Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The . gov means it’s official. Federal government websites often end in . gov or . mil VSports app下载. Before sharing sensitive information, make sure you’re on a federal government site. .

Https

The site is secure V体育官网. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely. .

. 2017 Jul 7:8:16003.
doi: 10.1038/ncomms16003.

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

Mi Yang  1   2 , Chang-Jun Li  1   2 , Xi Sun  1   3 , Qi Guo  1   4 , Ye Xiao  1 , Tian Su  1 , Man-Li Tu  1   2 , Hui Peng  1   3 , Qiong Lu  1 , Qing Liu  5 , Hong-Bo He  5 , Tie-Jian Jiang  1 , Min-Xiang Lei  1 , Mei Wan  2 , Xu Cao  2 , Xiang-Hang Luo  1
Affiliations

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

Mi Yang et al. Nat Commun. .

Erratum in

V体育官网 - Abstract

A specific bone vessel subtype, strongly positive for CD31 and endomucin (CD31hiEmcnhi), is identified as coupling angiogenesis and osteogenesis. The abundance of type CD31hiEmcnhi vessels decrease during ageing. Here we show that expression of the miR-497∼195 cluster is high in CD31hiEmcnhi endothelium but gradually decreases during ageing. Mice with depletion of miR-497∼195 in endothelial cells show fewer CD31hiEmcnhi vessels and lower bone mass VSports手机版. Conversely, transgenic overexpression of miR-497∼195 in murine endothelium alleviates age-related reduction of type CD31hiEmcnhi 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 CD31hiEmcnhi 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. .

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. MiR-497∼195 cluster was strongly expressed in CD31hiEmcnhi endothelial cells.
(a) Microarray profiling results of deregulated miRNAs in CD31hiEmcnhi and CD31loEmcnlo 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
Figure 2. CD31hiEmcnhi 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
Figure 3. Endothelial-cell-specific MiR-497∼195 cluster knockout mice showed less CD31hiEmcnhi 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∼195lox/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 CD31hiEmcnhi 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
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∼195lox/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∼195lox/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
Figure 5. MiR-497∼195 cluster transgenic mice showed increased CD31hiEmcnhi 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
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
Figure 7. Injection of aptamer-agomiR-195 increased CD31hiEmcnhi 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 CD31hiEmcnhi 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)).
See this image and copyright information in PMC

References

    1. Tashiro Y. et al. Inhibition of PAI-1 induces neutrophil-driven neoangiogenesis and promotes tissue regeneration via production of angiocrine factors in mice. Blood 119, 6382–6393 (2012). - PubMed
    1. Red-Horse K., Crawford Y., Shojaei F. & Ferrara N. Endothelium-microenvironment interactions in the developing embryo and in the adult. Dev. Cell 12, 181–194 (2007). - PubMed
    1. Ding L., Saunders T. L., Enikolopov G. & Morrison S. J. Endothelial and perivascular cells maintain haematopoietic stem cells. Nature 481, 457–462 (2012). - PMC - PubMed
    1. Butler J. M., Kobayashi H. & Rafii S. Instructive role of the vascular niche in promoting tumour growth and tissue repair by angiocrine factors. Nat. Rev. Cancer 10, 138–146 (2010). - PMC - PubMed
    1. Kusumbe A. P., Ramasamy S. K. & Adams R. H. Coupling of angiogenesis and osteogenesis by a specific vessel subtype in bone. Nature 507, 323–328 (2014). - PMC - PubMed

Publication types

MeSH terms

Substances