RESUMEN
Age-related macular degeneration (AMD) is the principal cause of blindness in the elderly population. A strong effect on AMD risk has been reported for genetic variants at the CFH locus, encompassing complement factor H (CFH) and the complement-factor-H-related (CFHR) genes, but the underlying mechanisms are not fully understood. We aimed to dissect the role of factor H (FH) and FH-related (FHR) proteins in AMD in a cohort of 202 controls and 216 individuals with AMD. We detected elevated systemic levels of FHR-1 (p = 1.84 × 10-6), FHR-2 (p = 1.47 × 10-4), FHR-3 (p = 1.05 × 10-5) and FHR-4A (p = 1.22 × 10-2) in AMD, whereas FH concentrations remained unchanged. Common AMD genetic variants and haplotypes at the CFH locus strongly associated with FHR protein concentrations (e.g., FH p.Tyr402His and FHR-2 concentrations, p = 3.68 × 10-17), whereas the association with FH concentrations was limited. Furthermore, in an International AMD Genomics Consortium cohort of 17,596 controls and 15,894 individuals with AMD, we found that low-frequency and rare protein-altering CFHR2 and CFHR5 variants associated with AMD independently of all previously reported genome-wide association study (GWAS) signals (p = 5.03 × 10-3 and p = 2.81 × 10-6, respectively). Low-frequency variants in CFHR2 and CFHR5 led to reduced or absent FHR-2 and FHR-5 concentrations (e.g., p.Cys72Tyr in CFHR2 and FHR-2, p = 2.46 × 10-16). Finally, we showed localization of FHR-2 and FHR-5 in the choriocapillaris and in drusen. Our study identifies FHR proteins as key proteins in the AMD disease mechanism. Consequently, therapies that modulate FHR proteins might be effective for treating or preventing progression of AMD. Such therapies could target specific individuals with AMD on the basis of their genotypes at the CFH locus.
Asunto(s)
Proteínas Inactivadoras del Complemento C3b/metabolismo , Factor H de Complemento/genética , Proteínas del Sistema Complemento/metabolismo , Predisposición Genética a la Enfermedad , Haplotipos , Degeneración Macular/patología , Polimorfismo de Nucleótido Simple , Estudios de Cohortes , Proteínas Inactivadoras del Complemento C3b/genética , Proteínas del Sistema Complemento/genética , Estudio de Asociación del Genoma Completo , Humanos , Degeneración Macular/etiología , Degeneración Macular/metabolismoRESUMEN
Age-related macular degeneration (AMD), one of the leading causes of blindness worldwide, causes personal suffering and high socioeconomic costs. While there has been progress in the treatments for the neovascular form of AMD, no therapy is yet available for the more common dry form, also known as geographic atrophy. We analysed the retinal tissue in a mouse model of retinal degeneration caused by sodium iodate (NaIO3)-induced retinal pigment epithelium (RPE) atrophy to understand the underlying pathology. RNA sequencing (RNA-seq), qRT-PCR, Western blot, immunohistochemistry of the retinas and multiplex ELISA of the mouse serum were applied to find the pathways involved in the degeneration. NaIO3 caused patchy RPE loss and thinning of the photoreceptor layer. This was accompanied by the increased retinal expression of complement components c1s, c3, c4, cfb and cfh. C1s, C3, CFH and CFB were complement proteins, with enhanced deposition at day 3. C4 was upregulated in retinal degeneration at day 10. Consistently, the transcript levels of proinflammatory ccl-2, -3, -5, il-1ß, il-33 and tgf-ß were increased in the retinas of NaIO3 mice, but vegf-a mRNA was reduced. Macrophages, microglia and gliotic Müller cells could be a cellular source for local retinal inflammatory changes in the NaIO3 retina. Systemic complement and cytokines/chemokines remained unaltered in this model of NaIO3-dependent retinal degeneration. In conclusion, systemically administered NaIO3 promotes degenerative and inflammatory processes in the retina, which can mimic the hallmarks of geographic atrophy.
