RESUMO
BACKGROUND: Attacks of hereditary angioedema are attributed to excessive plasma kallikrein (PKa) activity, which cleaves high-molecular-weight kininogen to generate the proinflammatory hormone bradykinin. OBJECTIVE: We evaluated the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of KVD900, an orally administered inhibitor of PKa in healthy adults. METHODS: KVD900 was administered in 2 clinical studies. In the first study, healthy adult men received single ascending doses (5-600 mg) of KVD900 capsule or placebo, single 100 mg doses of KVD900 tablet and KVD900 capsule (crossover), and single 600 mg doses of KVD900 (6 × 100 mg tablets) under fed and fasting conditions (crossover). In a second study, 3 cohorts of healthy adults were provided 600 mg of KVD900 tablets at 8-, 4-, and 2-hour intervals. RESULTS: Overall, 98 healthy participants received KVD900. All adverse events (AEs) were mild, except for a single moderate AE (headache). Exposure to KVD900 was proportional to dose. The PK parameters for KVD900 600 mg in tablet form under fasted conditions were mean (coefficient of variation) maximum plasma concentration of 6460 (22.0) ng/mL, mean (coefficient of variation) area under the curve (AUC0-24) of 18,600 (22.5) hâ ng/mL, and median (range) time to maximum plasma concentration of 0.5 (0.33-1.5) hours. Mean PKa inhibition was essentially complete (>98%) between 20 minutes and 3 hours, and >90% inhibition was maintained for at least 8 hours after dosing. High-molecular-weight kininogen cleavage protection at the 600 mg dose was attained within 20 minutes and maintained for 8 to 10 hours. CONCLUSION: These phase 1 studies evaluated the PK/PD profile of KVD900, showing that KVD900 rapidly achieves near-complete PKa inhibition and is generally safe and well tolerated. GOV IDENTIFIER: NCT04349800.
Assuntos
Angioedemas Hereditários , Administração Oral , Adulto , Angioedemas Hereditários/tratamento farmacológico , Área Sob a Curva , Estudos Cross-Over , Relação Dose-Resposta a Droga , Método Duplo-Cego , Voluntários Saudáveis , Humanos , Cininogênio de Alto Peso Molecular , Masculino , ComprimidosRESUMO
BACKGROUND: Hereditary angioedema (HAE) is a rare genetic disease that leads to recurrent episodes of swelling and pain caused by uncontrolled plasma kallikrein (PKa) activity. Current guidelines recommend ready availability of on-demand HAE treatments that can be administered early upon attack onset. This report describes the pharmacological and pharmacodynamic properties of the novel oral small-molecule PKa inhibitor KVD900 as a potential on-demand treatment for HAE. METHODS: Pharmacological properties of KVD900 on PKa and closely related serine proteases were characterized using kinetic fluorogenic substrate activity assays. Effects of KVD900 on PKa activity and kallikrein kinin system activation in whole plasma were measured in the presence of dextran sulphate (DXS)-stimulation using a fluorogenic substrate and capillary immunoassays to quantify high molecular weight kininogen (HK), plasma prekallikrein and Factor XII cleavage. Pharmacodynamic effects of orally administered KVD900 were characterized in plasma samples from six healthy controls in a first in human phase 1 clinical trial and from 12 participants with HAE in a phase 2 clinical trial. RESULTS: KVD900 is a selective, competitive and reversible inhibitor of human PKa enzyme with a Ki of 3.02 nM. The association constant (Kon ) of KVD900 for PKa is >10 × 106 M-1 s-1 . Oral administration of KVD900 in a first-in-human clinical trial achieved rapid and near complete inhibition of DXS-stimulated PKa enzyme activity and HK cleavage and reduced plasma prekallikrein and Factor XII activation in plasma. In individuals with HAE, orally administered KVD900 inhibited DXS-stimulated PKa activity in plasma by ≥95% from 45 min to at least 4 h post-dose and provided rapid protection of HK from cleavage. CONCLUSION: KVD900 is a fast-acting oral PKa inhibitor that rapidly inhibits PKa activity, kallikrein kinin system activation and HK cleavage in plasma. On-demand administration of KVD900 may provide an opportunity to halt the generation of bradykinin and reverse HAE attacks.
