Pericyte-Derived Dickkopf2 Regenerates Damaged PenileNeurovasculature Through an Angiopoietin-1-Tie2PathwayGuo Nan Yin,1 Hai-Rong Jin,1,2 Min-Ji Choi,1 Anita Limanjaya,1 Kalyan Ghatak,1 Nguyen Nhat Minh,1

Jiyeon Ock,1 Mi-Hye Kwon,1 Kang-Moon Song,1 Heon Joo Park,3 Ho Min Kim,4 Young-Guen Kwon,5

Ji-Kan Ryu,1,6 and Jun-Kyu Suh1

Diabetes 2018;67:1149–1161 | https://doi.org/10.2337/db17-0833

Penile erection requires well-coordinated interactions be-tween vascular and nervous systems. Penile neurovascu-lar dysfunction is amajor causeof erectile dysfunction (ED)in patients with diabetes, which causes poor response tooral phosphodiesterase-5 inhibitors. Dickkopf2 (DKK2),aWnt antagonist, is known topromote angiogenesis. Here,using DKK2-Tg mice or DKK2 protein administration, wedemonstrate that the overexpression of DKK2 in diabeticmice enhances penile angiogenesis and neural regenera-tion and restores erectile function. Transcriptome analysisrevealed that angiopoietin-1 and angiopoietin-2 are targetgenes for DKK2. Using an endothelial cell-pericyte co-culture system and ex vivo neurite sprouting assay, wefound thatDKK2-mediated juxtacrine signaling in pericyte-endothelial cell interactions promotes angiogenesis andneural regeneration through an angiopoietin-1-Tie2 path-way, rescuing erectile function in diabetic mice. The dualangiogenic and neurotrophic effects of DKK2, especiallyas a therapeutic protein, will open new avenues to treatingdiabetic ED.

Penile erection is a neurovascular phenomenon that requireswell-coordinated interactions among vascular endothelial(VE) cells, smooth muscle cells, pericytes, and neuronal cells(1,2). Erectile dysfunction (ED) affects more than half of

men 40–70 years of age (3). A variety of pathological con-ditions, including vascular risk factors or diseases, neurolog-ical abnormalities, and hormonal disturbances, are involvedin penile neurovascular dysfunction (4).

Phosphodiesterase type 5 inhibitors enhance the nitricoxide (NO)-cyclic guanosine monophosphate pathway andare currently used as a first-line therapy for ED (1). Thereduced responsiveness to phosphodiesterase type 5 inhib-itors in patients with neuropathy, severe angiopathy, or both,such as patients with diabetes, may be related to a decrease inthe endogenous NO released from the nerve terminal and/orendothelial cells of erectile tissue (5,6). Therefore, curativetherapy for advanced ED, including diabetic ED, requiresa new therapeutic strategy that re-establishes structural andfunctional penile neurovasculature and augments endoge-nous NO bioactivity.

Several proteins have been effective in targeting angio-genesis of the penis in diabetic or hypercholesterolemicanimal models of ED, including VE growth factor, angio-poietin (Ang)-1, and Ang4 (7–14). Targeting neural re-generation, brain-derived neurotrophic factor (BDNF) orneurotrophin-3 (NT-3) has shown some effectiveness byrestoring neuronal NO synthase (nNOS)-positive neurons(15,16). However, new therapeutic agents are far from de-velopment because of incomplete effects; potential adverse

1National Research Center for Sexual Medicine and Department of Urology, InhaUniversity School of Medicine, Incheon, Republic of Korea2Department of Urology, Yuhuangding Hospital, Yantai, Shandong Province,People’s Republic of China3Hypoxia-Related Disease Research Center, Inha University College of Medicine,Incheon, Republic of Korea4Graduate School of Medical Science and Engineering, Korea Advanced Institute ofScience and Technology (KAIST), Daejeon, Republic of Korea5Department of Biochemistry, College of Life Science and Biotechnology, YonseiUniversity, Seoul, Republic of Korea6Inha Research Institute for Medical Sciences, Inha University College of Medicine,Incheon, Republic of Korea

Corresponding author: Jun-Kyu Suh, jksuh@inha.ac.kr, or Ji-Kan Ryu, rjk0929@inha.ac.kr.

Received 14 July 2017 and accepted 2 March 2018.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db17-0833/-/DC1.

G.N.Y. and H.-R.J. contributed equally to this work.

© 2018 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, andthe work is not altered. More information is available at http://www.diabetesjournals.org/content/license.

Diabetes Volume 67, June 2018 1149

COMPLIC

ATIO

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effects, such as inflammation; and difficulties engineeringa protein formedicine.Moreover, there is no single candidateprotein that can solve the complicated underlying pathology,both angiopathy and neuropathy, in diabetic ED. Therefore,the development of a treatment modality targeting thesecomplicated pathologies in refractory ED would be ideal.

Dickkopf2 (DKK2) is a secreted protein containing twocysteine-rich regions that act as a Wnt antagonist by bindingLDL receptor–related protein 5/6 (17–19). Recently, DKK2was found to improve recovery from hind limb ischemia andmyocardial infarction by enhancing angiogenesis (20). More-over, in a corneal angiogenesis assay, DKK2-induced capil-laries hadmore coverage of endothelial cells by pericytes andwere less leaky than VE growth factor–induced vessels (20),which suggests that DKK2 promotes mature and stable bloodvessel formation. However, the role of DKK2 in the penishas not yet been explored. Therefore, we hypothesized thatDKK2 may be a potential target for therapeutic angiogen-esis, which ultimately leads to the restoration of physio-logical erection. We also determined the role of DKK2 inpenile neural regeneration.

