclear translocation, an indicator of cell migration, on a physiologically relevant extracellular matrix mimic. We found that nuclear translocation occurred in pulses whose magnitude was larger on the low ligand density surfaces than on the high ligand density surfaces. Large nuclear translocations only occurred on low ligand density surfaces when the rear traction stresses completely relocated to a posterior nuclear location, whereas such relocation took much longer time on high ligand density surfaces, probably due to the greater magnitude of traction stresses. Nuclear distortion was also observed as the traction stresses redistributed. Our results suggest that the reinforcement of the traction stresses around the nucleus as well as the relaxation of nuclear deformation are critical steps during fibroblast migration, serving as a speed regulator, which must be considered in any dynamic molecular reconstruction model of tissue cell migration. A traction gradient foreshortening model was proposed to explain how the relocation of rear traction stresses leads to pulsed fibroblast migration.