Page 1 of 84 Diabetes
Pericyte-derived Dickkopf2 regenerates damaged penile neurovasculature through an angiopoietin-1-Tie2 pathway
Guo Nan Yin1,*, Hai Rong Jin1,2,*, Min Ji Choi1, Anita Limanjaya1, Kalyan Ghatak1, 1 1 1 1 3 Nguyen Nhat Minh , Jiyeon Ock , Mi Hye Kwon , Kang Moon Song , Heon Joo Park , Ho Min Kim4, Young Guen Kwon5, Ji Kan Ryu1,6, †, Jun Kyu Suh1, †
1National Research Center for Sexual Medicine and Department of Urology, Inha University School of Medicine, Incheon 22332, Republic of Korea; 2Department of Urology, Yuhuangding Hospital, Yantai 264000, Shandong Province, China; 3Hypoxia related Disease Research Center, Inha University College of Medicine, Incheon 22212, Republic of Korea; 4Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Republic of Korea; 5Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 03722, Republic of Korea; 6Inha Research Institute for Medical Sciences, Inha University College of Medicine, Incheon 22212, Republic of Korea
*These authors contributed equally to this work.
†Correspondence and requests for materials should be addressed to J.K.S. or J.K.R. (email: [email protected] or [email protected])
Jun Kyu Suh, MD, PhD National Research Center for Sexual Medicine and Department of Urology Inha University College of Medicine 7 206, 3rd St, Shinheung Dong, Jung Gu Incheon 22332, Republic of Korea Tel: 82 32 890 3441, Fax: 82 32 890 3097 E mail: [email protected]
Diabetes Publish Ahead of Print, published online March 20, 2018 Diabetes Page 2 of 84
Ji Kan Ryu, MD, PhD National Research Center for Sexual Medicine and Department of Urology Inha University College of Medicine 7 206, 3rd ST, Shinheung Dong, Jung Gu, Incheon 22332 Republic of Korea Tel: 82 32 890 3505; Fax: 82 32 890 3099 E mail: [email protected]
Running title: Dickkopf2 regenerates damaged penile neurovasculature Word count (abstract): 140 Word count (main text): 4549 Number of Figures: 6 Number of Tables: 0 Number of Supplemental Figures: 12 Number of Supplemental Tables: 7
Page 3 of 84 Diabetes
Abstract
Penile erection requires well coordinated interactions between vascular and nervous
system. Penile neurovascular dysfunction is a major cause of erectile dysfunction (ED)
in patients with diabetes, which causes poor response to oral phosphodiesterase 5
inhibitors. Dickkopf2 (DKK2), a Wnt antagonist, is known to promote angiogenesis.
Here, using DKK2 Tg mice or DKK2 protein administration, we demonstrate that
overexpression of DKK2 in diabetic mice enhances penile angiogenesis and neural
regeneration and restores erectile function. Transcriptome analysis revealed that
angiopoietin 1 and angiopoietin 2 are target genes for DKK2. Using an endothelial
cell pericyte co culture system and ex vivo neurite sprouting assay, we found that
DKK2 mediated juxtacrine signaling in pericyte endothelial cell interactions promotes
angiogenesis and neural regeneration though an angiopoietin 1 Tie2 pathway, rescuing
erectile function in diabetic mice. The dual angiogenic and neurotrophic effects of
DKK2, especially as a therapeutic protein, will open new avenues to treating diabetic
ED.
Keywords: Dickkopf2, erectile dysfunction, diabetes mellitus, pericyte, angiopathy,
neuropathy Diabetes Page 4 of 84
Penile erection is a neurovascular phenomenon that requires well coordinated interactions between vascular endothelial cells, smooth muscle cells, pericytes, and neuronal cells (1, 2). Erectile dysfunction (ED) affects more than half of men aged 40 to 70 years (3). A variety of pathological conditions, including vascular risk factors or diseases, neurological abnormalities, and hormonal disturbances, are involved in penile neurovascular dysfunction (4).
Phosphodiesterase type 5 (PDE5) inhibitors enhance the nitric oxide (NO) cGMP pathway and are currently utilized as a first line therapy for ED (1). The reduced responsiveness to PDE5 inhibitors in patients with neuropathy, severe angiopathy, or both, such as diabetes, may be related to a decrease in the endogenous
NO released from the nerve terminal and/or endothelial cells of erectile tissue (5, 6).
Therefore, curative therapy for advanced ED, including diabetic ED, requires a new therapeutic strategy that re establishes structural and functional penile neurovasculature and augments endogenous NO bioactivity.
Several proteins have been effective in targeting angiogenesis of the penis in diabetic or hypercholesterolemic animal models of ED, including vascular endothelial growth factor (VEGF), angiopoietin 1 (Ang1), and angiopoietin 4 (7 14). Targeting neural regeneration, brain derived neurotrophic factor (BDNF) or neurotrophin 3 (NT
3) have shown some effectiveness by restoring neuronal nitric oxide synthase positive
(nNOS+) neurons (15, 16). However, new therapeutics are far from development because of incomplete effects, potential adverse effects, such as inflammation, and difficulties engineering protein for medicine. Moreover, there is no single candidate protein that can solve the complicated underlying pathology, both angiopathy and neuropathy, in diabetic ED. Therefore, the development of a treatment modality Page 5 of 84 Diabetes
targeting these complicated pathologies in refractory ED would be ideal.
Dickkopf 2 (DKK2) is a secreted protein containing two cysteine rich regions
that acts as a Wnt antagonist by binding low density lipoprotein (LDL) receptor related
protein 5/6 (17 19). Recently, DKK2 was found to improve recovery from hind limb
ischemia and myocardial infarction by enhancing angiogenesis (20). Moreover, in a
corneal angiogenesis assay, DKK2 induced capillaries had more coverage of
endothelial cells by pericytes and were less leaky than VEGF induced vessels (20),
which suggests that DKK2 promotes mature and stable blood vessel formation.
However, the role of DKK2 in the penis has not yet been explored. Therefore, we
hypothesized that DKK2 may be a potential target for therapeutic angiogenesis, which
ultimately leads to the restoration of physiologic erection. We also determined the role
of DKK2 in penile neural regeneration.
Here, we demonstrate that overexpression of DKK2 in DKK2 Tg mice that
express mouse DKK2 under the control of the endothelial cell specific Tie2
promoter/enhancer, or in wild type (WT) mice via local administration of DKK2
protein into the penis, rescues erectile function under diabetic conditions. This
recovery was accompanied by enhanced proliferation of cavernous endothelial cells
and pericytes; phosphorylation of endothelial NOS (eNOS), restoration of the integrity
of endothelial cell cell junctions, and decreased cavernous vascular permeability; and
enhanced neural regeneration through the secretion of neurotrophic factors.
Transcriptome analysis of DKK2 target genes in primary cultures of mouse cavernous
endothelial cells (MCECs) revealed that Ang1 expression is down regulated, and the
expression of angiopoietin 2 (Ang2), an endogenous antagonist of Ang1, up regulated
by high glucose (HG). This effect was reversed by treatment with DKK2 protein. Diabetes Page 6 of 84
Using an MCEC mouse cavernous pericyte (MCP) co culture system and an ex vivo neurite sprouting assay, we also found that DKK2 mediated juxtacrine signaling in
MCECs MCPs promotes angiogenesis and neural regeneration though an Ang1 Tie2 pathway. The recovery of erectile function mediated by DKK2 was abolished by inhibition of Ang1 Tie2 signaling with soluble Tie2 protein (sTie2 Fc). Page 7 of 84 Diabetes
Research design and Methods
Study design
The primary aim of the present study was to investigate the mechanisms through which
DKK2 restores diabetes induced ED. We used DKK2 Tg mice and administered
DKK2 protein into the penis of diabetic mice. Detailed mechanisms were evaluated
with WT or DKK2 Tg mice and primary cultures of MCECs, MCPs, and Neuro2A
cells. All parameters of genetically modified mice and diabetic mice were compared to
those of littermate controls.
Animals and treatments
Eight week old male C57BL/6 (Orient Bio) and DKK2 Tg mice (provided by Young
Guen Kwon, Yonsei University, Korea) were used in this study. DKK2 Tg mice were
backcrossed with C57BL/6 for at least seven generations (20). The experiments were
approved by the Institutional Animal Care and Use Committee of Inha University
(Assurance Number: INHA 140110 267 1). Diabetes was induced by intraperitoneal
injection of multiple low doses of STZ (50 mg/kg body weight in 0.1 M citrate buffer,
pH 4.5) for 5 consecutive days as described previously (21). Eight weeks after diabetes
was induced, the mice were anesthetized with intramuscular injections of ketamine
(100 mg/kg) and xylazine (5 mg/kg) and placed supine on a thermoregulated surgical
table.