Assuntos
Angioedemas Hereditários , Angioedemas Hereditários/tratamento farmacológico , Angioedemas Hereditários/prevenção & controle , Bradicinina , Proteína Inibidora do Complemento C1/genética , Fator XII , Corantes Fluorescentes/uso terapêutico , Humanos , Sistema Calicreína-Cinina , Calicreína Plasmática , Pré-Calicreína/metabolismoRESUMO
The plasma kallikrein-stimulated generation of bradykinin (BK) has been implicated in diabetic macular edema (DME). This study characterizes the effects of BK on the ultrastructure and proteome of the rat retina. The effects of intravitreal injection of BK on retinal thickness and vascular ultrastructure in Sprague Dawley rats were analyzed and compared with the effects of VEGF using spectral-domain optical coherence tomography. At 24â¯h post intravitreal injection of BK or saline vehicle retina were harvested and solubilized proteins were analyzed by mass spectrometry-based proteomics. Proteins were identified using X!Tandem and spectral counts were used as a semiquantitative measurement of protein abundance. Proteins identified from retinal extracts were annotated by Gene Ontology (GO) slim terms and compared with a human DME vitreous proteome. Intravitreal injection of BK and VEGF induced transient increases in retinal thickness of 46⯵m (24.6%, pâ¯=â¯0.015) and 39⯵m (20.3%, pâ¯=â¯0.004), respectively at 24â¯h, which were resolved to baseline thicknesses at 96â¯h post injection. BK and VEGF also increased retinal vessel diameters and tortuosity at 24â¯h post intravitreal injection. Proteomic analyses identified 1757 non-redundant proteins in the rat retina, including 1739 and 1725 proteins from BK- and saline control-injected eyes, respectively. Eighteen proteins, including two proteins associated with intercellular junctions, filamin A and actinin alpha 4, were decreased by at least 50% (pâ¯<â¯0.05) in retina from BK-injected eyes compare with retina from eyes injected with saline. In addition, 32 proteins were increased byâ¯>â¯2-fold (pâ¯<â¯0.05) in retina from BK-injected eyes. Eight proteins, including complement C3, were identified to be increased in both BK-stimulated rat retina and in human DME vitreous. Western blot analysis showed that Complement 3 levels in vitreous from BK-injected eyes in rats and clinical DME samples were increased by 6.6-fold (pâ¯=â¯0.039) and 4.3-fold (pâ¯=â¯0.02), compared with their respective controls. In summary, this study identifies protein changes in rat retina that are associated with BK-induced retinal thickening, including 8 proteins that were previously reported to be increased in the human DME vitreous proteome.
Assuntos
Bradicinina/farmacologia , Edema Macular/metabolismo , Proteoma/metabolismo , Retina/metabolismo , Vasodilatadores/farmacologia , Animais , Western Blotting , Injeções Intravítreas , Edema Macular/induzido quimicamente , Masculino , Calicreína Plasmática , Proteômica , Ratos , Ratos Sprague-Dawley , Retina/diagnóstico por imagem , Vasos Retinianos/metabolismo , Tomografia de Coerência Óptica , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
Retinal ischemia and hemorrhage are hallmarks of worsening diabetic retinopathy, which can lead to neovascularization, macular edema, and severe vision loss. Although diabetes alters expression of clotting factors and their activities, and increases retinal microthromboses, the effects of thrombotic processes on the pathogenesis of diabetic retinopathy are not fully understood. In addition to the roles of coagulation and fibrinolytic cascades in thrombosis and hemostasis, components in these systems also mediate multiple effects on the vasculature that promote inflammation. Plasma kallikrein, thrombin, and urokinase are increased in diabetic retinopathy, and exert proinflammatory effects that contribute to retinal vascular dysfunction. The accumulation and activation of these and additional coagulation factors in the vitreous due to hemorrhage and chronic retinal injury in the diabetic retina may contribute to worsening of retinal inflammation and capillary dysfunction, which lead to retinal ischemia and edema. Further understanding of the role for specific coagulation factors in diabetic retinopathy may suggest new therapeutic opportunities for this vision-threatening disease.
