Macrophage microRNA-150 promotes pathological angiogenesis as seen in age-related macular degeneration

Abstract

Macrophage aging is pathogenic in diseases of the elderly, including age-related macular degeneration (AMD), a leading cause of blindness in older adults. However, the role of microRNAs, which modulate immune processes, in regulating macrophage dysfunction and thereby promoting age-associated diseases is underexplored. Here, we report that microRNA-150 (miR-150) coordinates transcriptomic changes in aged murine macrophages, especially those associated with aberrant lipid trafficking and metabolism in AMD pathogenesis. Molecular profiling confirmed that aged murine macrophages exhibit dysregulated ceramide and phospholipid profiles compared with young macrophages. Of translational relevance, upregulation of miR-150 in human peripheral blood mononuclear cells was also significantly associated with increased odds of AMD, even after controlling for age. Mechanistically, miR-150 directly targets stearoyl-CoA desaturase-2, which coordinates macrophage-mediated inflammation and pathologic angiogenesis, as seen in AMD, in a VEGF-independent manner. Together, our results implicate miR-150 as pathogenic in AMD and provide potentially novel molecular insights into diseases of aging.

Keywords: Aging; Macrophages; Ophthalmology.

Figure 1. microRNA-150 is upregulated in aged macrophages of diverse origins.

(A) In young macrophages, 10 microRNAs were downregulated similarly in response to acetylated low-density lipoprotein (acLDL) and oxidized low-density lipoprotein (oxLDL). (B) In aged macrophages, 5 microRNAs were dysregulated similarly in response to acLDL and oxLDL. (C) In untreated (UT), acLDL-treated, and oxLDL-treated macrophages, 5 microRNAs were dysregulated similarly in aged and young macrophages under the same treatment conditions. microRNA-150 was upregulated in aged peritoneal macrophages (PM) (D; n = 12/group; 2-tailed Mann-Whitney U test), aged splenic macrophages (SM) (E; n = 13/group; 2-tailed Mann-Whitney U test), and aged BM-derived macrophages (BMDM) (F; n = 10/group; 2-tailed, unpaired Welch’s t test). (G) Upregulation of microRNA-150 in aged PMs was not affected by treatment with acLDL (n = 7/group; 2-tailed Mann-Whitney U test), oxLDL (n = 7/group; 2-tailed, unpaired Welch’s t test), or LPS (n = 5/group; 2-tailed Mann-Whitney U test). (H) Upregulation of microRNA-150 in aged BMDMs was not affected by treatment with oxLDL (n = 5/group; 2-tailed Mann-Whitney U test) or LPS (n = 5/group; 2-tailed Mann-Whitney U test). Open circles depict individual data points; graphs depict mean ± SEM (A–F) (*P < 0.05; **P < 0.01; ****P < 0.0001).

Figure 2. microRNA-150 (miR-150) regulates inflammation and lipid metabolism in macrophages.

(A) RNA-sequencing followed by hierarchical clustering revealed clear transcriptomic differences between macrophages transfected with miR-150 mimic versus those transfected with a nontargeting negative control. Pathway analysis of the dysregulated genes in miR-150–overexpressing macrophages that are also dysregulated in aged macrophages (Jonathan B. Lin, unpublished observations) suggested perturbations in numerous gene ontology (GO) processes (B), process networks (C), and pathway maps (D). The altered transcriptomic profile of miR-150–overexpressing macrophages suggested dysregulation of numerous inflammation and immune response process networks (C; brown and purple, respectively) and aberrant lipid trafficking and metabolism in age-related macular degeneration (D; purple).

Figure 3. Aged macrophages have altered ceramide and phospholipid profiles.

(A) Aged macrophages contained significantly more long-chain C16:0 than young macrophages but similar levels of very long-chain C22:0 and C24:0 (n = 5/group; 2-tailed, unpaired Welch’s t test), resulting in decreased C22:0/C16:0 and C24:0/C16:0 ratios (B; n = 5/group; 2-tailed, unpaired Welch’s t test). Young and aged macrophages had similar phosphatidylglycerol-D16:0-18:1 (PG-D16:0-18:1) content (C; n = 5/group; 2-tailed, unpaired student’s t test). Aged macrophages had higher total phosphatidylcholine (PC) (D; n = 5/group; 2-tailed, unpaired student’s t test) and higher total phosphatidylethanolamine (PE) (E; n = 5/group; 2-tailed, unpaired student’s t test), but they had similar total phosphatidylinositol (PI) (F; n = 5/group; 2-tailed, unpaired Welch’s t test) and similar total phosphatidylserine (PS) (G; n = 5/group; 2-tailed, unpaired Welch’s t test). Analysis of individual species revealed an interaction between age and species identity with increased levels of certain species but not others within each phospholipid class (H–K; n = 5/group; 2-way, repeated-measures ANOVA with Bonferroni post-hoc test). Open circles depict individual data points; graphs depict mean ± SEM (A–G) (*P < 0.05; **P < 0.01; ^#P < 0.0001).