For the DKK2 Tg study, the mice were distributed into four groups: WT
controls, DKK2 Tg mice, WT mice receiving STZ (50 mg/kg body weight for 5 days),
and DKK2 Tg mice receiving STZ (50 mg/kg body weight for 5 days). Eight weeks Diabetes Page 8 of 84
after the induction of diabetes, we measured erectile function during electrical stimulation of the cavernous nerve.
For the inhibition study with sTie2 Fc, the mice were distributed into four groups: DKK2 Tg mice, STZ induced WT diabetic mice, and STZ induced DKK2 Tg diabetic mice receiving subcutaneous injection of dimeric Fc or sTie2 Fc (4 µg/20 µl;
R&D Systems). The dose of sTie2 Fc was determined based on our previous report
(22). sTie2 Fc or dimeric Fc was administered 8 weeks after the induction of diabetes.
Two weeks after treatment, we measured erectile function, and then the penis was harvested for histological examination.
To test the efficacy of DKK2 protein, the mice were distributed into three groups: age matched controls and STZ induced WT diabetic mice receiving repeated intracavernous injections of PBS or DKK2 protein (days 3 and 0; 6 µg/20 µl; A&R
Therapeutics). For the inhibition study with sTie2 Fc, the mice were distributed into four groups: age matched controls and STZ induced WT diabetic mice receiving repeated intracavernous injections of PBS, DKK2 protein (days 3 and 0; 6 µg/20 µl) and dimeric Fc, or DKK2 protein (days 3 and 0; 6 µg/20 µl) and sTie2 Fc (4 µg/20
µl). sTie2 Fc or dimeric Fc was administered immediately before the injection of
DKK2 protein. We evaluated erectile function by cavernous nerve electrical stimulation 2 weeks after treatment. The penis was harvested for histological examination and biochemical study.
To examine the effect of insulin treatment on erectile function, the mice were distributed into three groups: age matched controls and STZ induced WT diabetic mice receiving repeated intraperitoneal injection of PBS or insulin (4 IU/day; Sigma
Aldrich) (23,24). Insulin treatment started 1 week after STZ injection (9 weeks of age) Page 9 of 84 Diabetes
and continued for 9 weeks (18 weeks of age). We measured erectile function during
electrical stimulation of the cavernous nerve.
Fasting and postprandial blood glucose levels were determined by an Accu
check blood glucose meter (Roche Diagnostics) before the mice were sacrificed. We
also measured glycosylated hemoglobin (HbA1c; A1C Now System, PTS Diagnostics)
and serum insulin levels by using a mouse insulin ELISA kit (Mercodia).
Human corpus cavernosum tissue
Human corpus cavernosum tissues were obtained from a 21 year old patient with
congenital penile curvature who had normal erectile function during reconstructive
penile surgery and a 56 year old patient with diabetic ED (type 2 DM; duration of DM,
2 22 years; HbA1c level, 8.8% [73 mmol/mol]; BMI, 23.1 kg/m ; comorbidity,
hypertension; medications, subcutaneous insulin and oral metformin) during penile
prosthesis implantation. All tissue donors provided informed consent, and the
experiments were approved by the internal review board of Inha University.
Measurement of erectile function
Measurement of erectile function was performed as described previously (21). Details
can be found in ‘Supplemental Methods’.
Cell culture experiments
The MCECs and MCPs were prepared and maintained as described previously (2, 25,
26). Tube formation assay, scratch wound healing assay, transfection assay, RT PCR,
and cDNA microarray were performed as described in ‘Supplemental Methods’. Diabetes Page 10 of 84
Aortic ring assay
Aortas were harvested from 8 week old C57BL/6 WT mice. The aortic rings were placed in the 8 well Nunc Lab Tek Chamber Slide System (Sigma Aldrich) and sealed in place with an overlay of 50 µl matrigel. The aortic rings were cultured in medium
199 with 20 ng/ml of basic fibroblast growth factor and 1% penicillin/streptomycin for
5 days. The aortic segments and sprouting cells were fixed in 4% paraformaldehyde for at least 30 minutes and used for immunofluorescent staining.
Ex vivo neurite sprouting assay
The mouse major pelvic ganglion (MPG) tissues were prepared and maintained as described previously (27), with minor modifications. The MPG tissues were isolated from male mice using a microscope, transferred into sterile vials containing Hank’s balanced salt solution (Gibco), and then rinsed and washed twice in PBS. The MPG tissues were cut into small pieces and the samples plated on poly D lysine hydrobromide coated (Sigma Aldrich) 12 well plate. The whole MPG tissue was covered with matrigel and the culture plate placed on ice for 5 minutes prior to incubation at 37°C for 10 15 minutes in a 5% CO2 atmosphere. We added 1 ml of complete Neurobasal medium (Gibco) supplemented with 2% serum free B 27
(Gibco) and 0.5 nM GlutaMAX™ I (Gibco). The dishes were then incubated at 37°C in a 5% CO2 atmosphere. Three days after incubation, we evaluated neurite outgrowth.
Establishment of in vitro or ex vivo experimental systems that mimic diabetic ED
To mimic an in vivo or ex vivo condition for diabetes induced angiopathy and Page 11 of 84 Diabetes
neuropathy, primary cultures or tissues were serum starved for 24 hours and then
exposed to normal glucose (NG, 5 mmol) or HG (30 mmol; Sigma Aldrich) conditions
for 2 days (MCECs, MCPs, and Neuro2A cells), 3 days (MPG tissue), or 5 days (aortic
ring).
Preparation of conditioned medium
To examine the effect of pericyte derived DKK2 on endothelial cells, the conditioned
medium (CM) derived from MCPs in the presence or absence of DKK2 depletion was
transferred to MCECs. In addition, CM derived from MCECs in the presence of
absence of DKK2 depletion was transferred to MCPs to determine the effect of
endothelial cell derived DKK2 on pericytes. To do this, MCECs and MCPs were
grown in 60 mm dishes until 80% confluence and the medium changed for an
additional 2 days. The culture supernatants were collected and centrifuged for 10
minutes at 200 × g to remove cell debris. For DKK2 depletion, an immunoprecipitation
protocol was used. Briefly, the CM was incubated with DKK2 antibody (1:200; Abcam,
Cat. Ab95274) or control rabbit IgG (1:100; Santa Cruz Biotechnology, Cat. sc 2027)
for 12 hours at 4°C. Protein G coupled Sepharose beads (Millipore) were added to the
medium and incubated for an additional 12 hours at 4°C to remove DKK2 antibody
and protein. MCP or MCEC complement medium was used as a control.
We also determined the effect of CM derived from MCEC MCP co culture on
neurite sprouting from MPG tissue. To do this, MCECs and MCPs were cultured and
treated under the following conditions: NG; HG + PBS; HG + DKK2 protein (200
ng/ml) + scrambled siRNA; HG + DKK2 protein (200 ng/ml) + Ang1 siRNA (200
pmol) transfection in MCECs; HG + DKK2 protein (200 ng/ml) + Ang1 siRNA (200 Diabetes Page 12 of 84
pmol) transfection in MCPs; and HG + DKK2 protein + Ang1 siRNA (200 pmol) transfection in both MCECs and MCPs. The culture supernatants were collected and centrifuged for 10 minutes at 200 × g to remove cell debris and then transferred to
MPG tissue.
Histological examinations
Histologic examinations and BrdU labeling were performed as described in
‘Supplemental Methods’.
Western blot
Western blot analysis was performed as described in ‘Supplemental Methods’.
Statistical analysis
The results are expressed as mean ± SE. Intergroup comparisons were made by Mann
Whitney U test or Kruskal Wallis test. P values < 5% were considered significant. We used SigmaStat 3.11 software (Systat Software) for statistical analyses. Page 13 of 84 Diabetes
Results
Metabolic variables
The fasting and postprandial blood glucose concentrations as well as HbA1c levels in
streptozotocin (STZ) treated WT or DKK2 Tg diabetic mice were significantly higher
than in control mice. In addition, body weight and serum insulin levels were
significantly lower in the STZ induced diabetic mice than in the controls. However, the
body weight and blood glucose levels of the diabetic mice did not differ significantly
regardless of treatment (Supplementary Table S1 S4).