Assuntos
Retinopatia Diabética/sangue , Inflamação/sangue , Hemorragia Retiniana/etiologia , Trombose/etiologia , Anti-Inflamatórios/uso terapêutico , Fatores de Coagulação Sanguínea/fisiologia , Permeabilidade Capilar , Citocinas/fisiologia , Retinopatia Diabética/complicações , Progressão da Doença , Humanos , Inflamação/etiologia , Isquemia/etiologia , Isquemia/fisiopatologia , Microcirculação , Modelos Biológicos , Neovascularização Retiniana/etiologia , Oclusão da Veia Retiniana/etiologia , Oclusão da Veia Retiniana/fisiopatologia , Vasos Retinianos/patologia , Trombofilia/sangue , Trombofilia/etiologiaRESUMO
Background: The kallikrein kinin system (KKS) is an established pharmacological target for the treatment and prevention of attacks in hereditary angioedema (HAE). Proteolytic activities of FXIIa and single-chain Factor XII (FXII) zymogen contribute to KKS activation and thereby may play roles in both initiating and propagating HAE attacks. In this report, we investigated the effects of potent small molecule FXIIa inhibitors on FXIIa and single chain FXII enzymatic activities, KKS activation, and angioedema in mice. Methods: We examined the effects of 29 structurally distinct FXIIa inhibitors on enzymatic activities of FXIIa and a mutant single chain FXII with R334A, R343A and R353A substitutions (rFXII-T), that does not undergo zymogen conversion to FXIIa, using kinetic fluorogenic substrate assays. We examined the effects of a representative FXIIa inhibitor, KV998086, on KKS activation and both carrageenan- and captopril-induced angioedema in mice. Results: FXIIa inhibitors designed to target its catalytic domain also potently inhibited the enzymatic activity of rFXII-T and the pIC50s of these compounds linearly correlated for rFXIIa and rFXII-T (R 2 = 0.93). KV998086, a potent oral FXIIa inhibitor (IC50 = 7.2 nM) inhibited dextran sulfate (DXS)-stimulated generation of plasma kallikrein and FXIIa, and the cleavage of high molecular weight kininogen (HK) in human plasma. KV998086 also inhibited rFXII-T mediated HK cleavage (p < 0.005) in plasma from FXII knockout mice supplemented with rFXII-T and stimulated with polyphosphate or DXS. Orally administered KV998086 protected mice from 1) captopril-induced Evans blue leakage in colon and laryngotracheal tissues and 2) blocked carrageenan-induced plasma HK consumption and paw edema. Conclusion: These findings show that small molecule FXIIa inhibitors, designed to target its active site, also inhibit the enzymatic activity of FXII zymogen. Combined inhibition of FXII zymogen and FXIIa may thereby suppress both the initiation and amplification of KKS activation that contribute to hereditary angioedema attacks and other FXII-mediated diseases.
RESUMO
The blood-brain barrier (BBB) refers to the network of microvessels that selectively restricts the passage of substances between the circulation and the central nervous system (CNS). This microvascular network is comprised of arterioles, capillaries and venules, yet the respective contribution of each of these to the BBB awaits clarification. In this regard, it has been postulated that brain microvascular endothelial cells (BMEC) from these different tributaries might exhibit considerable heterogeneity in form and function, with such diversity underlying unique roles in physiological and pathophysiological processes. Means to begin exploring such endothelial differences in situ, free from caveats associated with cell isolation and culturing procedures, are crucial to comprehending the nature and treatment of CNS diseases with vascular involvement. Here, the recently validated approach of immuno-laser capture microdissection (immuno-LCM) coupled to quantitative real-time PCR (qRT-PCR) was used to analyze gene expression patterns of BMEC retrieved in situ from either capillaries or venules. From profiling 87 genes known to play a role in BBB function and/or be enriched in isolated brain microvessels, results imply that most BBB properties reside in both segments, but that capillaries preferentially express some genes related to solute transport, while venules tend toward higher expression of an assortment of genes involved in inflammatory-related tasks. Fuller appreciation of such heterogeneity will be critical for efficient therapeutic targeting of the endothelium and the management of CNS disease.
