Inhibition of Bone Morphogenic Protein 4 Restores Endothelial Function in db/db Diabetic Mice Yang Zhang,* Jian Liu,* Xiao Yu Tian, Wing Tak Wong, Yangchao Chen, Li Wang, Jiangyun Luo, Wai San Cheang, Chi Wai Lau, Kin Ming Kwan, Nanping Wang, Xiaoqiang Yao, Yu Huang Objective—Bone morphogenic protein 4 (BMP4) is involved in the development of endothelial dysfunction in hypertension. This study investigated whether the inhibition of BMP4 signaling improves endothelial function in db/db diabetic mice. Approach and Results—Male db/db mice were treated with noggin via osmotic pump infusion (1 µg/[h·kg–1]) for 2 weeks. Adenovirus BMP4-short hairpin RNA was introduced via tail vein injection at a dosage of 109 pfu/mouse and its effects were examined 7 days after. Vasoreactivity was studied on wire and pressure myograph. Both noggin treatment and adenovirus BMP4-short hairpin RNA transduction improved endothelium-dependent relaxations in aortae and flowmediated dilatation in mesenteric arteries of db/db mice. Ex vivo treatment with BMP4 inhibitors and adenovirus BMP4short hairpin RNA rescued the impaired endothelium-dependent relaxations in db/db mouse aortae and reduced reactive oxygen species overproduction determined by dihydroethidium staining, CM-H2DCFDA fluorescence imaging, and chemiluminescence assay in db/db mouse aortae, and also in ex vivo cultured C57BL/6 mouse aortae or primary mouse aortic endothelial cells treated with high glucose. Likewise, activin receptor–like kinase 3 silencing by short hairpin RNA lentivirus improved endothelium-dependent relaxations in db/db mouse aortae accompanied by reactive oxygen species inhibition in endothelial cells. In addition, noggin reduced BMP4 upregulation in high-glucose–treated endothelial cells and in C57BL/6 mouse aortae and in aortae from db/db mice. Conclusions—Inhibition of BMP4/activin receptor–like kinase 3/reactive oxygen species signaling improved endothelial function in diabetic mice through limiting oxidative stress in endothelium. Inhibiting BMP4 cascade can become another potential therapeutic strategy against diabetic vascular dysfunction.   (Arterioscler Thromb Vasc Biol. 2014;34:152-159.) Key Words: bone morphogenetic protein 4, mouse ◼ diabetes mellitus ◼ endothelial dysfunction ◼ reactive oxygen species

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proatherogenic effects of BMP4 in endothelial cells.16 BMP4 is also upregulated by saturated free fatty acid or in obese mice.17 However, the involvement of BMP4 in diabetic vascular dysfunction is less clear. BMPs bind to 2 types of serine threonine kinase receptor: type I and type II.18 BMP type I receptors can be divided into 2 subgroups: activin receptor–like kinase 3 (ALK3 or BMPR1a) and ALK6 (BMPR1b) group and the ALK1 and ALK2 group.3 BMP4 interacts with ALK3/6 with much higher affinity to form a binding complex and to trigger the downstream signaling.18 The activation of BMP4 signaling leads to Smad-dependent and Smad-independent pathways, including the activation of NADPH oxidases, which results in elevated production of reactive oxygen species (ROS),19 p38 mitogen-activated protein kinases activation,20 and ­cyclooxygenase-2 upregulation.13

one morphogenic protein 4 (BMP4), a member of BMPs belonging to the transforming growth factors-β superfamily, plays an essential role in embryonic development, cartilage formation, and bone mineralization.1,2 In the cardiovascular system, mutations or anomalies of BMPmediated signals are related with many vascular diseases,3 including pulmonary hypertension,4,5 cardiovascular calcification,6–8 stroke,9 and type 2 diabetes mellitus.10 As a proinflammatory cytokine, BMP4 can be induced under the oscillatory shear stress stimulation.11 BMP4 is upregulated in vasculature of atherosclerosis and hypertension.12 BMP4 impairs endothelium-dependent vasodilatation.13 Noggin is a negative regulator in bone development and neuronal differentiation, which binds to and inactivates BMP4.14,15 Noggin improves endothelial function in hypertension, inhibits BMP4-induced hypertension in mice, and counteracts the

Received on: August 22, 2012; final version accepted on: October 21, 2013. From the Institute of Vascular Medicine, Li Ka Shing Institute of Health Sciences (Y.Z., L.W., J.L., W.S.C., C.W.L., X.Y., Y.H.), School of Biomedical Sciences (J.L., Y.C., X.Y., Y.H.), School of Life Sciences (K.M.K.), Chinese University of Hong Kong, Hong Kong SAR, China; Department of Cardiovascular Sciences, Houston Methodist Research Institute, Houston, TX (X.Y.T., W.T.W.); and Institute of Cardiovascular Sciences, Peking University Health Science Center, Beijing, China (N.W.). *These authors contributed equally to this article. The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.113.302696/-/DC1. Correspondence to Yu Huang, PhD, School of Biomedical Sciences, Room 222, Lo Kwee-Seong Integrated Biomedical Sciences Bldg, Chinese University of Hong Kong, Hong Kong, China (e-mail [email protected]); or Xiao Yu Tian, PhD, Department of Cardiovascular Sciences, Houston Methodist Research Institute, Houston, TX. (e-mail [email protected]). © 2013 American Heart Association, Inc. Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org

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DOI: 10.1161/ATVBAHA.113.302696

Zhang et al   BMP4 and Endothelial Dysfunction in Diabetes  153

Nonstandard Abbreviations and Acronyms ALK3 BMP4 DHE EDR NO ROS shRNA SNP

activin receptor–like kinase 3 bone morphogenic protein 4 dihydroethidium endothelium-dependent relaxation nitric oxide reactive oxygen species short hairpin RNA sodium nitroprusside

In type 2 diabetes mellitus, endothelial dysfunction is both the trigger and the hallmark of cardiovascular complications. Oxidative stress is one of the major factors that cause endothelial dysfunction in hyperglycemic conditions and insulin resistance.21 Vascular expression of BMPs and Smad activation is enhanced by hyperglycemic stimulation in type 2 diabetic mice.10 However, the role of BMP4 to mediate endothelial dysfunction is largely unknown. In the present study, we hypothesize that inhibition of BMP4 protects against endothelial dysfunction in db/db diabetic mice through limiting ROS production in endothelial cells and thus increases nitric oxide (NO) bioavailability in blood vessels.

Materials and Methods Materials and Methods are available in the online-only Supplement.

Results Inhibition of BMP4 In Vivo Improves Endothelial Function of db/db Mice Acetylcholine-induced endothelium-dependent relaxations (EDRs) in db/db mouse aortae were diminished compared with those in db/m+ mouse aortae. Noggin infusion to db/db mice restored the impaired EDRs in both aortae (Figure 1A and 1B) and mesenteric arteries (Figure 1C). By contrast, sodium nitroprusside (SNP)–induced endothelium-independent relaxations were unchanged in the 3 groups (Figure IIA in the online-only Data Supplement). Noggin treatment reduced systolic blood pressure from 120.9±3.1 to 101.7±4.0 mm Hg (P