DKK2 is mainly expressed in pericytes
The expression of DKK2 mRNA and protein was significantly higher in primary
cultures of MCPs and human brain microvascular pericytes (HBMPs) than in MCECs
and human umbilical vein endothelial cells (HUVECs), respectively (Fig. 1A D and
Supplementary Fig. 1). Similarly, immunohistochemical staining of normal human or
mouse erectile tissue revealed that a significant proportion of the DKK2 expression
overlapped with pericytes and partially co localized with endothelial cells (Fig. 1E to
H).
Cavernous expression of DKK2 is decreased under diabetic conditions
Western blot analysis revealed a decrease in DKK2 expression in the penis tissue of
diabetic mice in vivo and in MCECs or MCPs exposed to HG conditions in vitro (Fig.
1I N). Immunofluorescent staining also revealed that the expression of DKK2 protein
was significantly lower in cavernous tissue from diabetic patients or diabetic mice than
in controls (Fig. 1O Q). These findings gave us a rationale to use DKK2 for the Diabetes Page 14 of 84
treatment of diabetic ED.
Overexpression of DKK2 preserves the regenerative potential of endothelial cells and pericytes under diabetic conditions
Eight weeks after the injection of STZ into WT or DKK2 Tg mice and the induction of diabetes, the cavernous endothelial cell and pericyte content was significantly lower in
WT mice that received STZ than in untreated WT mice, whereas cavernous endothelial cell and pericyte content was relatively preserved in DKK2 Tg mice that received STZ
(Fig. 2A and G). Moreover, DKK2 profoundly enhanced the proliferation of endothelial cells and pericytes under diabetic conditions both in vivo and in vitro (Fig.
2B and H and Supplementary Fig. 2). Overexpression of DKK2 significantly induced the phosphorylation of Akt and eNOS (Supplementary Fig. 3), restored cavernous endothelial cell cell junction proteins claudin 5 and vascular endothelial (VE) cadherin
(Supplementary Fig. 4), and decreased the extravasation of oxidized LDL
(Supplementary Fig. 5) under diabetic conditions compared to WT littermates that received STZ.
We further examined the role of DKK2 in MCEC and MCP monoculture or direct mixed co culture. The mixture of MCECs and MCPs formed well organized capillary like structures at the ratio of 3:1 (Supplementary Fig. 6). In vitro matrigel assays revealed impaired tube formation in MCEC and MCP monoculture or co culture exposed to HG, and these impairments were completely restored by treatment with
DKK2 protein (200 ng/ml) (Fig. 2 C and I). DKK2 protein also promoted MCEC, MCP,
HUVEC, and HBMP migration under HG conditions (Fig. 2D and J and
Supplementary Fig. 7). In an ex vivo aortic ring assay, the average length and branch Page 15 of 84 Diabetes
number of outgrowing microvessels were significantly lower in aortic segments
exposed to HG than segments exposed to NG conditions. Moreover, DKK2 protein
significantly enhanced the outgrowth of microvessels from aortic rings under HG
conditions (Fig. 2E and K). We observed higher DKK2 expression in the sprouting
front than the aortic ring (Fig. 2F).
Overexpression of DKK2 decreases cavernous ROS production under diabetic
conditions
Cavernous inducible NOS (iNOS) protein expression was significantly higher in the
PBS treated diabetic mice than in the age matched controls. Repeated intracavernous
injections of DKK2 protein (days 3 and 0; 6 µg/20 µl) significantly decreased
cavernous iNOS expression in the diabetic mice (Supplementary Fig. 8A-C). We also
performed immunohistochemical localization of nitrotyrosine to determine
peroxynitrite generation, which is derived from NO and superoxide anion, and in situ
analysis of superoxide anion production. Nitrotyrosine expression and the fluorescent
products of oxidized hydroethidine in endothelial cells of the corpus cavernosum were
significantly higher in WT mice that received STZ than in untreated WT mice, whereas
the generation of superoxide anion and nitrotyrosine was profoundly decreased in
DKK2 Tg mice that received STZ. DKK2 protein also significantly decreased
cavernous ROS production in diabetic condition (Supplementary Fig. 8D to I).
Transmigration of DKK2 from pericytes to endothelial cells promotes
angiogenesis
Immunocytochemical staining revealed higher DKK2 expression in MCPs than Diabetes Page 16 of 84
MCECs. In contrast, after cultivation of MCECs and MCPs using an indirect non contact co culture system, DKK2 expression was higher in MCECs than in MCPs (Fig.
3A). To confirm whether pericyte derived DKK2 migrates into the endothelial cells,
MCPs were transfected with DKK2 RFP DNA. We observed DKK2 RFP expression in
MCECs after co culture with DKK2 RFP transfected MCPs (Fig. 3B).
To test the functional role of pericyte derived DKK2 on endothelial cells,
MCECs were treated with CM derived from MCPs in the presence or absence of
DKK2. We observed enhanced tube formation in MCECs treated with MCP CM compared to cells treated with complement medium for MCPs. However, MCECs treated with DKK2 depleted MCP CM had profoundly impaired tube formation (Fig.
3C and D). Although DKK2 depleted MCEC CM slightly decreased tube formation in
MCPs compared to cells treated with DKK2 containing CM, it was not significant (Fig.
3E and F).
Overexpression of DKK2 preserves neurotrophic function under diabetic conditions
The expression of neurofilament, βIII tubulin, and nNOS in dorsal nerve bundle or corpus cavernosum was significantly lower in WT mice that received STZ than in untreated WT mice, whereas the neuronal cell content was completely restored in
DKK2 Tg mice that received STZ (Fig. 4A, D, E, F). DKK2 protein also significantly enhanced neurite sprouting in an ex vivo MPG tissue culture exposed to HG (Fig. 4B and G).
Next, we asked whether the effects of DKK2 were mediated by the production of neurotrophic factors and their receptors. The cavernous expression of nerve growth Page 17 of 84 Diabetes
factor (NGF), BDNF, and TrkA 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 mouse albino neuroblastoma
(Neuro2A) cells in vitro (Fig. 4C, H, I, J, K). The expression of TrkB and TrkC was not
detectable in the penis or Neuro2A cells.
Overexpression of DKK2 preserves erectile function under diabetic conditions
In accordance with DKK2 mediated angiogenesis and neural regeneration, the ratio of
maximal intracavernous pressure (ICP) or total ICP to mean systolic blood pressure
(MSBP) was significantly lower in WT mice that received STZ than in untreated WT
mice, whereas erectile function was relatively preserved in DKK2 Tg mice that
received STZ (Supplementary Fig. 9A, C, D).
Two weeks after treatment, repeated intracavernous injections of DKK2
protein (days 3 and 0; 6 µg/20 µl) significantly induced recovery of erection
parameters in WT mice that received STZ (Supplementary Fig. 9B, E, F). No
detectable differences in MSBP were found among the experimental groups
(Supplementary Table S1 and S2). The dosage of DKK2 protein was determined based
on our pilot study. We obtained the highest erectile function recovery at a
concentration of 6 µg/20 µl (Supplementary Fig. 10).
Transcriptome analysis of DKK2 target genes in MCECs
To identify the genes regulated by DKK2, microarray analysis was performed. We
selected genes for which the ratios changed more than 2 fold in both conditions, i.e.,
MCECs exposed to NG conditions compared to those exposed to HG conditions + PBS, Diabetes Page 18 of 84
and MCECs exposed to HG conditions + DKK2 protein compared to those exposed to
HG conditions + PBS. After filtering data from 39,429 genes, 201 genes were changed more than 2 fold. Of these genes, only three were down regulated under HG conditions compared to NG conditions. These levels were reversed after treatment with DKK2 protein (Supplementary Table S5 and S6). These genes included Angpt1 (Ang1) and
Angpt2 (Ang2) (Fig. 5A).
DKK2-mediated angiogenesis and neural regeneration and the recovery of erectile function are dependent on the Ang1-Tie2 signaling pathway
We further confirmed that Ang1 mRNA and protein expression were significantly lower and Ang2 expression significantly higher in MCECs or MCPs exposed to HG conditions than in cells exposed to NG conditions. The expression of both Ang1 and
Ang2 returned to baseline values after treatment with DKK2 protein (Fig. 5B D).
Physiological erection studies revealed that inhibition of the Ang1 Tie2 pathway by sTie2 Fc (4 µg/20 µl) abolished DKK2 mediated erectile function recovery in both diabetic DKK2 Tg mice and diabetic WT mice treated with DKK2 protein (Fig. 5E J). No detectable differences in MSBP were found among the experimental groups (Supplementary Table S3 and S4).