Assuntos
Barreira Hematoencefálica/metabolismo , Encéfalo/irrigação sanguínea , Artérias Cerebrais/fisiologia , Células Endoteliais/metabolismo , Regulação da Expressão Gênica/fisiologia , Microcirculação/fisiologia , Animais , Barreira Hematoencefálica/ultraestrutura , Capilares/metabolismo , Capilares/ultraestrutura , Artérias Cerebrais/ultraestrutura , Circulação Cerebrovascular/genética , Células Endoteliais/ultraestrutura , Perfilação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microdissecção , Proteínas/genética , Proteínas/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vênulas/metabolismo , Vênulas/ultraestruturaRESUMO
Laser capture microdissection (LCM) holds great potential for analyzing gene expression profiles in situ. Most recently, this laboratory employed a novel immunostain-based LCM protocol (immuno-LCM) to selectively retrieve brain microvascular endothelial cells (BMEC) from intimately associated perivascular cells. However, before this protocol can be confidently coupled to downstream analytical platforms, it must be demonstrated that any variability associated with it is minimal, so as not to obscure data interpretation. As various factors could contribute to variability, this study focused on determining whether technical inconsistency and/or biological diversity of sample populations, played such a role. Specifically, two separate immuno-LCM-derived BMEC samples derived from adjacent tissue sections of a single mouse (to detect only technical variability), and from analogous tissue sections of three different mice (to detect technical and biological variability) were compared for their relative expression of 16 genes, using quantitative-RT-PCR (qRT-PCR). Both significant linear and rank-order correlations were observed between different sections from the same animal, underscoring lack of technical variability in this LCM application. Furthermore, a three-dimensional scatter plot of gene expression profiles from the three animals was linear, and ANOVA showed absence of statistically significant differences between any of the animals, confirming lack of biological variability. These findings argue that immuno-LCM coupled to qRT-PCR affords a reproducible means to assay gene expression in situ.
Assuntos
Barreira Hematoencefálica/fisiologia , Perfilação da Expressão Gênica/métodos , Lasers , Microdissecção/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Animais , Células Endoteliais/fisiologia , Expressão Gênica , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reprodutibilidade dos TestesRESUMO
PURPOSE: Plasma kallikrein is a serine protease and circulating component of inflammation, which exerts clinically significant effects on vasogenic edema. This study examines the role of plasma kallikrein in VEGF-induced retinal edema. METHODS: Intravitreal injections of VEGF and saline vehicle were performed in plasma prekallikrein-deficient (KLKB1-/-) and wild-type (WT) mice, and in both rats and mice receiving a selective plasma kallikrein inhibitor, VA999272. Retinal vascular permeability (RVP) and retinal thickness were measured by Evans blue permeation and optical coherence tomography, respectively. The retinal kallikrein kinin system was examined by Western blotting and immunohistochemistry. Retinal neovascularization was investigated in KLKB1-/- and WT mice subjected to oxygen-induced retinopathy. RESULTS: Vascular endothelial growth factor-induced RVP and retinal thickening were reduced in KLKB1-/- mice by 68% and 47%, respectively, compared to VEGF responses in WT mice. Plasma kallikrein also contributes to TNFα-induced retinal thickening, which was reduced by 52% in KLKB1-/- mice. Systemic administration of VA999272 reduced VEGF-induced retinal thickening by 57% (P < 0.001) in mice and 53% (P < 0.001) in rats, compared to vehicle-treated controls. Intravitreal injection of VEGF in WT mice increased plasma prekallikrein in the retina, which was diffusely distributed throughout the inner and outer retinal layers. Avascular and neovascular areas induced by oxygen-induced retinopathy were similar in WT and KLKB1-/- mice. CONCLUSIONS: Vascular endothelial growth factor increases extravasation of plasma kallikrein into the retina, and plasma kallikrein is required for the full effects of VEGF on RVP and retinal thickening in rodents. Systemic plasma kallikrein inhibition may provide a therapeutic opportunity to treat VEGF-induced retina edema.
Assuntos
Edema Macular/metabolismo , Calicreína Plasmática/metabolismo , Retina/patologia , Animais , Western Blotting , Permeabilidade Capilar , Injeções Intravítreas , Edema Macular/induzido quimicamente , Edema Macular/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Calicreína Plasmática/antagonistas & inibidores , Ratos , Ratos Sprague-Dawley , Retina/metabolismo , Retina/fisiopatologia , Tomografia de Coerência Óptica , Fator A de Crescimento do Endotélio Vascular/administração & dosagem , Fator A de Crescimento do Endotélio Vascular/intoxicaçãoRESUMO
This study characterizes the kallikrein-kinin system in vitreous from individuals with diabetic macular edema (DME) and examines mechanisms contributing to retinal thickening and retinal vascular permeability (RVP). Plasma prekallikrein (PPK) and plasma kallikrein (PKal) were increased twofold and 11.0-fold (both P < 0.0001), respectively, in vitreous from subjects with DME compared with those with a macular hole (MH). While the vascular endothelial growth factor (VEGF) level was also increased in DME vitreous, PKal and VEGF concentrations do not correlate (r = 0.266, P = 0.112). Using mass spectrometry-based proteomics, we identified 167 vitreous proteins, including 30 that were increased in DME (fourfold or more, P < 0.001 vs. MH). The majority of proteins associated with DME displayed a higher correlation with PPK than with VEGF concentrations. DME vitreous containing relatively high levels of PKal and low VEGF induced RVP when injected into the vitreous of diabetic rats, a response blocked by bradykinin receptor antagonism but not by bevacizumab. Bradykinin-induced retinal thickening in mice was not affected by blockade of VEGF receptor 2. Diabetes-induced RVP was decreased by up to 78% (P < 0.001) in Klkb1 (PPK)-deficient mice compared with wild-type controls. B2- and B1 receptor-induced RVP in diabetic mice was blocked by endothelial nitric oxide synthase (NOS) and inducible NOS deficiency, respectively. These findings implicate the PKal pathway as a VEGF-independent mediator of DME.
