MiR-497∼195 cluster regulates angiogenesis during coupling with osteogenesis by maintaining endothelial Notch and HIF-1α activity

Abstract

A specific bone vessel subtype, strongly positive for CD31 and endomucin (CD31^hiEmcn^hi), is identified as coupling angiogenesis and osteogenesis. The abundance of type CD31^hiEmcn^hi vessels decrease during ageing. Here we show that expression of the miR-497∼195 cluster is high in CD31^hiEmcn^hi endothelium but gradually decreases during ageing. Mice with depletion of miR-497∼195 in endothelial cells show fewer CD31^hiEmcn^hi vessels and lower bone mass. Conversely, transgenic overexpression of miR-497∼195 in murine endothelium alleviates age-related reduction of type CD31^hiEmcn^hi vessels and bone loss. miR-497∼195 cluster maintains the endothelial Notch activity and HIF-1α stability via targeting F-box and WD-40 domain protein (Fbxw7) and Prolyl 4-hydroxylase possessing a transmembrane domain (P4HTM) respectively. Notably, endothelialium-specific activation of miR-195 by intravenous injection of aptamer-agomiR-195 stimulates CD31^hiEmcn^hi vessel and bone formation in aged mice. Together, our study indicates that miR-497∼195 regulates angiogenesis coupled with osteogenesis and may represent a potential therapeutic target for age-related osteoporosis.

Figure 1. MiR-497∼195 cluster was strongly expressed in CD31^hiEmcn^hi endothelial cells.

(a) Microarray profiling results of deregulated miRNAs in CD31^hiEmcn^hi and CD31^loEmcn^lo endothelial cells (Type H ECs and Type L ECs). (n=3 per group). (b) qRT-PCR analysis of miR-195/miR-497 level in Type H ECs and Type L ECs. (n=5 in each group from three independent experiments). (c,d) qRT-PCR analysis of the levels of miR-195 (c) and miR-497 (d) expression in endothelial cells derived from the mice at different ages. (n=5 mice in each group from three independent experiments). (e,f) Age-associated changes of miR-195 (e) and miR-497 (f) levels in endothelial cells from 33 human females. Data shown as mean±s.d. *P<0.05, (b, Student’s t-test; (c,d), analysis of variance (ANOVA)).

Figure 2. CD31^hiEmcn^hi vessels and bone formation decreased during ageing.

(a) Representative images of CD31 (green), Emcn (red) and Osterix (white) immunostaining. Nuclei, DAPI (blue). Scale bar, 100 μm. (b) Quantitative analysis of Osterix⁺ osteoprogenitors in distal femora. (n=5 mice in each group from three independent experiments). (c,d) FACS analysis dot plot (c) and quantitation of Type H ECs (d) from long bone of the indicated age group. (n=6 mice in each group from three independent experiments). (e–i) Representative microcomputed tomography (μCT) images (e) and quantitative μCT analysis (f–i) of trabecular bone microarchitecture in femora from 1-, 3- and 12-month-old WT mice. (Tb. BV/TV, trabecular bone volume per tissue volume; Tb.N, trabecular number; Tb.Th, trabecular thickness; Tb.Sp, trabecular separation. n=5 mice in each group from three independent experiments). Data shown as mean±s.d. *P<0.05, (analysis of variance (ANOVA)).

Figure 3. Endothelial-cell-specific MiR-497∼195 cluster knockout mice showed less CD31^hiEmcn^hi vessels formation.

(a) Representative images of CD31 (green), Emcn (red) and Osterix (white) immunostaining in femora from 1-, 3- and 12-month-old MiR-497∼195^lox/lox and miR-497∼195 knockout (miR-497∼195^−/−) mice. Scale bar, 100 μm. (b) Quantification of number of Osterix⁺ osteoprogenitors in distal femora. (n=5 mice in each group from three independent experiments). (c–e) FACS analysis dot plot (c) and quantitation of CD31^hiEmcn^hi endothelial cells (Type H ECs) (d) and total endothelial cells (Total ECs) (e) from long bone of the indicated age group. (n=6 mice in each group from three independent experiments). Data shown as mean±s.d. *P<0.05, (Student’s t-test).

Figure 4. Endothelial-cell-specific MiR-497∼195 cluster knockout mice showed less bone formation.

(a–e) Representative microcomputed tomography (μCT) images (a) and quantitative μCT analysis (b–e) of trabecular bone microarchitecture in femora from 1-, 3- and 12-month-old MiR-497∼195^lox/lox and miR-497∼195^−/− mice. (n=5 mice in each group from three independent experiments). (f–h) Representative images of calcein double labelling of trabecular bone (f) with quantification of BFR per bone surface (BFR/BS) (g) and mineral apposition rate (MAR) (h) in femora of 1-, 3- and 12-month-old MiR-497∼195^lox/lox and miR-497∼195^−/− mice. (Scale bar, 25 μm. n=5 mice in each group from three independent experiments). Data shown as mean±s.d. *P<0.05, (Student’s t-test).