Asunto(s)
Proteínas del Sistema Complemento/inmunología , Proteínas del Sistema Complemento/metabolismo , Susceptibilidad a Enfermedades , Yodatos/efectos adversos , Degeneración Retiniana/etiología , Degeneración Retiniana/metabolismo , Animales , Apoptosis/genética , Apoptosis/inmunología , Proteínas del Sistema Complemento/genética , Modelos Animales de Enfermedad , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica/efectos de los fármacos , Inmunidad Innata , Inmunohistoquímica , Ratones , Degeneración Retiniana/patologíaRESUMEN
Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in the elderly population. Variants in the HTRA1-ARMS2 locus have been linked to increased AMD risk. In the present study we investigated the impact of elevated HtrA1 levels on the retina pigment epithelial (RPE) secretome using a polarized culture system. Upregulation of HtrA1 alters the abundance of key proteins involved in angiogenesis and extracellular matrix remodeling. Thrombospondin-1, an angiogenesis modulator, was identified as a substrate for HtrA1 using terminal amine isotope labeling of substrates in conjunction with HtrA1 specificity profiling. HtrA1 cleavage of thrombospondin-1 was further corroborated by in vitro cleavage assays and targeted proteomics together with small molecule inhibition of HtrA1. While thrombospondin-1 is anti-angiogenic, the proteolytically released N-terminal fragment promotes the formation of tube-like structure by endothelial cells. Taken together, our findings suggest a mechanism by which increased levels of HtrA1 may contribute to AMD pathogenesis. The proteomic data has been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier. For quantitative secretome analysis, project accession: PXD007691, username: reviewer45093@ebi.ac.uk, password: 1FUpS6Yq. For TAILS analysis, project accession: PXD007139, username: reviewer76731@ebi.ac.uk, password: sNbMp7xK.
Asunto(s)
Inductores de la Angiogénesis/química , Serina Peptidasa A1 que Requiere Temperaturas Altas/metabolismo , Degeneración Macular/metabolismo , Fragmentos de Péptidos/química , Pigmentos Retinianos/metabolismo , Trombospondina 1/química , Anciano , Secuencia de Aminoácidos , Inductores de la Angiogénesis/aislamiento & purificación , Inductores de la Angiogénesis/farmacología , Medios de Cultivo Condicionados/química , Cámaras de Difusión de Cultivos , Impedancia Eléctrica , Células Epiteliales/metabolismo , Células Epiteliales/patología , Matriz Extracelular/metabolismo , Matriz Extracelular/patología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Serina Peptidasa A1 que Requiere Temperaturas Altas/genética , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , Degeneración Macular/genética , Degeneración Macular/patología , Modelos Moleculares , Fragmentos de Péptidos/aislamiento & purificación , Fragmentos de Péptidos/farmacología , Cultivo Primario de Células , Proteolisis , Proteoma/genética , Proteoma/metabolismo , Pigmentos Retinianos/genética , Trombospondina 1/genética , Trombospondina 1/metabolismoRESUMEN
Age-related macular degeneration (AMD) is the leading cause of irreversible vision loss. The protein HtrA1 is enriched in retinal pigment epithelial (RPE) cells isolated from AMD patients and in drusen deposits. However, it is poorly understood how increased levels of HtrA1 affect the physiological function of the RPE at the intracellular level. Here, we developed hfRPE (human fetal retinal pigment epithelial) cell culture model where cells fully differentiated into a polarized functional monolayer. In this model, we fine-tuned the cellular levels of HtrA1 by targeted overexpression. Our data show that HtrA1 enzymatic activity leads to intracellular degradation of tubulin with a corresponding reduction in the number of microtubules, and consequently to an altered mechanical cell phenotype. HtrA1 overexpression further leads to impaired apical processes and decreased phagocytosis, an essential function for photoreceptor survival. These cellular alterations correlate with the AMD phenotype and thus highlight HtrA1 as an intracellular target for therapeutic interventions towards AMD treatment.