Here, we demonstrate that the overexpression of DKK2in DKK2-Tg mice that express mouse DKK2 under thecontrol of the endothelial cell–specific Tie2 promoter/enhancer, or in wild-type (WT) mice via local administrationof DKK2 protein into the penis, rescues erectile functionunder diabetic conditions. This recovery was accompaniedby enhanced proliferation of cavernous endothelial cells andpericytes; phosphorylation of endothelial NO synthase, res-toration of the integrity of endothelial cell-cell junctions, anddecreased cavernous vascular permeability; and enhancedneural regeneration through the secretion of neurotrophicfactors. Transcriptome analysis of DKK2 target genes in pri-mary cultures of mouse cavernous endothelial cells (MCECs)revealed that Ang1 expression is downregulated, and theexpression of Ang2, an endogenous antagonist of Ang1, isupregulated by high glucose (HG). This effect was reversedby treatment with DKK2 protein. Using an MCEC-mousecavernous pericyte (MCP) coculture system and an ex vivoneurite sprouting assay, we also found that DKK2-mediatedjuxtacrine signaling in MCECs-MCPs promotes angiogenesisand neural regeneration though an Ang1-Tie2 pathway. Therecovery of erectile function mediated by DKK2 was abol-ished by the inhibition of Ang1-Tie2 signaling with solubleTie2 protein (sTie2-Fc).

RESEARCH DESIGN AND METHODS

Study DesignThe primary aim of the current study was to investigate themechanisms through which DKK2 restores diabetes-inducedED.We used DKK2-Tgmice and administered DKK2 proteininto the penis of diabetic mice. Detailed mechanisms wereevaluated withWT or DKK2-Tgmice and primary cultures ofMCECs, MCPs, and mouse albino neuroblastoma (Neuro2A)cells. All parameters of geneticallymodifiedmice and diabeticmice were compared with those of littermate controls.

Animals and TreatmentsEight-week-old male C57BL/6 (Orient Bio) and DKK2-Tgmice (provided by Young-Guen Kwon, Yonsei University,Republic of Korea) were used in this study. DKK2-Tg micewere backcrossed with C57BL/6 mice for at least seven gen-erations (20). The experiments were approved by the In-stitutional Animal Care andUse Committee of InhaUniversity(Assurance Number INHA 140110–267–1). Diabetes wasinduced by intraperitoneal injection of multiple low dosesof streptozotocin (STZ) (50 mg/kg body wt in 0.1 mol/Lcitrate buffer, pH 4.5) for 5 consecutive days, as describedpreviously (21). Eight weeks after diabetes was induced, themice were anesthetized with intramuscular injections ofketamine (100 mg/kg) and xylazine (5 mg/kg) and placedin the supine position on a thermoregulated surgical table.

For the DKK2-Tg study, the mice were distributed intothe following four groups: WT controls, DKK2-Tg mice, WTmice receiving STZ (50 mg/kg body wt for 5 days), andDKK2-Tg mice receiving STZ (50 mg/kg body wt for 5 days).Eight weeks after the induction of diabetes, we measurederectile function during electrical stimulation of the cavern-ous nerve.

For the inhibition study with sTie2-Fc, the mice weredistributed into the following four groups: DKK2-Tg mice,STZ-induced WT diabetic mice, and STZ-induced DKK2-Tgdiabetic mice receiving subcutaneous injection of dimeric-Fc or sTie2-Fc (4 mg/20 mL; R&D Systems). The dose ofsTie2-Fc was determined based on our previous report (22).sTie2-Fc or dimeric-Fc was administered 8 weeks after theinduction of diabetes. Two weeks after treatment, we mea-sured erectile function, and then the penis was harvested forhistological examination.

To test the efficacy of DKK2 protein, the mice weredistributed into the following three groups: age-matchedcontrols and STZ-induced WT diabetic mice receiving re-peated intracavernous injections of PBS or DKK2 protein(days 23 and 0; 6 mg/20 mL; A&R Therapeutics). For theinhibition study with sTie2-Fc, the mice were distributedinto the following four groups: age-matched controls andSTZ-induced WT diabetic mice receiving repeated intraca-vernous injections of PBS, DKK2 protein (days 23 and 0;6 mg/20 mL) and dimeric-Fc, or DKK2 protein (days23 and0; 6 mg/20 mL) and sTie2-Fc (4 mg/20 mL). sTie2-Fc ordimeric-Fc was administered immediately before the in-jection of DKK2 protein. We evaluated erectile function bycavernous nerve electrical stimulation 2 weeks after treat-ment. The penis was harvested for histological examinationand biochemical study.

To examine the effect of insulin treatment on erectilefunction, the mice were distributed into the following threegroups: age-matched controls and STZ-induced WT diabeticmice receiving repeated intraperitoneal injection of PBS orinsulin (4 international units/day; Sigma-Aldrich) (23,24).Insulin treatment started 1week after STZ injection (9weeksof age) and continued for 9 weeks (18 weeks of age). Wemeasured erectile function during electrical stimulation ofthe cavernous nerve.

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Fasting and postprandial blood glucose levels were de-termined by an Accu-Check blood glucose meter (RocheDiagnostics) before the mice were sacrificed. We also mea-sured glycosylated hemoglobin (HbA1c; A1C Now System;PTS Diagnostics) and serum insulin levels by using a mouseinsulin ELISA kit (Mercodia).

Human Corpus Cavernosum TissueHuman corpus cavernosum tissue samples were obtainedfrom a 21-year-old patient with congenital penile curvaturewho had normal erectile function during reconstructivepenile surgery and a 56-year-old patient with diabetic ED(type 2 diabetes mellitus, with a duration of 22 years; HbA1clevel 8.8% [73 mmol/mol]; BMI, 23.1 kg/m2; comorbidityhypertension; medications subcutaneous insulin and oralmetformin) during penile prosthesis implantation. All tissuedonors provided informed consent, and the experimentswere approved by the internal review board of Inha Uni-versity.