Immunofluorescent staining of penis tissue revealed that the enhanced cavernous angiogenesis and neural regeneration were profoundly diminished in STZ injected DKK2 Tg mice treated with sTie2 Fc (Fig. 5K, N, O). Treatment of MCEC
MCP co culture or MPG tissue with sTie2 Fc also abolished the DKK2 mediated enhancement of tube formation and neurite sprouting under HG conditions (Fig. 5I, M,
P, Q). Page 19 of 84 Diabetes
We also examined the cellular sources of Ang1 that may play a major role in
DKK2 mediated angiogenesis and neural regeneration. Compared to MCECs
transfected with Ang1 siRNA, the transfection of Ang1 siRNA into MCPs or both
MCECs and MCPs profoundly diminished the DKK2 mediated enhancement of tube
formation in mixed MCEC MCP co culture exposed to HG (Supplementary Fig. 11A
and B). We also determined whether CM derived from MCECs or MCPs cultivated
under the aforementioned conditions affects neural regeneration. Similar to the results
of the tube formation assays, DKK2 mediated neurite sprouting was more impaired in
MPG tissue treated with CM derived from Ang1 siRNA transfected MCPs than in the
tissue treated with CM derived from Ang1 siRNA transfected MCECs (Supplementary
Fig. 11C and D). These findings suggest that pericyte derived Ang1 plays a crucial role
in DKK2 mediated angiogenesis and neural regeneration.
Insulin treatment does not prevent the deterioration of erectile function under
diabetic conditions
We finally examined whether insulin treatment rescues erectile function in diabetic
condition. The treatment of diabetic mice with insulin did not restore erectile function
(Supplementary Fig. 12A F). Insulin treatment also failed to restore cavernous DKK2
expression in the diabetic mice (Supplementary Fig. 12G and H). Metabolic and
physiologic variables, including body weight, blood glucose concentrations, and
systemic blood pressure, are summarized in Supplementary Table S7. Diabetes Page 20 of 84
Discussion
Here, we investigated whether DKK2 plays a role as a positive regulator of angiogenesis and neural regeneration, exerting beneficial effects in diabetic ED.
DKK2 mediated interactions between pericytes and endothelial cells promoted angiogenesis and neural regeneration through an Ang1 Tie2 pathway, rescuing erectile function in diabetic animals. The detailed mechanisms of action by which DKK2 restores erectile function are illustrated in Fig. 6.
To test whether DKK2 induces cavernous angiogenesis under pathological conditions, we immunohistochemically evaluated the expression of CD31 and platelet derived growth factor receptor β (PDGFRβ). Similar to the results of previous studies in STZ induced diabetic rats (28, 29) and mice (2, 9, 11), the cavernous endothelial cell and pericyte area was significantly smaller in WT diabetic mice than in control mice. BrdU labeling revealed increased endothelial cell and pericyte proliferation, and these cellular contents were relatively well preserved in STZ treated DKK2 Tg mice.
DKK2 protein also promoted tube formation, proliferation, and migration by endothelial cells and pericytes, and enhanced microvessel sprouting from the aortic ring under HG conditions.
Endothelial cell cell junctions serve as a barrier by regulating paracellular permeability and play a crucial role in vascular formation, the vascular network, and remodeling of blood vessels (30). Diabetes mellitus promotes LDL oxidation and extravasation, which in turn induces vascular inflammatory responses and endothelial cell apoptosis (31). We recently revealed impaired cavernous endothelial cell cell junctions and increased cavernous endothelial permeability to oxidized LDL in diabetic mice (32). In the present study, cavernous endothelial cell cell junction Page 21 of 84 Diabetes
proteins were well preserved and less oxidized LDL extravasated in STZ injected
DKK2 Tg mice than in STZ injected WT littermates. The restoration of endothelial
cell cell junctions and enhanced pericyte coverage on endothelial cells by DKK2 may
be attributable to a decrease in cavernous vascular permeability.
The interaction between endothelial cells and pericytes plays a crucial role in
blood vessel formation and vascular maturation (33). The close anatomical relationship
between endothelial cells and pericytes implicates paracrine or juxtacrine signaling, i.e.,
endothelial cell pericyte signaling. Using human and mouse erectile tissues in vivo,
and endothelial cells (MCECs and HUVECs) and pericytes (MCPs and HBMPs) in
vitro, we found that pericytes are the major source of DKK2 expression. In indirect
non contact MCEC and DKK2 RFP transfected MCP co culture experiments, we
confirmed that pericyte derived DKK2 migrates into endothelial cells. Moreover, we
found profound tube formation impairment in MCECs treated with DKK2 depleted
MCP CM, whereas the treatment of MCPs with DKK2 depleted MCEC CM did not
significantly affect tube formation. These findings suggest that pericyte derived DKK2
may play a crucial role in DKK2 mediated angiogenesis.
The number of functional nNOS+ neurons is critical for physiological penile
erection (34). In the present study, DKK2 Tg mice that received STZ had preserved
nNOS, neurofilament, and βIII tubulin expression in the corpus cavernosum and dorsal
nerve bundle. Moreover, direct administration of DKK2 protein or DKK2 protein
treated CM derived from MCEC MCP co culture profoundly enhanced neurite
sprouting in MPG tissue under HG conditions. In penis tissue from WT diabetic mice
and Neuro2A cells exposed to HG, the expression of NGF, BDNF, and TrkA was
greatly restored by treatment with DKK2 protein. Given that the activation of Wnt Diabetes Page 22 of 84
signaling has neuroprotective effects (35, 36), the Wnt signaling antagonist DKK2 may exert its neurotrophic effects independent of the Wnt pathway.
Transcriptome analysis showed that Ang1 and Ang2 are target genes of DKK2.
Inhibition of the Ang1 Tie2 pathway with sTie2 Fc diminished DKK2 mediated angiogenesis and neural regeneration in STZ injected DKK2 Tg mice and MCECs or
MPG tissue exposed to HG. Pre treatment with sTie2 Fc also abolished DKK2 mediated erectile function recovery in both STZ injected DKK2 Tg mice and STZ injected WT mice treated with DKK2 protein. These findings suggest that the Ang1
Tie2 pathway is crucial for DKK2 mediated restoration of the penile neurovascular structure and erectile function. Although the Ang1 Tie2 pathway is well known to play an important role in generating a stable and functional vasculature (37), its role in the nervous system is largely unknown. However, Ang1 has been reported to promote neurite outgrowth in dorsal root ganglion cells positive for Tie2 receptor through transactivation of TrkA receptor (38).
For clinical applications, a GLP preclinical study of DKK2 protein has begun.
An interim report revealed no toxicity in C.B 17 SCID mice (i.e., no unscheduled death, clinical signs, changes in body weight, or abnormal necropsy findings) after a single intravenous injection of DKK2 protein at a dosage of up to 90 mg/kg body weight (Supporting Data 1). Also, no tumorigenesis related to DKK2 protein was noted in breast cancer cell lines (MDA MB 231, MCF 7) or a lung cancer cell line (A549) in vitro (Supporting Data 2) or in vivo after implantation of a lung cancer cell line (A549) in nude mice (Supporting Data 3). It was reported that DKK2 Tg mice exhibited increased retinal blood vessel formation during developmental period (20). DKK2 Tg mice also showed increased blood vessel density and decreased hemorrhage in the Page 23 of 84 Diabetes
oxygen induced retinopathy model (39), suggesting clinical utility of DKK2 in diabetic
retinopathy.
Our study has some limitations. First, this study did not explain how DKK2
regulates the expression of Ang1 and Ang2. Second, the mechanisms by which the
Ang1 Tie2 pathway is involved in neural regeneration need to be documented in detail.
Finally, we measured systemic blood pressure by use of a non invasive tail cuff system,
not by direct carotid artery cannulation, because of concerns about the viability of
animals and the reliability of the ICP results with significant bleeding during the carotid
artery cannulation. Systemic blood pressure and ICP were not measured simultaneously,
because vibrations occurring from electrical stimulation of the cavernous nerve can
impede the accurate measurement of systemic blood pressure by use of the tail cuff
system. Thus, we measured systemic blood pressure immediately before electrical
stimulation of the cavernous nerve.
Taken together, our findings reveal a unique function of DKK2 in
reprogramming damaged erectile tissue toward neurovascular repair through an Ang1
Tie2 pathway. Dual angiogenic and neurotrophic effects of DKK2, especially in the
form of locally injectable protein, may provide a paradigm shift in the development of
novel therapeutics not only for ED, but also for other ischemic vascular diseases or
neurological disorders. Diabetes Page 24 of 84
Acknowledgements. This work was supported by a grant from the Korean Health
Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (J. K.S.,
A110076; J. K.R. and H.M.K., H15C0508), by a Medical Research Center Grant (J.