Asunto(s)
Polaridad Celular , Serina Peptidasa A1 que Requiere Temperaturas Altas/metabolismo , Degeneración Macular/metabolismo , Degeneración Macular/patología , Modelos Biológicos , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patología , Tubulina (Proteína)/metabolismo , Uniones Adherentes/metabolismo , Adulto , Feto/metabolismo , Serina Peptidasa A1 que Requiere Temperaturas Altas/genética , Humanos , Microtúbulos/metabolismo , Mutación/genética , Nanopartículas/química , Fagocitosis , Polimerizacion , Agregado de Proteínas , Unión Proteica , Transcripción GenéticaRESUMEN
The human protease family HtrA is responsible for preventing protein misfolding and mislocalization, and a key player in several cellular processes. Among these, HtrA1 is implicated in several cancers, cerebrovascular disease and age-related macular degeneration. Currently, HtrA1 activation is not fully characterized and relevant for drug-targeting this protease. Our work provides a mechanistic step-by-step description of HtrA1 activation and regulation. We report that the HtrA1 trimer is regulated by an allosteric mechanism by which monomers relay the activation signal to each other, in a PDZ-domain independent fashion. Notably, we show that inhibitor binding is precluded if HtrA1 monomers cannot communicate with each other. Our study establishes how HtrA1 trimerization plays a fundamental role in proteolytic activity. Moreover, it offers a structural explanation for HtrA1-defective pathologies as well as mechanistic insights into the degradation of complex extracellular fibrils such as tubulin, amyloid beta and tau that belong to the repertoire of HtrA1.
Asunto(s)
Serina Peptidasa A1 que Requiere Temperaturas Altas/química , Multimerización de Proteína , Proteolisis , Regulación Alostérica , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/metabolismo , Serina Peptidasa A1 que Requiere Temperaturas Altas/genética , Serina Peptidasa A1 que Requiere Temperaturas Altas/metabolismo , Humanos , Dominios Proteicos , Relación Estructura-Actividad , Tubulina (Proteína)/química , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Proteínas tau/química , Proteínas tau/genética , Proteínas tau/metabolismoRESUMEN
BACKGROUND AND OBJECTIVE: Elevated levels of platelet-leukocyte aggregates (PLAs) have been reported in several cardiovascular diseases and suggested to contribute to disease pathology. Our aim was to characterize the effects of inclacumab, a novel human anti-P-selectin antibody, on the interactions between leukocytes and platelets in preclinical and clinical studies. EXPERIMENTAL APPROACHES: Dual-label flow cytometry was used to detect the effect of inclacumab on agonist-induced platelet-leukocyte/platelet-monocyte aggregates in cynomolgus monkeys and humans, following ex vivo and in vivo administration. Platelet-dependent leukocyte activation and leukocyte adhesion to a platelet monolayer were also investigated after ex vivo administration of inclacumab to human blood. RESULTS: Treatment of cynomolgus monkeys with inclacumab profoundly inhibited thrombin receptor-activating peptide (TRAP) or adenosine diphosphate (ADP)-induced PLAs with an IC50 (<2 µg/mL) similar to the in vitro spiking experiments. Maximal inhibition of PLAs persisted for ≥28 days following single dose of inclacumab. In human blood, inclacumab was about 2-fold more potent in inhibiting TRAP-induced PLAs (IC50: 0.7 µg/mL) compared to monkeys. PLA formation was suppressed independently of the inducing platelet agonist. Inclacumab also inhibited the activation of the leukocyte integrin Mac-1 and leukocyte adhesion to a platelet monolayer under flow conditions. In clinical studies, inclacumab inhibited TRAP-induced PLA formation in a dose-dependent manner following single and multiple dose administration to healthy volunteers. It also reduced elevated circulating PLA levels in patients with peripheral arterial disease. CONCLUSION: By inhibiting platelet-leukocyte interactions, demonstrated in multiple preclinical and clinical studies, inclacumab may provide an effective treatment for cardiovascular diseases.