Measurement of Erectile FunctionMeasurement of erectile function was performed as de-scribed previously (21). Details can be found in the Supple-mentary Data.

Cell Culture ExperimentsThe MCECs and MCPs were prepared and maintained asdescribed previously (2,25,26). Tube formation assay, scratchwound-healing assay, transfection assay, RT-PCR, and cDNAmicroarray were performed as described in the Supplemen-tary Data.

Aortic Ring AssayAortas were harvested from 8-week-old C57BL/6 WT mice.The aortic rings were placed in the eight-well Nunc Lab-TekChamber Slide System (Sigma-Aldrich) and sealed in placewith an overlay of 50 mL of Matrigel. The aortic rings werecultured in medium 199 with 20 ng/mL basic fibroblastgrowth factor and 1%penicillin/streptomycin for 5 days. Theaortic segments and sprouting cells were fixed in 4% para-formaldehyde for at least 30 min and used for immunoflu-orescent staining.

Ex Vivo Neurite Sprouting AssayThe mouse major pelvic ganglion (MPG) tissues were pre-pared and maintained as described previously (27), withminor modifications. The MPG tissues were isolated frommale mice using a microscope, were transferred into sterilevials containing Hank’s balanced salt solution (Gibco), andthen rinsed and washed twice in PBS. The MPG tissue wascut into small pieces, and the samples were plated on a poly-D-lysine hydrobromide–coated (Sigma-Aldrich) 12-well plate.The whole MPG tissue sample was covered with Matrigel,and the culture plate was placed on ice for 5 min prior toincubation at 37°C for 10–15 min in a 5% CO2 atmosphere.We added 1 mL of complete Neurobasal Medium (Gibco)supplemented with 2% serum-free B-27 (Gibco) and0.5 nmol/L GlutaMAX-I (Gibco). The dishes were then

incubated at 37°C in a 5% CO2 atmosphere. Three daysafter incubation, we evaluated neurite outgrowth.

Establishment of In Vitro or Ex Vivo ExperimentalSystems That Mimic Diabetic EDTomimic an in vivo or ex vivo condition for diabetes-inducedangiopathy and neuropathy, primary cultures or tissues wereserum starved for 24 h and then exposed to normal glucose(NG; 5 mmol) or HG (30 mmol; Sigma-Aldrich) conditionsfor 2 days (MCECs, MCPs, and Neuro2A cells), 3 days (MPGtissue), or 5 days (aortic ring).

Preparation of Conditioned MediumTo examine the effect of pericyte-derived DKK2 on endo-thelial cells, the conditioned medium (CM) derived fromMCPs in the presence or absence of DKK2 depletion wastransferred toMCECs. In addition, CM derived fromMCECsin the presence or absence of DKK2 depletion was trans-ferred to MCPs to determine the effect of endothelial cell–derived DKK2 on pericytes. To do this, MCECs and MCPswere grown in 60-mm dishes until 80% confluence, and themedium was changed for an additional 2 days. The culturesupernatants were collected and centrifuged for 10 min at200g to remove cell debris. For DKK2 depletion, an immu-noprecipitation protocol was used. Briefly, the CM was in-cubated with DKK2 antibody (1:200; catalog #Ab95274;Abcam) or control rabbit IgG (1:100; catalog #sc-2027; SantaCruz Biotechnology) for 12 h at 4°C. Protein G-coupledSepharose beads (Millipore) were added to the medium andincubated for an additional 12 h at 4°C to remove DKK2antibody and protein. MCP or MCEC complement mediumwas used as a control.

We also determined the effect of CMderived fromMCEC-MCP coculture on neurite sprouting fromMPG tissue. To dothis, MCECs and MCPs were cultured and treated under thefollowing conditions: NG; HG + PBS; HG + DKK2 protein(200 ng/mL) + scrambled small interfering RNA (siRNA);HG + DKK2 protein (200 ng/mL) + Ang1 siRNA (200 pmol)transfection in MCECs; HG + DKK2 protein (200 ng/mL) +Ang1 siRNA (200 pmol) transfection in MCPs; and HG +DKK2 protein + Ang1 siRNA (200 pmol) transfection in bothMCECs and MCPs. The culture supernatants were collectedand centrifuged for 10 min at 200g to remove cell debris andthen transferred to MPG tissue.

Histological ExaminationsHistological examinations and BrdU labeling were performedas described in the Supplementary Data.

Western BlotWestern blot analysis was performed as described in theSupplementary Data.

Statistical AnalysisThe results are expressed as the mean 6 SE. Intergroupcomparisons weremade byMann-WhitneyU test or Kruskal-Wallis test. P values ,5% were considered significant. Weused SigmaStat 3.11 software (Systat Software) for statisticalanalyses.

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RESULTS

Metabolic VariablesThe fasting and postprandial blood glucose concentrationsas well as HbA1c levels in STZ-treated WT or DKK2-Tg dia-betic mice were significantly higher than in control mice. Inaddition, body weight and serum insulin levels were sig-nificantly lower in the STZ-induced diabetic mice than in thecontrols. However, the body weight and blood glucose levelsof the diabetic mice did not differ significantly regardless oftreatment (Supplementary Tables 1–4).

DKK2 Is Mainly Expressed in PericytesThe expression of DKK2 mRNA and protein was signifi-cantly higher in primary cultures of MCPs and human brainmicrovascular pericytes (HBMPs) than in MCECs and hu-man umbilical vein endothelial cells (HUVECs), respectively(Fig. 1A–D and Supplementary Fig. 1). Similarly, immuno-histochemical staining of normal human or mouse erectiletissue revealed that a significant proportion of the DKK2expression overlapped with pericytes and partially colocal-ized with endothelial cells (Fig. 1E–H).