K.R. and H.J.P., 2014R1A5A2009392), and by the National Research Foundation of
Korea (NRF) grant (J. K.R., 2016R1A2B2010087) funded by the Korean government
(Ministry of Science, ICT and Future Planning). Professor Ji Kan Ryu is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Duality of interest. No potential conflicts of interest relevant to this article were reported.
Author contributions. G.N.Y., H. R.J., J. K.R., and J. K.S. designed the experiments and wrote the manuscript; G.N.Y., H. R.J., M. J.C., A.L., K.G., N.N.M., J.O., K. M.H., and K. M.S. performed the experiments; Y. G.K. and H.M.K. contributed essential reagents; H.J.P. analyzed and critically discussed the data; and J. K.R. and J. K.S. supervised the project. Page 25 of 84 Diabetes
References
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Page 29 of 84 Diabetes
Figure legends
Figure 1. Decreased DKK2 expression under diabetic conditions. A and B:
Representative RT PCR and Western blots for DKK2 in mouse cavernous endothelial
cells (MCECs) and mouse cavernous pericytes (MCPs). C and D: Normalized band
intensity values (n = 4). *P < 0.05 vs. MCEC group. The relative ratio of the MCEC
group was arbitrarily set to 1. E and F: VWF (green)/CD31(green) and DKK2 (red) or
PDGFRβ (green) and DKK2 (red) staining in normal human or mouse cavernous tissue.
Nuclei were labeled with DAPI (blue). Scale bar = 100 µm. G and H: The DKK2
immunopositive area in cavernous endothelial cells and pericytes was quantified by
Image J. 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 MCPs exposed to normal glucose (NG) or high glucose (HG)
conditions for 48 hours. L-N: Normalized band intensity values (n = 4). *P< 0.05 vs.
Control or NG group. O: DKK2 (red) staining in cavernous tissue from a patient with
diabetic erectile dysfunction or diabetic mice and age matched controls. Scale bar =
100 µm. P and Q: The DKK2 immunopositive area was quantified by Image J. n = 1
human sample; n = 4 mouse samples. In human tissues, images were obtained for four
different regions per group. *P < 0.01 vs. control group. The P values were determined
by Mann Whitney U test. Data in graphs are presented as mean ± SE. The relative ratio
in the control or NG groups was arbitrarily set to 1. DM, diabetes mellitus; PDGFRβ,
platelet derived growth factor receptor β; VWF, von Willebrand factor.
Diabetes Page 30 of 84
Figure 2. DKK2 overexpression is resistant to diabetes induced angiopathy. A: CD31
(green) and PDGFRβ (red) staining in cavernous tissue from wild type (WT) and
DKK2 Tg mice, WT mice receiving STZ, and DKK2 Tg mice receiving STZ. Scale bar = 100 µm. B: CD31 (red) and BrdU (green, arrow head) staining in penis tissue from each group. Nuclei were labeled with DAPI (blue). Scale bars = 50 µm (merged) and 25 µm (magnification). C: Tube formation assay in mouse cavernous endothelial cell (MCEC) or mouse cavernous pericyte (MCP) mono culture and MCEC MCP mixed co culture exposed to normal glucose (NG) or high glucose (HG) conditions for
48 hours and treated with PBS or DKK2 protein (200 ng/ml). 40× magnification. D:
Scratch wound healing assay in MCECs 24 hours after treatment. 40× magnification. E and F: Ex vivo aortic ring assay. The images were taken 5 days after treatment. 40× magnification. (F) DKK2 (red) staining in aortic ring. Nuclei were labeled with DAPI
(blue). The sprouting front and aortic tissue are demarcated by the white dashed line.
100× magnification. Results were similar in three independent experiments. G:
Quantification of cavernous endothelial cell and pericyte content by Image J (n = 6).
*P < 0.001 vs. WT and DKK2 Tg groups. #P < 0.001 vs. WT + STZ group. The relative ratio of the WT or DKK2 Tg group was arbitrarily set to 1. H: Number of
BrdU positive endothelial cells per high power field (n = 6). *P < 0.05 vs. WT and
DKK2 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 of outgrowing microvessels 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 mean ± SE. PDGFRβ, platelet Page 31 of 84 Diabetes
derived growth factor receptor β; STZ, streptozotocin.
Figure 3. Pericyte derived DKK2 migrates into endothelial cells and regulates
angiogenesis. A: DKK2 (red) staining in mouse cavernous endothelial cell (MCEC)
and mouse cavernous pericyte (MCP) mono culture and indirect non contact MCEC
MCP co culture. Nuclei were labeled with DAPI (blue). Note the increased DKK2
expression in MCECs after co culture. Scale bar = 50 µm. B: MCPs were transfected
with DKK2 RFP DNA then co cultured with MCECs. Note the DKK2 RFP expression
in MCECs after co culture. Images are representative of four independent experiments.
Scale bar = 100 µm. C and D: Tube formation assay in MCECs exposed to MCP
complement medium (control) or MCP conditioned culture medium (CM) with or
without deletion of DKK2 with neutralizing antibody. 40× magnification. Number of
tubes per high power field (n = 4). *P < 0.05 vs. control group. #P < 0.05 vs. untreated
CM group. E and F: Tube formation assay in MCPs exposed to MCEC complement
medium (control) or MCEC conditioned CM with or without deletion of DKK2 with
neutralizing antibody. 40× magnification. Number of tubes per high power field (n =
4). *P < 0.001 vs. control group. P values were determined by Kruskal Wallis test.
Data in graphs are presented as mean ± SE. RFP, red fluorescent protein.
Figure 4. DKK2 overexpression is resistant to diabetes induced neuropathy. A:
Neurofilament (NF; red), neuronal nitric oxide synthase (nNOS; red in dorsal nerve
and green in cavernosum), and βIII tubulin (red) staining in penis tissue from wild type
(WT) and DKK2 Tg mice, WT mice receiving streptozotocin (STZ), and DKK2 Tg
mice receiving STZ. Nuclei were labeled with DAPI (blue). Scale bars = 25 m (dorsal Diabetes Page 32 of 84
nerve bundle) or 50 m (cavernosum). B: βIII tubulin (red) staining in mouse pelvic ganglion (MPG) tissue culture exposed to normal glucose (NG) or high glucose (HG) conditions for 72 hours and treated with PBS or DKK2 protein (200 ng/ml). Scale bar
= 200 µm. C: Representative Western blot for neurotrophic factors (nerve growth factor [NGF], brain derived neurotrophic factor [BDNF], and neurotrophin 3 [NG3]) and tropomyosin receptor kinase A (TrkA) in penis tissue from age matched control or diabetic mice 2 weeks after repeated intracavernous injections of PBS (20 µl) or DKK2 protein (days 3 and 0; 6 µg/20 µl), and in Neuro2A cells exposed to NG or HG conditions for 48 hours and treated with PBS or DKK2 protein (200 ng/ml). D-F: The
NF, nNOS, and βIII tubulin immunopositive areas quantified in dorsal nerve bundle or cavernous tissue by Image J (n = 6). *P < 0.05 vs. WT and DKK2 Tg groups. #P <
0.05 vs. WT + STZ group. G: Quantification of neurite length by Image J (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 are presented as mean ± SE. The relative ratio in the WT, C, or NG group was arbitrarily set to 1.