Assuntos
Complicações do Diabetes/etiologia , Sistema Calicreína-Cinina/fisiologia , Calicreínas/metabolismo , Cininas/metabolismo , Edema Macular/etiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Bovinos , Células Cultivadas , Diabetes Mellitus Experimental/complicações , Células Endoteliais/fisiologia , Regulação da Expressão Gênica/fisiologia , Humanos , Camundongos , Ratos , Vasos Retinianos/patologia , Corpo Vítreo/químicaRESUMO
Immuno-laser capture microdissection (immuno-LCM) enables highly selective retrieval of designated cell populations from their in situ locations in complex tissue like the brain. However, the amount of tissue acquired by immuno-LCM is extremely limited, and the RNA purification, amplification and labeling steps necessary for expression analysis by hybridization microarray are tedious and time consuming. This report therefore describes a protocol in which these RNA steps are eliminated altogether, yet allows for global gene profiling. Specifically, immuno-LCM tissue was solubilized and the extract directly subjected to reverse transcription to generate cDNA. Pre-amplification of cDNA was performed next, and then relative expression of 96 different immune-related genes simultaneously determined by quantitative real-time PCR using a microfluidic card TaqMan(®) Low Density Array (TLDA). This protocol was highly reproducible and extremely sensitive, demonstrating high correlation of raw Ct values among both technical and biological replicate samples when using only 1/32 of total pre-amplified cDNA obtained from as little as 500 LCM 'shots.' As this abridged protocol takes only approximately 7h from LCM tissue acquisition to analysis by TLDA, it can prove a very effective tool for both screening and validation purposes when investigating gene regulation in health and disease of the nervous system and other tissues.
Assuntos
Encéfalo/metabolismo , Células Endoteliais/metabolismo , Perfilação da Expressão Gênica/métodos , Microdissecção e Captura a Laser/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Animais , Encéfalo/irrigação sanguínea , Endotélio Vascular/metabolismo , Feminino , CamundongosRESUMO
Given strong regional specialization of the brain, cerebral angiogenesis may be regionally modified during normal aging. To test this hypothesis, expression of a broad cadre of angiogenesis-associated genes was assayed at the neurovascular unit (NVU) in discrete brain regions of young versus aged mice by laser capture microdissection coupled to quantitative real-time polymerase chain reaction (PCR). Complementary quantitative capillary density/branching studies were performed as well. Effects of physical exercise were also assayed to determine if age-related trends could be reversed. Additionally, gene response to hypoxia was probed to highlight age-associated weaknesses in adapting to this angiogenic stress. Aging impacted resting expression of angiogenesis-associated genes at the NVU in a region-dependent manner. Physical exercise reversed some of these age-associated gene trends, as well as positively influenced cerebral capillary density/branching in a region-dependent way. Lastly, hypoxia revealed a weaker angiogenic response in aged brain. These results suggest heterogeneous changes in angiogenic capacity of the brain during normal aging, and imply a therapeutic benefit of physical exercise that acts at the level of the NVU.