Cavernous Expression of DKK2 Is Decreased UnderDiabetic ConditionsWestern blot analysis revealed a decrease in DKK2 expres-sion in the penis tissue of diabeticmice in vivo and inMCECsor MCPs exposed to HG conditions in vitro (Fig. 1I–N).Immunofluorescent staining also revealed that the expres-sion of DKK2 protein was significantly lower in cavernoustissue from patients with diabetes or diabetic mice thanin the control groups (Fig. 1O–Q). These findings gave usa rationale to use DKK2 for the treatment of diabetic ED.

Overexpression of DKK2 Preserves the RegenerativePotential of Endothelial Cells and Pericytes UnderDiabetic ConditionsEight weeks after the injection of STZ into WT or DKK2-Tgmice and the induction of diabetes, the cavernous endothe-lial cell and pericyte content was significantly lower in WTmice that received STZ than in untreated WT mice, whereascavernous endothelial cell and pericyte content was relativelypreserved in DKK2-Tg mice that received STZ (Fig. 2A andG). Moreover, DKK2 profoundly enhanced the proliferationof endothelial cells and pericytes under diabetic conditionsboth in vivo and in vitro (Fig. 2B and H and SupplementaryFig. 2). Overexpression of DKK2 significantly induced thephosphorylation of Akt and endothelial NO synthase (Sup-plementary Fig. 3), restored cavernous endothelial cell-celljunction proteins claudin-5 and VE-cadherin (Supplemen-tary Fig. 4), and decreased the extravasation of oxidized LDL(Supplementary Fig. 5) under diabetic conditions comparedwith WT littermates that received STZ.

We further examined the role of DKK2 in MCEC andMCPmonoculture or directmixed coculture. Themixture ofMCECsand MCPs formed well-organized capillary-like structures ata ratio of 3:1 (Supplementary Fig. 6). In vitro Matrigel assaysrevealed impaired tube formation in MCEC and MCP mono-culture or coculture exposed to HG, and these impairments

were completely restored by treatment with DKK2 protein(200 ng/mL) (Fig. 2C and I). DKK2 protein also promotedMCEC, MCP, HUVEC, and HBMP migration under HGconditions (Fig. 2D and J and Supplementary Fig. 7). Inan ex vivo aortic ring assay, the average length and branchnumber of outgrowing microvessels were significantly lowerin aortic segments exposed to HG than segments exposed toNG conditions. Moreover, DKK2 protein significantly en-hanced the outgrowth of microvessels from aortic rings underHG conditions (Fig. 2E and K). We observed higher DKK2 ex-pression in the sprouting front than the aortic ring (Fig. 2F).

Overexpression of DKK2 Decreases Cavernous ReactiveOxygen Species Production Under Diabetic ConditionsCavernous inducible NO synthase protein expression wassignificantly higher in the PBS-treated diabetic mice than inthe age-matched controls. Repeated intracavernous injectionsof DKK2 protein (days 23 and 0; 6 mg/20 mL) significantlydecreased cavernous inducible NO synthase expression in thediabetic mice (Supplementary Fig. 8A–C). We also performedimmunohistochemical localization of nitrotyrosine to de-termine peroxynitrite generation, which is derived from NOand superoxide anion, and in situ analysis of superoxide anionproduction. Nitrotyrosine expression and the fluorescent prod-ucts of oxidized hydroethidine in endothelial cells of the corpuscavernosum were significantly higher in WTmice that receivedSTZ than in untreated WT mice, whereas the generation ofsuperoxide anion and nitrotyrosine was profoundly decreasedin DKK2-Tg mice that received STZ. DKK2 protein alsosignificantly decreased cavernous reactive oxygen species pro-duction under diabetic conditions (Supplementary Fig. 8D–I).

Transmigration of DKK2 From Pericytes to EndothelialCells Promotes AngiogenesisImmunocytochemical staining revealed higher DKK2 expres-sion in MCPs than MCECs. In contrast, after cultivationof MCECs and MCPs using an indirect noncontact cocul-ture system, DKK2 expression was higher inMCECs than inMCPs (Fig. 3A). To confirm whether pericyte-derived DKK2migrates into the endothelial cells, MCPs were transfectedwith DKK2-red fluorescent protein (RFP) DNA.We observedDKK2-RFP expression in MCECs after coculture with DKK2-RFP–transfected MCPs (Fig. 3B).

To test the functional role of pericyte-derived DKK2 onendothelial cells, MCECs were treated with CM derived fromMCPs in the presence or absence of DKK2. We observedenhanced tube formation in MCECs treated with MCP-CMcompared with cells treated with complement medium forMCPs. However, MCECs treated with DKK2-depleted MCP-CM had profoundly impaired tube formation (Fig. 3C and D).Although DKK2-depleted MCEC-CM slightly decreased tubeformation in MCPs compared with cells treated with DKK2containing CM, it was not significant (Fig. 3E and F).

Overexpression of DKK2 Preserves NeurotrophicFunction Under Diabetic ConditionsThe expression of neurofilament, bIII tubulin, and nNOS indorsal nerve bundle or corpus cavernosum was significantly

1152 Dickkopf2 and Damaged Penile Neurovasculature Diabetes Volume 67, June 2018

lower in WT mice that received STZ than in untreatedWT mice, whereas the neuronal cell content was completelyrestored in DKK2-Tg mice that received STZ (Fig. 4A andD–F). DKK2 protein also significantly enhanced neuritesprouting in an ex vivo MPG tissue culture exposed to HG(Fig. 4B and G).

Next, we asked whether the effects of DKK2 were mediatedby the production of neurotrophic factors and their receptors.The cavernous expression of nerve growth factor (NGF), BDNF,and tropomyosin receptor kinase (Trk) A was significantly

higher in diabetic mice receiving DKK2 protein than in PBS-treated diabetic mice and comparable to the level found in age-matched controls. We observed similar results in Neuro2A cellsin vitro (Fig. 4C and H–K). The expression of TrkB and TrkCwas not detectable in the penis or Neuro2A cells.