Figure 5. DKK2 mediated recovery of erectile function is dependent on the Ang1 Tie2 signaling pathway. A: Microarray analysis using total RNA from mouse cavernous endothelial cells (MCECs) exposed to normal glucose (NG) or high glucose (HG) conditions for 48 hours and treated with PBS or DKK2 protein (200 ng/ml). B:
Representative RT PCR and Western blot for Ang1 and Ang2 in MCECs and mouse cavernous pericytes (MCPs) exposed to NG or HG conditions for 48 hours and treated Page 33 of 84 Diabetes
with PBS or DKK2 protein (200 ng/ml). C and D: Normalized band intensity values (n
= 4). *P < 0.05 vs. NG group. #P < 0.05 vs. PBS treated group. The relative ratio in the
NG group was arbitrarily set to 1. E-G: Representative intracavernous pressure (ICP)
responses for the DKK2 Tg mice, wild type (WT) mice receiving streptozotocin (STZ),
DKK2 Tg mice receiving STZ and dimeric Fc (4 µg), and DKK2 Tg mice receiving
STZ and soluble Tie2 antibody (sTie2; 4 µg) (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 after repeated intracavernous injections of PBS (20 µl), DKK2 protein (days
3 and 0; 6 µg/20 µl) 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 + Fc group. K:
CD31 (green), PDGFR β (red), and βIII tubulin (red) staining in penis tissue from each
group (n = 6). Scale bar = 100 µm (top), 25 µm (middle), and 50 µm (bottom). L: Tube
formation assay in MCECs exposed to NG or HG conditions for 48 hours and treated
with PBS, DKK2 protein (200 ng/ml), or DKK2 protein + sTie2 (100 ng/ml). 40×
magnification. M: βIII tubulin (red) staining in mouse pelvic ganglion (MPG) tissue
culture. N and O: The CD31, PDGFR β, and βIII tubulin immunopositive areas
quantified by Image J (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 relative ratio 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 Image J (n = 4). *P < 0.001 vs. NG group. #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. Diabetes Page 34 of 84
Data in graphs are presented as mean ± SE. PDGFRβ, platelet derived growth factor receptor β.
Figure 6. Schematic diagram of a proposed mechanism in which DKK2 preserves erectile function in diabetic mice. Pericyte derived DKK2 migrates into endothelial cells (ECs). The DKK2 mediated interaction between pericytes and endothelial cells then promotes angiogenesis and neural regeneration through an Ang1 Tie2 pathway, rescuing erectile function in diabetic condition. Ang, angiopoietin; BDNF, brain derived neurotrophic factor; ECJ, endothelial cell cell junction; eNOS, endothelial nitric oxide synthase; MCECs, mouse cavernous endothelial cells; MCPs, mouse cavernous pericytes; NGF, nerve growth factor; nNOS, neuronal nitric oxide synthase;
NT 3, neurotrophin 3; PC, pericyte; sTie2 = soluble Tie2 protein. Pagea 35 of 84 b Diabetesi j k MCECs MCPs MCECs MCPs Control DM NG HG NG HG DKK2 DKK2 DKK2 Penis WB MCPs -actin MCECs RT-PCR GAPDH -actin
l 1.5 m 1.5 n 1.5 c 2.5 d 4 * * 2.0 1.0 1.0 1.0
3 -actin) -actin) -actin)
1.5 -actin)
2 * 1.0 0.5 * 0.5 * 0.5 Relative ratio ratio Relative 1 ratio Relative Relative ratio ratio Relative (DKK2 (DKK2 / 0.5 (DKK2 / (DKK2 (DKK2 / Relative ratio ratio Relative Relative ratio ratio Relative 0.0 0.0 0.0 (DKK2 (DKK2 / GAPDH) 0.0 (DKK2 / 0 C DM NG HG NG HG MCECs MCPs MCECs MCPs Mouse MCECs MCPs o DKK2 DAPI Merged Magnification e VWF / Control Human Human DKK2 PDGFRβ DM / DKK2 f CD31 / Control Mouse Mouse DKK2 DM PDGFRβ / DKK2 g h p q 70 60 1.5 1.5 60 * 50 * 50 40 1.0 1.0 40 30 30 20 0.5 0.5 20 * /Cavernosum /Cavernosum DKK2 DKK2 (+) area 10 DKK2 (+) area
10 (%) cavernosum
cavernosum (%) cavernosum *
Merged area/Mouse area/Mouse Merged 0 0.0 0.0 Merged area/Human area/Human Merged 0 VWF PDGFRβ CD31 PDGFRβ C DM C DM Human Mouse a g b Endothelium or Pericyte Magnification Merged BrdU CD31 Merged PDGFRβ CD31 area/Cavernosum 0.0 0.5 1.0 1.5 TWT WT WT WT PDGFRβ CD31 DKK2 -Tg * * STZ DKK2 # -Tg DKK2-Tg # DKK2-Tg h No.BrdU (+) endothelial
cells/HPF 10 0 2 4 6 8 TWT WT DKK2 -Tg WT+STZ DKK2-Tg+ STZ DKK2-Tg+ WT+STZ WTSTZ + * STZ DKK2 -Tg # i
Branch points/Field STZ + DKK2-Tg 100 20 40 60 80 0 MCECs-MCPs MCPs MCECs GPSDKK2 PBS NG Diabetes * * c * e f d HG Aortic ring Aortic ring MCEC-MCP mix
# assay assay MCECs ed co-culture MCPs MCECs Sprouting Sprouting # front # DKK2 NG NG NG
Number of j migrated cells 100 150 200
50 Aortic 0 ring GPSDKK2 PBS NG * HG + PBS HG + DKK2 + HG PBS + HG HG + PBS HG + DKK2 + HG PBS + HG HG+PBS HG DAPI # k Vascular area (%) 0.5 1.0 1.5 2.0 2.5 0 GPSDKK2 PBS NG HG+DKK2 Merged * HG 100x # 40x 40x 40x Page 36of84 Page 37of84 b a e c MCPs MCECs DKK2/DAPI MCPs Lipofectamine2000 MCEC complementMCEC MCPcomplement MCECs MCECs MCPs medium medium DKK2-RFPtransfection Mono-culture MCPs MCP-conditionedculture MCEC-conditioned DKK2-RFP culturemedium MCPs MCPs medium MCPs Diabetes MCECs DKK2 (-) MCEC-conditioned(-) DKK2 Lipofectamine2000 DKK2 (-) MCP-conditioned(-) DKK2 Co-culturewith MCPs for 24 hours culturemedium MCECs culturemedium MCECs MCPs Co-culture MCECs MCECs f d
Branch points/Field Branch points/Field DKK2-RFP 100 120 100 120 20 40 60 80 20 40 60 80 MCECs MCPs 0 MCPs 0 MCPs C NT DKK2 (-) DKK2 NT C C NT DKK2 (-) DKK2 NT C * * CM CM # a c Cavernosum Cavernosum Dorsal nerve Dorsal nerve Dorsal nerve βIII tubulin nNOS/DAPI βIII tubulin nNOS/DAPI NF/DAPI oto PSDKK2 PBS Control WT Cavernosum STZ K2T T+SZDKK2-TgSTZ + WTSTZ + DKK2-Tg GPSDKK2 PBS NG N2Acell HG -actin TrkA NT-3 BDNF NGF Diabetes i f Relative ratio βIII tubulin (+) area/Dorsal d
(BDNF/-actin) NF (+) area/Dorsal nerve b 0.0 0.5 1.0 1.5 0.0 0.5 1.0 1.5 2.0 nerve or Cavernosum 0.0 0.5 1.0 1.5 2.0 2.5 Merged Phase image βIII tubulin G P M /GPSDKK2 PBS C/NG TWT WT WT N2A cell N2A Cavernosum Cavernosum DNB DKK2 -Tg * DKK2 NG -Tg STZ/HG * * STZ # * # DKK2 WT -Tg # * STZ DKK2 # -Tg G P M j
Relative ratio # (NT-3/-actin) 0.0 0.5 1.0 1.5 2.0 G+PSHGDKK2 + HGPBS + g nNOS (+) area/Dorsal e Neurite length 0.0 0.5 1.0 1.5 nerve or Cavernosum /GPSDKK2 PBS C/NG 0.0 0.5 1.0 1.5 2.0 N2A cell N2A Cavernosum GPBS NG * WT Cavernosum DNB STZ/HG * * HG DKK2 DKK2 -Tg # G P M h * k Relative ratio Relative ratio WT
(TrkA/-actin) (NGF/-actin) * 0.0 0.5 1.0 1.5 2.0 0.0 0.5 1.0 1.5 2.0 STZ DKK2 # -Tg /GPSDKK2 PBS C/NG /GPBS C/NG N2A cell N2A Cavernosum N2A cell N2A Cavernosum # * * Page 38of84 STZ/HG STZ/HG * * DKK2 # # # # Page 39 of 84 Diabetes Diabetes Page 40 of 84
MCPs Ang1 DKK2 Cavernous nerve Ang2 DKK2
MCECs Ang1 DKK2 Ang2
Neurotrophic factors (NGF, BDNF, NT-3) EC regeneration sTie2 PC regeneration • Proliferation • Proliferation Neural regeneration sTie2 • Migration • Migration
eNOS activation Penile erection sTie2 Vessel ECJ proteins permeability MCPs-DKK2 Ang1 Page 41 of 84 Diabetes
Supplemental Materials, Tables, and Figures
Pericyte-derived Dickkopf2 regenerates damaged penile neurovasculature through an angiopoietin-1-Tie2 pathway
Guo Nan Yin1,*, Hai Rong Jin1,2,*, Min Ji Choi1, Anita Limanjaya1, Kalyan Ghatak1, Nguyen Nhat Minh1, Jiyeon Ock1, Mi Hye Kwon1, Kang Moon Song1, Heon Joo 3 4 5 1,6, † 1, † Park , Ho Min Kim , Young Guen Kwon , Ji Kan Ryu , Jun Kyu Suh
1National Research Center for Sexual Medicine and Department of Urology, Inha University School of Medicine, Incheon 22332, Republic of Korea; 2Department of Urology, Yuhuangding Hospital, Yantai 264000, Shandong Province, China; 3Hypoxia related Disease Research Center, Inha University College of Medicine, Incheon 22212, Republic of Korea; 4Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Republic of Korea; 5Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 03722, Republic of Korea; 6Inha Research Institute for Medical Sciences, Inha University College of Medicine, Incheon 22212, Republic of Korea
*These authors contributed equally to this work.