Figure 5. MiR-497∼195 cluster transgenic mice showed increased CD31^hiEmcn^hi vessels and bone formation.

(a) Representative images of CD31 (green), Emcn (red) and Osterix (white) immunostaining in femora from 1-, 3- and 12-month-old WT and miR-497∼195 transgenic (Tg) mice. Scale bar, 100 μm. (b) Quantification of number of Osterix⁺ osteoprogenitors in distal femora. (n=5 mice in each group from three independent experiments). (c–e) FACS analysis dot plot (c) and quantitation of Type H ECs (d) and total endothelial cells (Total ECs) (e) from long bone of the indicated age group (n=6 mice in each group from three independent experiments). (f–j) Representative microcomputed tomography (μCT) images (f) and quantitative μCT analysis (g–j) of trabecular bone microarchitecture in femora from 1-, 3- and 12-month-old WT and miR-497∼195 Tg mice. (n=5 mice in each group from three independent experiments). (k,l) quantification of BFR per bone surface (BFR/BS) (k) and mineral apposition rate (MAR) (l) in femora of 1-, 3- and 12-month-old WT and miR-497∼195 Tg mice. (n=5 mice in each group from three independent experiments). Data shown as mean±s.d. *P < 0.05, (Student’s t-test).

Figure 6. MiR-497∼195 cluster targets Fbxw7 and P4HTM to maintain endothelial Notch and HIF-α activity.

(a,b) qRT-PCR analysis of the relative levels of miR-195 (a) and miR-497 (b) expression in BMECs transfected with agomiR-497∼195, antagomiR-497∼195 or their negative controls. NC, negative control. (c,d) qRT-PCR analysis of the relative levels of CD31 (c) and Emcn (d) mRNA expression in endothelial cells. (e–i) Western blot analysis (e) and the quantitation of the relative levels of NICD (f), HIFα (g), Fbxw7 (h) and P4HTM (i) protein expression in BMECs transfected with agomiR-497∼195, antagomiR-497∼195 or control. (n=3 in each group from three independent experiments). (j,k) ECs were transfected with luciferase reporter carrying WT or MUT 3′-UTR of the Fbxw7 or P4HTM gene, WT-pGL3-Fbxw7 or MUT-pGL3-Fbxw7 (j) WT-pGL3-P4HTM and MUT-pGL3-P4HTM (k) respectively, and cotransfected with agomiR-497∼195 or agomiR-NC. Effects of miR-497∼195 on the reporter constructs were determined at 48 h after transfection. Firefly luciferase values, normalized for renilla luciferase, are presented. (n=3 in each group from three independent experiments). (l,m) Western blot analysis (l) and the quantitation (m) of the relative levels of Fbxw7, NICD protein expression in BMECs transfected with Fbxw7 siRNA or control. (n,o) Western blot analysis (n) and the quantitation (o) of the relative levels of P4HTM, HIFα protein expression in BMECs transfected with P4HTM siRNA or control. (n=3 in each group from three independent experiments). Data shown as mean±s.d. *P<0.05, **P<0.01. (j,k,m and o Student’s t-test; a–d,f–i, analysis of variance (ANOVA)).

Figure 7. Injection of aptamer-agomiR-195 increased CD31^hiEmcn^hi vessel and bone formation.

Aptamer-angomiR-195 was injected via tail vein of 12-month-old mice once per week for 3 months. NC, negative control. (a) Representative images of CD31 (green), Emcn (red) and Osterix (white) immunostaining in femora from aptamer-agomiR-195 treated mice and their control group. Scale bar, 100 μm. (b) Quantification of number of Osterix⁺ osteoprogenitors in distal femora. (n=5 mice in each group from three independent experiments). (c–e) FACS analysis dot plot (c) and quantitation of CD31^hiEmcn^hi endothelial cells (Type H ECs) (d) and total endothelial cells (Total ECs) (e) from long bone of the aptamer-agomiR-195 treated mice. (n=6 mice in each group from three independent experiments). (f–j) Representative microcomputed tomography (μCT) images (f) and quantitative μCT analysis (g–j) of trabecular bone microarchitecture in femora from aptamer-agomiR-195 treated mice and their control group. (k–m) Representative images of calcein double labelling of trabecular bone (k) with quantification of BFR per bone surface (BFR/BS) (l) and mineral apposition rate (MAR) (m). (Scale bar, 25 μm. n=5 mice in each group from three independent experiments). (n,o) Three-point bending measurement of tibia maximum load. (n=5 mice in each group from three independent experiments). Data shown as mean±s.d. *P<0.05, (analysis of variance (ANOVA)).