Overexpression of DKK2 Preserves Erectile FunctionUnder Diabetic ConditionsIn accordance with DKK2-mediated angiogenesis and neuralregeneration, the ratio of maximal intracavernous pressure(ICP) or total ICP to mean systolic blood pressure (MSBP)

Figure 1—Decreased DKK2 expression under diabetic conditions. A and B: Representative RT-PCR and Western blots for DKK2 in MCECs andMCPs.C andD: Normalized band intensity values (n = 4). *P, 0.05 vs. MCEC group. The relative ratio of theMCEC group was arbitrarily set to 1.E andF: VWF (green)/CD31 (green) andDKK2 (red) or PDGFRb (green) andDKK2 (red) staining in normal humanormouse cavernous tissue. Nucleiwere labeled with DAPI (blue). Scale bar = 100 mm. G and H: The DKK2-immunopositive area in cavernous endothelial cells and pericytes wasquantified by ImageJ. n = 1 human sample; n = 6 mouse samples. In human tissue, images were obtained for four different regions. *P, 0.01 vs.VWF or CD31 group. I–K: Representative Western blots for DKK2 in age-matched control and diabetic mouse penis and in MCECs and MCPsexposed toNG or HG conditions for 48 h. L–N: Normalized band intensity values (n = 4). *P, 0.05 vs. Control or NG group.O: DKK2 (red) stainingin cavernous tissue from a patient with diabetic ED or diabetic mice and age-matched control groups. Scale bar = 100 mm (left) or 50 mm(magnification image). P andQ: The DKK2-immunopositive area was quantified by ImageJ. n = 1 human sample; n = 4mouse samples. In humantissues, imageswere obtained for four different regions per group. *P, 0.01 vs. control group. The P values were determined byMann-WhitneyUtest. Data in graphs are presented as the mean6 SE. The relative ratio in the control or NG groups was arbitrarily set to 1. C, control; DM, type 2diabetes for human data and type 1 diabetes for mouse data; VWF, von Willebrand factor; WB, Western blot.

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Figure 2—DKK2overexpression is resistant to diabetes-induced angiopathy.A: CD31 (green) andPDGFRb (red) staining in cavernous tissue fromWT and DKK2-Tg mice, WT mice receiving STZ, and DKK2-Tg mice receiving STZ. Scale bar = 100 mm. B: CD31 (red) and BrdU (green, arrowhead) staining in penis tissue from each group. Nuclei were labeled with DAPI (blue). Scale bars = 50 mm (top) and 25 mm (bottom). C: Tubeformation assay in MCEC or MCPmonoculture andMCEC-MCPmixed coculture exposed to NG or HG conditions for 48 h and treated with PBSor DKK2 protein (200 ng/mL). Original magnification 340. D: Scratch wound-healing assay in MCECs 24 h after treatment. Originalmagnification 340. E and F: Ex vivo aortic ring assay. The images were taken 5 days after treatment. E: Original magnification 340. F:DKK2 (red) staining in aortic ring. Nuclei were labeled with DAPI (blue). The sprouting front and aortic tissue are demarcated by the white dashedline. Original magnification 3100. Results were similar in three independent experiments. G: Quantification of cavernous endothelial cell and

1154 Dickkopf2 and Damaged Penile Neurovasculature Diabetes Volume 67, June 2018

was significantly lower inWTmice that received STZ than inuntreated WT mice, whereas erectile function was relativelypreserved in DKK2-Tg mice that received STZ (Supplemen-tary Fig. 9A, C, and D).

Two weeks after treatment, repeated intracavernousinjections of DKK2 protein (days 23 and 0; 6 mg/20 mL)significantly induced the recovery of erection parameters inWT mice that received STZ (Supplementary Fig. 9B, E, andF). No detectable differences in MSBP were found amongthe experimental groups (Supplementary Tables 1 and 2).The dosage of DKK2 protein was determined based on thefindings of our pilot study. We obtained the highest erectilefunction recovery at a concentration of 6 mg/20 mL (Sup-plementary Fig. 10).

TranscriptomeAnalysis of DKK2 Target Genes inMCECsTo identify the genes regulated byDKK2,microarray analysiswas performed. We selected genes for which the ratioschanged more than twofold in both conditions (i.e., MCECsexposed to NG conditions compared with those exposed toHG conditions + PBS, and MCECs exposed to HG condi-tions + DKK2 protein compared with those exposed to HGconditions + PBS). After filtering data from 39,429 genes,201 genes were changedmore than twofold. Of these genes,only three were downregulated under HG conditions com-pared with NG conditions. These levels were reversed aftertreatment with DKK2 protein (Supplementary Tables 5 and6). These genes included Angpt1 (Ang1) and Angpt2 (Ang2)(Fig. 5A).

DKK2-Mediated Angiogenesis and Neural Regenerationand the Recovery of Erectile Function Are Dependent onthe Ang1-Tie2 Signaling PathwayWe further confirmed that Ang1 mRNA and protein expres-sion were significantly lower and Ang2 expression signifi-cantly higher in MCECs or MCPs exposed to HG conditionsthan in cells exposed toNG conditions. The expression of bothAng1 and Ang2 returned to baseline values after treatmentwith DKK2 protein (Fig. 5B–D).

Physiological erection studies revealed that inhibition ofthe Ang1-Tie2 pathway by sTie2-Fc (4 mg/20 mL) abolishedDKK2-mediated erectile function recovery in both diabeticDKK2-Tg mice and diabetic WT mice treated with DKK2protein (Fig. 5E–J). No detectable differences in MSBPwere found among the experimental groups (SupplementaryTables 3 and 4).