†Correspondence and requests for materials should be addressed to J.K.S. or J.K.R. (email: [email protected] or [email protected])
Jun Kyu Suh, MD, PhD National Research Center for Sexual Medicine and Department of Urology Inha University College of Medicine 7 206, 3rd St, Shinheung Dong, Jung Gu Incheon 22332, Republic of Korea Tel: 82 32 890 3441, Fax: 82 32 890 3097 Diabetes Page 42 of 84
E mail: [email protected]
Ji Kan Ryu, MD, PhD National Research Center for Sexual Medicine and Department of Urology Inha University College of Medicine 7 206, 3rd ST, Shinheung Dong, Jung Gu, Incheon 22332 Republic of Korea Tel: 82 32 890 3505; Fax: 82 32 890 3099 E mail: [email protected] Page 43 of 84 Diabetes
Supplemental Materials
Measurement of erectile function
Bipolar platinum wire electrodes were placed around the cavernous nerve.
Stimulation parameters were 5 V at a frequency of 12 Hz, a pulse width of 1 ms, and
duration of 1 minute. The maximal intracavernous pressure (ICP) was recorded
during tumescence. The total ICP was determined by the area under the curve from
the beginning of cavernous nerve stimulation to a point 20 s after stimulus
termination. Two electrostimulations were replicated at intervals of 10 minutes.
Systemic blood pressure was measured using a non invasive tail cuff system
(Visitech systems). The validity of this system was demonstrated previously (1).
Systemic blood pressure was measured prior to the measurement of ICP, because
vibration during electrical stimulation the cavernous nerve did not allow accurate
assessment of blood pressure. The ratio of maximal ICP and total ICP to mean
systolic blood pressure was calculated to adjust for variations in systemic blood
pressure.
Cell culture
Penis tissue was harvested and transferred into sterile vials containing Hank’s
balanced salt solution (Gibco) and washed twice in PBS. The glans penis, urethra,
and dorsal neurovascular bundle were removed from the penis, and only the corpus
cavernosum tissue was used for primary cell culture. For primary culture of mouse
cavernous endothelial cells (MCEC), the corpus cavernosum tissue was cut into two
or three pieces and the samples plated on matrigel coated (Becton Dickinson) 60 Diabetes Page 44 of 84
mm cell culture dishes. The matrigel was polymerized with a 5 minute incubation period at 37°C; 3 ml of complement medium 199 (Gibco) supplemented with 20% fetal bovine serum (FBS), 1% penicillin/streptomycin, 0.5 mg/ml heparin (Sigma
Aldrich), and 5 ng/ml vascular endothelial growth factor (VEGF, R&D Systems) was added to the cell culture dish. The dishes were incubated at 37°C in a 5% CO2 atmosphere. After the cells were confluent and spread over the bottom of the dish
(~2 to 3 weeks after the start of culture), only sprouting cells were used for subcultivation. The sprouting cells were seeded onto dishes coated with 0.2% gelatin
(Sigma Aldrich).
For primary culture of mouse cavernous pericytes (MCP), the corpus cavernosum tissue was cut into several 1 mm pieces and the fragmented pieces settled by gravity into collagen I coated 35 mm cell culture dishes (BD Biosciences).
After 30 minutes of incubation at 37°C with 300 µl complement Dulbecco’s modified Eagle Medium (DMEM; Gibco) supplemented with 10% FBS, 1% penicillin/streptomycin, and 10 nM human pigment epithelium derived factor
(PEDF; Sigma Aldrich), we added an additional 900 µl complement medium and incubated the samples at 37°C in a 5% CO2 atmosphere. The medium was changed every 2 days. After the cells were confluent and spread over the bottom of the dish
(~2 weeks after the start of culture), only sprouting cells were used for subcultivation. The sprouting cells were seeded onto dishes coated with 50 µl/ml collagen I (Advanced BioMatrix). Cells between passages 2 and 3 were used for experiments.
Human umbilical vein endothelial cells (HUVEC) were cultured in complement medium 199 (Gibco) supplemented with 20% FBS, 1% Page 45 of 84 Diabetes
penicillin/streptomycin, 0.5 mg/ml heparin (Sigma Aldrich), and 5 ng/ml VEGF
(R&D Systems). Human brain microvascular pericytes (HBMPs) and Neuro2A cells
were cultured in DMEM supplemented with 10% FBS and 1%
penicillin/streptomycin.
Establishment of MCEC-MCP co-culture system
We established indirect non contact and direct mixed co culture systems to examine
the interaction between endothelial cells and pericytes. For the indirect non contact
co culture system, the inside of the insert (1.0 µm pore size Transwells; Becton
Dickinson) was coated with 0.2% gelatin and the bottom of the culture plate with 50
µl collagen I. MCECs were then plated on the inside of the insert and MCPs on the
bottom of the culture plate until cells were confluent. To determine the optimal ratio
of MCECs and MCPs for direct mixed co culture, the cells were cultivated at
different ratios (MCEC:MCP = 10:1, 5:1, 3:1, 2:1, 1:1) in 50% DMEM and 50%
M199 complement medium.
Tube formation assay
Approximately 50 µl of growth factor reduced matrigel (Collaborative Biomedical
Products) was dispensed into 96 well tissue culture plates at 4°C. After gelling at
37°C for at least 30 minutes, the MCECs or MCPs were seeded onto the gel at 2 ×
104 cells/well in 200 µl of M199 or DMEM medium. The assay was performed in a
CO2 incubator and the plates incubated at 37°C for 24 hours. Images were obtained
with a phase contrast microscope and the number of tubes in each well of the plate
counted at a screen magnification of ×40. Only integrated tubes were counted. Diabetes Page 46 of 84
Scratch wound-healing assay
MCECs, MCPs, HVECs, or HBMPs were plated on 60 mm culture dishes at 95% confluence, wounded with a 2 mm wide razor blade, and marked at the injury line.
After wounding, the cultures were washed with serum free medium and further incubated in M199 media or DMEM with 2% FBS for 24 hours. Phase contrast images were taken 24 hours after scratching. Migration was quantitated by counting the number of cells that moved beyond the reference line.
DNA transfection
The mouse DKK2 (NM_020265.3) ORF mammalian expression plasmid (C
OFPSpark/RFP tag cDNA) was purchased from Sino Biological. Inc. (SB, Beijing,
China). Transient transfection was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. MCPs were transfected with 1 µg of
DKK2 RFP at a density of 5 × 105 cells/wells.
RNA interference
MCECs or MCPs at a density of 2 × 106 cells/well were transfected with siRNA against Ang1 (200 pmol; Santa Cruz Biotechnology) using Lipofectamine 2000
(Invitrogen).