Assuntos
Envelhecimento/fisiologia , Artérias Cerebrais/crescimento & desenvolvimento , Circulação Cerebrovascular/fisiologia , Terapia por Exercício/métodos , Neovascularização Fisiológica/fisiologia , Condicionamento Físico Animal/métodos , Animais , Artérias Cerebrais/fisiologia , Transtornos Cerebrovasculares/genética , Transtornos Cerebrovasculares/fisiopatologia , Transtornos Cerebrovasculares/terapia , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: There is increasing awareness that, aside from producing cerebrospinal fluid, the choroid plexus (CP) might be a key regulator of immune activity in the central nervous system (CNS) during neuroinflammation. Specifically, the CP has recently been posited to control entry of sentinel T cells into the uninflamed CNS during the early stages of neuroinflammatory diseases, like multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). As the CP is compartmentalized into a stromal core containing fenestrated capillaries devoid of typical blood-brain barrier properties, surrounded by a tight junction-expressing choroidal epithelium, each of these compartments might mount unique responses that instigate the neuroinflammatory process. METHODS: To discern responses of the respective CP stromal capillary and choroidal epithelial tissues during evolving neuroinflammation, we investigated morphology and in situ expression of 93 immune-related genes during early stages of EAE induced by immunization with myelin oligodendrocyte glycoprotein peptide (MOG35-55). Specifically, 3-D immunofluorescent imaging was employed to gauge morphological changes, and laser capture microdissection was coupled to an Immune Panel TaqMan Low Density Array to detail alterations in gene expression patterns at these separate CP sites on days 9 and 15 post-immunization (p.i.). To resolve CP effects due to autoimmunity against MOG peptide, from those due to complete Freund's adjuvant (CFA) and pertussis toxin (PTX) included in the immunization, analysis was performed on MOG-CFA/PTX-treated, CFA/PTX-treated, and naïve cohorts. RESULTS: The CP became swollen and displayed significant molecular changes in response to MOG-CFA/PTX immunization. Both stromal capillary and choroidal epithelial tissues mounted vigorous, yet different, changes in expression of numerous genes over the time course analyzed - including those encoding adhesion molecules, cytokines, chemokines, statins, interleukins, T cell activation markers, costimulatory molecules, cyclooxygenase, pro-inflammatory transcription factors and pro-apoptotic markers. Moreover, CFA/PTX-treatment, alone, resulted in extensive, though less robust, alterations in both CP compartments. CONCLUSIONS: MOG-CFA/PTX immunization significantly affects CP morphology and stimulates distinct expression patterns of immune-related genes in CP stromal capillary and epithelial tissues during evolving EAE. CFA/PTX treatment, alone, causes widespread gene alterations that could prime the CP to unlock the CNS to T cell infiltration during neuroinflammatory disease.
RESUMO
Laser-capture microdissection (LCM) allows for retrieval of distinct populations of cells from their closely surrounding neighbors in situ. As such, LCM is highly advantageous for investigating gene expression along the central nervous system (CNS) microvascular endothelium, a tissue that shows both -considerable segmental and regional heterogeneity. Combining immunohistochemical staining of CNS microvascular endothelial cells with immunofluorescent staining of perivascular astrocytes or smooth muscle cells, immune-guided LCM, immuno-LCM, may be coupled to downstream qRT-PCR to probe varied expression of the endothelium along the CNS microvascular tree during health and disease. Immuno-LCM/qRT-PCR has been used to highlight contributions of the respective segments of the CNS microvasculature to the blood-brain barrier (BBB), and can be employed to examine changes in BBB gene expression -during pathology.
Assuntos
Encéfalo/irrigação sanguínea , Endotélio Vascular/citologia , Lasers , Microdissecção/métodos , Microvasos/citologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Animais , Criopreservação/métodos , Endotélio Vascular/metabolismo , Imunofluorescência/métodos , Perfilação da Expressão Gênica/métodos , Camundongos , Microvasos/metabolismo , RNA/genética , RNA/isolamento & purificação , Fixação de Tecidos/métodosRESUMO
The blood-brain barrier (BBB) has been well studied in terms of its pharmacological properties. However, for a better understanding of the molecular mechanisms regulating these activities, means to thoroughly investigate the BBB at the genomic and proteomic levels are essential. Global gene expression analysis platforms have, in fact, provided a venue for cataloguing the BBB transcriptome. By comparison, and largely because of technical issues, there have been few comprehensive studies of the cerebral microvasculature at the protein level. Recent advances in both microdissection techniques and proteomic analytical tools have nonetheless circumvented many of these obstacles, allowing for isolation of relatively pure cell populations from complex tissues in situ and profiling of cellular proteomes. For example, immunohistochemistry-guided laser capture microdissection (immuno-LCM) provides the unique opportunity to selectively remove brain microvascular endothelial cells from the surrounding cell populations at the BBB, while supporting downstream proteomic analysis. In this chapter, we describe the use of immuno-LCM coupled with a sensitive, high resolution, hybrid linear ion trap coupled with Fourier transform mass spectrometry (FTMS) for proteomic profiling of mouse brain microvascular endothelium, a crucial cellular component of the BBB. We provide details of the quick double-immunostaining protocol for immuno-LCM, laser capture process, sample pooling, and protein recovery followed by in-gel digestion of protein sample, mass spectrometric analysis, and protein identification. Using such an approach to obtain comprehensive protein expression profiles of the cerebral endothelium in situ will enable detailed understanding of the crucial mediators of brain microvascular signaling and BBB function in both normal and pathophysiological conditions.