Figure 4. Upregulation of microRNA-150 in human peripheral blood mononuclear cells (PBMCs) is associated with age-related macular degeneration (AMD).

(A) AMD patients (n = 43) had higher PBMC microRNA-150 copy numbers compared with controls (n = 63; 2-tailed Mann-Whitney U test). (B) Both early AMD (n = 20) and wet AMD patients (n = 23) had higher PBMC microRNA-150 copy numbers compared with controls (n = 63), but there was no significant difference between early and wet AMD patients (Kruskal-Wallis test with Dunn’s multiple comparison post-hoc test). (C) There was no correlation between PBMC microRNA-150 copy number and age in AMD patients or controls. Patients in the highest tertile of microRNA-150 copy number and above median age, as indicated by the dashed rectangle, had the highest prevalence of AMD (87.5%). Open circles depict individual human subjects (A–C); horizontal lines depict medians (A and B) (**P < 0.01; ***P < 0.001; ****P < 0.0001).

Figure 5. microRNA-150 modulates fatty acid synthase (Fasn) and stearoyl-CoA desaturase-2 (Scd2) expression.

(A and B) Eight of the 26 putative microRNA-150 targets had decreased expression in aged macrophages (n = 6-12/group; 2-tailed, unpaired Welch’s t test). (C and D) microRNA-150 mimic–transfected macrophages had reduced expression of Fasn and Scd2 compared with nontargeting negative control-transfected (NC-transfected) macrophages (n = 12/group; 2-tailed, unpaired student’s t test). (E and F) Macrophages transfected with Fasn- and Scd2-targeting small-interfering RNA (siRNA) had reduced expression of target genes (n = 4-5/group; 2-tailed, unpaired student’s t test; KD, knock down). (G and H) Fasn-deficient (Fasn^KD) macrophages were somewhat abnormally activated but had normal expression of proangiogenic factors (n = 14/group; 2-tailed, unpaired Welch’s t test). (I and J) Scd2-deficient (Scd2^KD) macrophages were abnormally activated and had increased expression of proangiogenic factors (n = 10/group; 2-tailed, unpaired Welch’s t test). Open circles depict individual data points; graphs depict mean ± SEM (C–F) (*P < 0.05; **P < 0.01; ***P < 0.001; ^#P < 0.0001).

Figure 6. microRNA-150 directly targets Scd2 and thereby promotes pathological angiogenesis.

(A) The 3′ untranslated region (UTR) of Scd2 contains canonical 7mer-A1 and offset 6mer microRNA-150 binding sites. (B) Cotransfection of a dual-reporter plasmid with the Scd2 3′ UTR inserted downstream of a secreted Gaussia luciferase (GLuc) reporter gene and microRNA-150 mimic led to reduced GLuc activity compared with cotransfection of the same plasmid with a negative control (NC) mimic (n = 10/group; 2-tailed, unpaired student’s t test). (C) Removing the 7mer-A1 target site (mutant 1) but not the offset 6mer target site (mutant 2) reduced the extent to which microRNA-150 cotransfection inhibited GLuc activity (n = 10/group; Kruskal-Wallis test with Dunn’s multiple comparison post-hoc test). (D and E) Adoptively transferred Scd2-deficient (Scd2^KD) macrophages were less able to inhibit laser injury–induced choroidal neovascularization (CNV) compared with NC-transfected macrophages (D, representative images from n = 8–11 burns/group; 2-tailed, unpaired Welch’s t test). (F) In vitro VEGF secretion was not significantly increased in Scd2-deficient macrophages (n = 16/group; 2-tailed, unpaired Welch’s t test). (G and H) Scd2^–m/–m mice exhibited larger CNV complexes after laser injury compared with Scd2^f/f mice (G, representative images from n = 8–9 burns/group; 2-tailed Mann-Whitney U test). (I and J) Fasn^–m/–m and Fasn^f/f mice had similarly sized CNV complexes after laser injury (I, representative images from n = 8 burns/group; 2-tailed Mann-Whitney U test). Scale bars: 100 μm (D, G, I). Open circles depict individual data points; graphs depict mean ± SEM (B, E, F, H, J) (*P < 0.05; ***P < 0.001).

Figure 7. Upregulation of miR-150 in aged macrophages causes stearoyl-CoA desaturase-2 deficiency and dysregulated lipid metabolism, thereby promoting pathological angiogenesis, as seen in age-related macular degeneration.