Immunofluorescent staining of penis tissue revealed thatthe enhanced cavernous angiogenesis and neural regenera-tion were profoundly diminished in STZ-injected DKK2-Tg

mice treated with sTie2-Fc (Fig. 5K, N, and O). Treatmentof MCEC-MCP coculture or MPG tissue with sTie2-Fc alsoabolished the DKK2-mediated enhancement of tube forma-tion and neurite sprouting under HG conditions (Fig. 5I,M,P, and Q).

We also examined the cellular sources of Ang1 that mayplay a major role in DKK2-mediated angiogenesis and neuralregeneration. Compared with MCECs transfected with Ang1siRNA, the transfection of Ang1 siRNA into MCPs or bothMCECs andMCPs profoundly diminished the DKK2-mediatedenhancement of tube formation in mixed MCEC-MCPcoculture exposed to HG (Supplementary Fig. 11A and B).We also determined whether CM derived from MCECsor MCPs cultivated under the aforementioned conditions af-fects neural regeneration. Similar to the results of the tube for-mation assays, DKK2-mediated neurite sprouting was moreimpaired in MPG tissue treated with CM derived from Ang1siRNA-transfected MCPs than in the tissue treated with CMderived fromAng1 siRNA-transfectedMCECs (SupplementaryFig. 11C andD). These findings suggest that pericyte-derivedAng1 plays a crucial role in DKK2-mediated angiogenesis andneural regeneration.

Insulin Treatment Does Not Prevent the Deterioration ofErectile Function Under Diabetic ConditionsWe finally examined whether insulin treatment rescueserectile function under diabetic conditions. The treatmentof diabetic mice with insulin did not restore erectile function(Supplementary Fig. 12A–F). Insulin treatment also failed torestore cavernous DKK2 expression in the diabetic mice(Supplementary Fig. 12G and H). Metabolic and physiolog-ical variables, including body weight, blood glucose con-centrations, and systemic blood pressure, are summarizedin Supplementary Table 7.

DISCUSSION

Here, we investigatedwhetherDKK2plays a role as a positiveregulator of angiogenesis and neural regeneration, exertingbeneficial effects in diabetic ED. DKK2-mediated inter-actions between pericytes and endothelial cells promotedangiogenesis and neural regeneration through an Ang1-Tie2 pathway, rescuing erectile function in diabetic animals.The detailed mechanisms of action by which DKK2 restoreserectile function are illustrated in Fig. 6.

To test whether DKK2 induces cavernous angiogenesisunder pathological conditions, we immunohistochemicallyevaluated the expression of CD31 and platelet-derivedgrowth factor receptor-b (PDGFRb). Similar to the results of

pericyte content by ImageJ (n = 6). *P , 0.001 vs. WT and DKK2-Tg groups. #P , 0.001 vs. WT + STZ group. The relative ratio of the WT orDKK2-Tg group was arbitrarily set to 1. H: Number of BrdU-positive endothelial cells per high-power field (HPF) (n = 6). *P , 0.05 vs. WT andDKK2-Tg groups. #P, 0.01 vs.WT + STZ group. I: Number of branch points per high-power field (n = 4). *P, 0.001 vs. NG group. #P, 0.001 vs.PBS-treated group. J: Number of migrated endothelial cells (n = 6). *P , 0.001 vs. NG group. #P , 0.001 vs. PBS-treated group. K: Area ofoutgrowingmicrovessels from aortic ring (n = 6). *P, 0.05 vs. NG group. #P, 0.05 vs. PBS-treated group. P values were determined by Kruskal-Wallis test. Data in graphs are presented as the mean 6 SE.

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Figure 3—Pericyte-derived DKK2 migrates into endothelial cells and regulates angiogenesis. A: DKK2 (red) staining in MCEC and MCPmonoculture and indirect noncontactMCEC-MCPcoculture. Nuclei were labeledwithDAPI (blue). Note the increasedDKK2expression inMCECsafter coculture. Scale bar = 50mm.B: MCPswere transfectedwith DKK2-RFPDNA then coculturedwithMCECs. Note the DKK2-RFP expressionin MCECs after coculture. Images are representative of four independent experiments. Scale bar = 100 mm. C and D: Tube formation assay inMCECs exposed to MCP complement medium (control) or MCP-conditioned CM with or without deletion of DKK2 with neutralizing antibody.Original magnification340. Number of tubes per high-power field (n = 4). *P, 0.05 vs. control group. #P, 0.05 vs. untreated CMgroup. E and F:Tube formation assay in MCPs exposed toMCEC complement medium (control) or MCEC-conditioned CMwith or without deletion of DKK2withneutralizing antibody. Original magnification 340. Number of tubes per high-power field (n = 4). *P , 0.001 vs. control group. P values weredetermined by Kruskal-Wallis test. Data in graphs are presented as the mean 6 SE. C, control; NT, no treatment.

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previous studies in STZ-induced diabetic rats (28,29) andmice (2,9,11), the cavernous endothelial cell and pericytearea was significantly smaller in WT diabetic mice than incontrol mice. BrdU labeling revealed increased endothelialcell and pericyte proliferation, and these cellular contentswere relatively well preserved in STZ-treated DKK2-Tg mice.DKK2 protein also promoted tube formation, proliferation,

and migration by endothelial cells and pericytes, and en-hanced microvessel sprouting from the aortic ring under HGconditions.