Assuntos
Encéfalo/irrigação sanguínea , Endotélio Vascular/metabolismo , Lasers , Microcirculação , Microdissecção/métodos , Proteômica , Animais , Encéfalo/metabolismo , Análise de Fourier , Espectrometria de Massas , CamundongosRESUMO
While the expression of the C-C chemokine ligand 2 (CCL2) in the central nervous system (CNS) is associated with numerous neuroinflammatory conditions, the critical cellular sources of this chemokine, which is responsible for disease processes-as well as associated pathogenic mechanisms, remain unresolved. As the potential for anti-CCL2 therapeutics in treating neuroinflammatory disease is likely to be contingent upon effective drug delivery to the source(s) and/or target(s) of CCL2 action in the CNS, tools to highlight the course of CCL2 action during neuroinflammation are imperative. In response to this need, we used the Cre/loxP and FLP-FRT recombination system to develop the first two, cell-conditional CCL2 knockout mice-separately targeting CCL2 gene elimination to astrocytes and endothelial cells, both of which have been considered to play crucial though undefined roles in neuroinflammatory disease. Specifically, mice containing a floxed CCL2 allele were intercrossed with GFAP-Cre or Tie2-Cre transgenic mice to generate mice with CCL2-deficient astrocytes (astrocyte KO) or endothelial cells (endothelial KO), respectively. Polymerase chain reaction, reverse transcription polymerase chain reaction/quantitative reverse transcriptase polymerase chain reaction, and enzyme-linked immunosorbent assay of CCL2 gene, RNA, and protein, respectively, from cultured astrocytes and brain microvascular endothelial cells (BMEC) established the efficiency and specificity of the CCL2 gene deletions and a CCL2 null phenotype in these CNS cells. Effective cell-conditional knockout of CCL2 was also confirmed in an in vivo setting, wherein astrocytes and BMEC were retrieved by immune-guided laser capture microdissection from their in situ positions in the brains of mice experiencing acute, lipopolysaccharide-mediated endotoxemia to induce CCL2 gene expression. In vivo analysis further revealed apparent cross-talk between BMEC and astrocytes regarding the regulation of astrocyte CCL2 expression. Use of astrocyte KO and endothelial KO mice should prove critical in elaborating the pathogenic mechanisms of and optimizing the treatments for neuroinflammatory disease.
Assuntos
Astrócitos/metabolismo , Sistema Nervoso Central , Quimiocina CCL2 , Células Endoteliais/metabolismo , Inflamação/fisiopatologia , Animais , Astrócitos/citologia , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/fisiopatologia , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Selectina E/genética , Selectina E/metabolismo , Células Endoteliais/citologia , Genótipo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Selectina-P/genética , Selectina-P/metabolismoRESUMO
The purpose of this study was to identify the protein profile of the mouse brain microvascular endothelium in situ. This involved coupling of a double-label, immuno-laser capture microdissection (LCM) process with LTQ-FT mass spectrometry to perform the in situ proteomic analysis. LCM was utilized to isolate cells from frozen mouse brain tissue sections. Following cell capture, samples were solubilized and proteins separated by gel electrophoresis in preparation for enzymatic digestion and LC-MS analysis. Processed samples were subsequently analyzed using a linear IT coupled with a Fourier transform mass spectrometer (LTQ-FT MS). Overall, in this study, 881 proteins were identified from a specific cell category using immuno-guided LCM to probe these cell types along the entirety of the cerebral microvascular tree. The identification of sufficient numbers of proteins with high biological interest should allow us to study protein expression by specific cell types - as defined by certain cell markers - in complex tissues.