Endothelial cell-cell junctions serve as a barrier by regu-lating paracellular permeability and play a crucial role invascular formation, the vascular network, and remodelingof blood vessels (30). DM promotes LDL oxidation and

Figure 4—DKK2 overexpression is resistant to diabetes-induced neuropathy. A: Neurofilament (NF; red), nNOS (red in dorsal nerve and green incavernosum), and bIII tubulin (red) staining in penis tissue from WT and DKK2-Tg mice, WT mice receiving STZ, and DKK2-Tg mice receiving STZ.Nuclei were labeled with DAPI (blue). Scale bars = 25 mm (dorsal nerve bundle) or 50 mm (cavernosum). B: bIII tubulin (red) staining in MPG tissueculture exposed to NG or HG conditions for 72 h and treated with PBS or DKK2 protein (200 ng/mL). Scale bar = 200mm.C: RepresentativeWesternblot for neurotrophic factors (NGF, BDNF, and NT-3) and TrkA in penis tissue from age-matched control or diabetic mice 2 weeks after repeatedintracavernous injections of PBS (20 mL) or DKK2 protein (days23 and 0; 6 mg/20 mL), and in Neuro2A cells exposed to NG or HG conditions for48 h and treatedwith PBS or DKK2 protein (200 ng/mL).D–F: The NF, nNOS, and bIII tubulin immunopositive areaswere quantified in dorsal nervebundle (DNB) or cavernous tissue by ImageJ (n = 6). *P, 0.05 vs. WT and DKK2-Tg groups. #P, 0.05 vs. WT + STZ group. G: Quantification ofneurite length by ImageJ (n = 4). *P, 0.001 vs. NG group. #P, 0.001 vs. PBS-treated group.H–K: Normalized band intensity values (n = 4). *P,0.05 vs. control (C) and NG groups. #P , 0.05 vs. PBS-treated groups. P values were determined by Kruskal-Wallis test. Data in graphs arepresented as the mean 6 SE. The relative ratio in the the WT, C, or NG group was arbitrarily set to 1.

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Figure 5—DKK2-mediated recovery of erectile function is dependent on the Ang1-Tie2 signaling pathway.A: Microarray analysis using total RNAfromMCECsexposed toNGorHGconditions for 48 h and treatedwithPBSor DKK2protein (200 ng/mL).B: Representative RT-PCRandWesternblot for Ang1 andAng2 inMCECsandMCPs exposed toNGorHGconditions for 48 h and treatedwith PBSorDKK2protein (200 ng/mL).C andD:Normalized band intensity values (n=4). *P,0.05 vs. NGgroup. #P, 0.05 vs. PBS-treated group. The relative ratio in theNGgroupwas arbitrarilyset to 1. E–G: Representative ICP responses for the DKK2-Tg mice, WTmice receiving STZ, DKK2-Tg mice receiving STZ and dimeric-Fc (4 mg),and DKK2-Tg mice receiving STZ and sTie2 antibody (sTie2; 4 mg) (n = 6). *P, 0.001 vs. DKK2-Tg group. #P, 0.001 vs. WT + STZ group. †P,0.001 vs. DKK2-Tg + STZ + Fc group. H–J: Representative ICP responses for age-matched control (C) or diabetic mice stimulated 2 weeks afterrepeated intracavernous injections of PBS (20 mL), DKK2 protein (days23 and 0; 6 mg/20 mL) and dimeric-Fc, or DKK2 protein and sTie2 (n = 5).*P, 0.001 vs. control group. #P, 0.001 vs. PBS-treated group. †P, 0.001 vs. DKK2+Fcgroup.K: CD31 (green), PDGFR-b (red), andbIII tubulin

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extravasation, which in turn induces vascular inflam-matory responses and endothelial cell apoptosis (31). Werecently revealed impaired cavernous endothelial cell-celljunctions and increased cavernous endothelial permeabilityto oxidized LDL in diabetic mice (32). In the current study,cavernous endothelial cell-cell junction proteins were wellpreserved, and less oxidized LDL was extravasated in STZ-injected DKK2-Tg mice than in STZ-injected WT litter-mates. The restoration of endothelial cell-cell junctions andenhanced pericyte coverage on endothelial cells byDKK2 may be attributable to a decrease in cavernousvascular permeability.

The interaction between endothelial cells and pericytesplays a crucial role in blood vessel formation and vascularmaturation (33). The close anatomical relationship betweenendothelial cells and pericytes implicates paracrine or juxta-crine signaling (i.e., endothelial cell-pericyte signaling). Using

human and mouse erectile tissues in vivo, and endothelialcells (MCECs and HUVECs) and pericytes (MCPs andHBMPs) in vitro, we found that pericytes are the majorsource of DKK2 expression. In indirect noncontact MCECand DKK2-RFP–transfected MCP coculture experiments, weconfirmed that pericyte-derived DKK2 migrates into endo-thelial cells. Moreover, we found profound tube formationimpairment in MCECs treated with DKK2-depleted MCP-CM, whereas the treatment of MCPs with DKK2-depletedMCEC-CM did not significantly affect tube formation. Thesefindings suggest that pericyte-derived DKK2 may play a cru-cial role in DKK2-mediated angiogenesis.

The number of functional nNOS-positive neurons iscritical for physiological penile erection (34). In the currentstudy, DKK2-Tg mice that received STZ had preservednNOS, neurofilament, and bIII tubulin expression in the cor-pus cavernosum and dorsal nerve bundle. Moreover, direct

(red) staining in penis tissue from each group (n = 6). Scale bar = 100 mm (top), 25 mm (middle), and 50 mm (bottom). L: Tube formation assay inMCECs exposed to NG or HG conditions for 48 h and treated with PBS, DKK2 protein (200 ng/mL), or DKK2 protein + sTie2 (100 ng/mL). Originalmagnification 340. M: bIII tubulin (red) staining in MPG tissue culture. N and O: The CD31, PDGFR-b, and bIII tubulin immunopositive areasquantified by ImageJ (n = 6). *P, 0.05 vs. DKK2-Tg group. #P, 0.05 vs.WT + STZ group. †P, 0.01 vs. DKK2-Tg + STZ + Fc group. The relativeratio in the DKK2-Tg group was arbitrarily set to 1. P: Number of tubes per high-power field (n = 4). *P, 0.001 vs. NG group. #P, 0.01 vs. PBS-treated group. †P, 0.01 vs. DKK2 + Fc group.Q: Quantification of neurite length by ImageJ (n = 4). *P, 0.001 vs. NGgroup. #P, 0.001 vs. PBS-treated group. †P, 0.001 vs. DKK2 + Fc group. The relative ratio in the NG group was arbitrarily set to 1. P values were determined by Kruskal-Wallis test. Data in graphs are presented as the mean 6 SE. DNB, dorsal nerve bundle; WB, Western blot.

Figure 6—Schematic diagram of a proposed mechanism in which DKK2 preserves erectile function in diabetic mice. Pericyte-derived DKK2migrates into endothelial cells (ECs). The DKK2-mediated interaction between pericytes and endothelial cells then promotes angiogenesis andneural regeneration through an Ang1-Tie2 pathway, rescuing erectile function under diabetic conditions. ECJ, endothelial cell-cell junction; eNOS,endothelial NO synthase; PC, pericyte.

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administration of DKK2 protein or DKK2 protein–treatedCM derived from MCEC-MCP coculture profoundly en-hanced neurite sprouting in MPG tissue under HG condi-tions. In penis tissue from WT diabetic mice and Neuro2Acells exposed to HG, the expression of NGF, BDNF, and TrkAwas greatly restored by treatment with DKK2 protein. Giventhat the activation of Wnt signaling has neuroprotective ef-fects (35,36), the Wnt signaling antagonist DKK2 may exertits neurotrophic effects independent of the Wnt pathway.

Transcriptome analysis showed that Ang1 and Ang2 aretarget genes of DKK2. Inhibition of the Ang1-Tie2 pathwaywith sTie2-Fc diminished DKK2-mediated angiogenesisand neural regeneration in STZ-injected DKK2-Tg mice andMCECs or MPG tissue exposed to HG. Pretreatment withsTie2-Fc also abolished DKK2-mediated erectile functionrecovery in both STZ-injected DKK2-Tg mice and STZ-injectedWTmice treated withDKK2 protein. These findingssuggest that the Ang1-Tie2 pathway is crucial for DKK2-mediated restoration of the penile neurovascular structureand erectile function. Although the Ang1-Tie2 pathway iswell known to play an important role in generating a stableand functional vasculature (37), its role in the nervous sys-tem is largely unknown. However, Ang1 has been reportedto promote neurite outgrowth in dorsal root ganglion cellspositive for Tie2 receptor through the transactivation ofTrkA receptor (38).

For clinical applications, a Good Laboratory Practicepreclinical study of DKK2 protein has begun. An interimreport revealed no toxicity in C.B-17 SCID mice (i.e., nounscheduled death, clinical signs, changes in body weight, orabnormal necropsy findings) after a single intravenous in-jection of DKK2 protein at a dosage of up to 90 mg/kg bodywt (Supporting Data 1 in the Supplementary Data). Also,no tumorigenesis related to DKK2 protein was noted inbreast cancer cell lines (MDA-MB-231, MCF-7) or a lungcancer cell line (A549) in vitro (Supporting Data 2 in theSupplementary Data) or in vivo after implantation of a lungcancer cell line (A549) in nude mice (Supporting Data 3 inthe Supplementary Data). It was reported that DKK2-Tgmice exhibited increased retinal blood vessel formationduring the developmental period (20). DKK2-Tg mice alsoshowed increased blood vessel density and decreased hem-orrhage in the oxygen-induced retinopathy model (39),suggesting the clinical utility of DKK2 in diabetic retinopathy.

Our study has some limitations. First, this study did notexplain how DKK2 regulates the expression of Ang1 andAng2. Second, the mechanisms by which the Ang1-Tie2pathway is involved in neural regeneration need to be doc-umented in detail. Finally, we measured systemic blood pres-sure by use of a noninvasive tail-cuff system, not by directcarotid artery cannulation, because of concerns about the vi-ability of animals and the reliability of the ICP results withsignificant bleeding during the carotid artery cannulation.Systemic blood pressure and ICP were not measured simulta-neously because vibrations occurring fromelectrical stimulationof the cavernous nerve can impede the accurate measurementof systemic blood pressure by use of the tail-cuff system.

Thus, we measured systemic blood pressure immediately be-fore electrical stimulation of the cavernous nerve.

Taken together, our findings reveal a unique function ofDKK2 in reprogramming damaged erectile tissue towardneurovascular repair through an Ang1-Tie2 pathway. Dualangiogenic and neurotrophic effects of DKK2, especially inthe formof locally injectable protein,may provide a paradigmshift in the development of novel therapeutics not only forED, but also for other ischemic vascular diseases or neuro-logical disorders.

Funding. This work was supported by a grant from the Korean Health TechnologyR&D Project; the Ministry of Health & Welfare, Republic of Korea (grants A110076 [toJ.-K.S.] and H15C0508 [to J.-K.R. and H.M.K.]); a Medical Research Center Grant(grant 2014R1A5A2009392 [to J.-K.R. and H.J.P.]); and the National ResearchFoundation of Korea (NRF), funded by the Korean government (Ministry of Science,ICT and Future Planning, grant 2016R1A2B2010087 [to J.-K.R.]).Duality of Interest. No potential conflicts of interest relevant to this article werereported.Author Contributions. G.N.Y. and H.-R.J. designed and performed theexperiments and wrote the manuscript. M.-J.C., A.L., K.G., N.N.M., J.O., M.-H.K.,and K.-M.S. performed the experiments. H.J.P. analyzed and critically discussed thedata. H.M.K. and Y.-G.K. contributed essential reagents. J.-K.R. and J.-K.S. designedthe experiments, supervised the project, and wrote the manuscript. J.-K.R. is theguarantor of this work and, as such, had full access to all the data in the study andtakes responsibility for the integrity of the data and the accuracy of the data analysis.

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