RT-PCR
Total RNA was extracted from cultured cells using Trizol (Thermo Fisher Scientific) following the manufacturer’s protocols. Reverse transcription was performed using 1 Page 47 of 84 Diabetes
µg of RNA in 20 µl of reaction buffer with oligo dT primer and AccuPower RT
Premix (Bioneer). We used the following primers to measure relative changes in
mRNA levels: mouse Dkk2 forward, 5′ TGA TGG TGG AGA GCT CAC AG 3′;
mouse Dkk2 reverse, 5′ GTA GGC ATG GGT CTC CTT CA 3′; mouse Ang1 forward,
5′ AAA CAG CAA ATG GGA ACA GG 3′; mouse Ang1 reverse, 5′ GGG CAG GTG
AAC TCC ACT AA 3′; mouse Ang2 forward, 5′ CAA GGC ACT GAG AGA CAC
CA 3′; mouse Ang2 reverse, 5′ CTG AAC TCC CAC GGA ACA TT 3′; human
DKK2 forward, 5′ TAG AGA TTG AGT TTG AGC CT 3′; human DKK2 reverse, 5′
AAA GGG TGG ACA TAA GAA A 3′; GAPDH forward, 5′ CCA CTG GCG TCT
TCA CCA C 3′; GAPDH reverse, 5′ CCT GCT TCA CCA CCT TCT TG 3′. The
PCR reaction was performed with denaturation at 94°C for 30 s, annealing at 60°C
for 30 s, and extension at 72°C for 1 minute in a DNA Engine Tetrad Peltier Thermal
Cycler (MJ Research). For the analysis of PCR products, 10 µl of each PCR reaction
was electrophoresed on 1% agarose gel and detected under ultraviolet light. GAPDH
was used as an internal control.
cDNA microarray
For the microarray study, the MCECs were cultured and treated (n = 3 per group)
with normal glucose (NG, 5 mmol), high glucose (HG, 30 mmol) and PBS, or HG
(30 mmol) and DKK2 protein (200 ng/ml). The cDNA microarray analysis was
performed 48 hours after treatment. For the RNAs isolated from the three groups, the
synthesis of target cRNA probes and hybridization were performed using Agilent’s
Low RNA Input Linear Amplification kit (Agilent Technology) according to the
manufacturer’s instructions. The hybridized images were scanned using an Agilent Diabetes Page 48 of 84
DNA microarray scanner and quantified by Feature Extraction Software (Agilent
Technology). All data normalization and gene selection were performed using
GeneSpringGX 7.3 (Agilent Technology). The average normalized ratios were calculated by dividing the average normalized signal channel intensity by the average normalized control channel intensity. Functional gene annotation was performed according to the Gene OntologyTM Consortium
(http://www.geneontology.org/index.shtml) using GeneSpringGX 7.3. gene classification based on searches performed by BioCarta (http://www.biocarta.com/),
GenMAPP (http://www.genmapp.org/), DAVID (http://david.abcc.ncifcrf.gov/), and
Medline databases (http://www.ncbi.nlm.nih.gov/). The cDNA microarray data have been deposited in the Gene Expression Omnibus database
(www.ncbi.nlm.nih.gov/geo accession no. GSE95178).
Histological examinations
The penis tissue and cultured major pelvic ganglion or aortic ring were fixed in 4% paraformaldehyde for 24 hours at 4°C and frozen tissue sections (8 m [thin cut] or 60
m [thick cut]) incubated with antibodies to DKK2 (1:100; Abcam, Cat. ab95274),
DKK2 (1:50; Santa Cruz Biotechnology, Cat. sc 25517), CD31 (1:50; Millipore, Cat.
MAB1398Z), von Willebrand factor (VWF; 1:50; Santa Cruz Biotechnology, Cat. sc
59810), fluorescein isothiocyanate (FITC) conjugated antibody to smooth muscle α actin (1:200; Sigma Aldrich, Cat. F3777), PDGFR β (1:50; Santa Cruz Biotechnology,
Cat. sc 1627), neurofilament (1:100; Sigma Aldrich, Cat. N 5389), nNOS (1:100;
Santa Cruz Biotechnology, Cat. sc 5302), βIII tubulin (1:200; Abcam, Cat. ab107216),
5′ bromo 2′ deoxyuridine (BrdU, 1:50; AbD Serotec, Cat. MCA2060), phospho eNOS Page 49 of 84 Diabetes
(1:100; Cell Signaling, Cat. 9571), claudin 5 (1:100; Thermo Fisher Scientific, Cat.
352500), VE cadherin (1:100; Millipore, Cat. AB1955), nitrotyrosine (1:50; Millipore,
Cat. 06 284), or oxidized LDL (1:400; Abcam, Cat. ab14519) at 4°C overnight.
Control sections were incubated without the primary antibody at this step. After
several washes with PBS, the sections were incubated with tetramethyl rhodamine
isothiocyanate (TRITC) conjugated (Jackson ImmunoResearch, Inc., Cat. 127 025
160 for Hamster; Cat. 711 026 152 for Rabbit; Cat. 315 025 003 for Mouse; Cat.
705 025 147 for Goat; Cat. 703 025 155 for Chicken.) or FITC conjugated
secondary antibodies (Jackson ImmunoResearch, Inc., Cat. 127 095 160 for
Hamster; Cat. 711 096 152 for Rabbit; Cat. 715 545 150 for Mouse; Cat. 705 545
147 for Goat) or DyLight TM 405 (Jackson ImmunoResearch, Inc., Cat. 127 475
160) for 2 hours at room temperature. Signals were visualized and digital images
obtained with a confocal microscope (FV1000, Olympus). For BrdU labeling
experiments, an additional antigen retrieval step was performed. Briefly, following
fixation, the sections were washed with PBS three times and incubated for 10
minutes in 1M HCl on ice and for 20 minutes in 2M HCl at 37°C to allow DNA
denaturation. The sections were then washed three times (5 minutes each) in 0.1M
sodium borate buffer (Na2B4O7). Histological examinations were quantitated using
an image analyzer system (National Institutes of Health [NIH] Image J 1.34,
http://rsbweb.nih.gov/ij/).
In situ detection of superoxide anion
Hydroethidine (1:5000; Molecular Probes, Cat. D 23107), an oxidative fluorescent
dye, was used to evaluate levels of superoxide anion in situ as previously described Diabetes Page 50 of 84
(2, 3). After immunofluorescent staining with antibody to CD31 and FITC conjugated secondary antibodies, hydroethidine (2 µmol/l) was applied to each tissue section and the samples were coverslipped. The slides were incubated in a light protected humidified chamber at 37°C for 30 minutes. The number of ethidium bromide fluorescence positive endothelial cells was counted at a screen magnification of ×400 in 6 or 8 different regions. Values were expressed per high power field.
BrdU labeling
The mice from each group were given intraperitoneal injections of BrdU (50 mg/kg of body weight; Sigma Aldrich) once a day for three consecutive days and sacrificed
1 day after BrdU injection. The MCECs, MCPs, HUVECs, or HBMPs were incorporated with BrdU at a final concentration of 10 µM at 37°C for 1 hour. An additional antigen retrieval step was performed as described above. The number of
BrdU positive endothelial cells or pericytes was counted at a screen magnification of
400× in six or eight different regions. Values were expressed per high power field.
Western blot
The collected penile tissues were immediately stored within liquid nitrogen. The tissues were then homogenized with small volume of liquid nitrogen. This experimental step was repeated several times till changing to the complete powder of tissues. And then, the RAPI buffer (100 l, Sigma Aldrich) was added into the homogenates. After incubating on ice for 60 minutes, the homogenates were centrifuged at 13,000 g for 20 minutes at 4°C and the concentrations of each protein Page 51 of 84 Diabetes
in the supernatants were measured using the Universal Microplate Reader ELx800G
(BioTek Instruments Inc.). Equal amounts of protein (50 µg per lane) were
electrophoresed on 8% to 15% sodium dodecylsulfate polyacrylamide gels and
transferred to nitrocellulose membranes. After blocking with 5% nonfat dry milk for
1 hour at room temperature, the membrane was incubated at room temperature with
antibodies to DKK2 (1:500; Abcam, Cat. ab95274), BDNF (1:500; Santa Cruz
Biotechnology, Cat. sc 546), NGF (1:500; Santa Cruz Biotechnology, Cat. sc 548),
NT 3 (1:500; Santa Cruz Biotechnology, Cat. sc 547), TrkA (1:500; Abcam, Cat.
ab76291), Ang1 (1:500; NOVUS Biologicals, Cat. NB600 629), Ang2 (1:500;
NOVUS Biologicals, Cat. NB110 85467), iNOS (1:1000; Becton Dickinson, Cat.
610332), Akt (1:1000; Cell Signaling, Cat. 9272), phospho Akt (1:1000; Cell
Signaling, Cat. 9271), eNOS (1:1000; Becton Dickinson, Cat. 610297), phospho
eNOS (1:1000; Cell Signaling, Cat. 9571), or β actin (1:6000; Abcam, Cat. ab8226)
for 2 hour and washed 3 times with PBS T. The signals were visualized by using an
ECL (Amersham Pharmacia Biotech.) detection system. The results were quantified
by densitometry.
Immunoprecipitation
We performed additional experiments to confirm the Western blot data. Shortly,
immunoprecipitation was performed in lysates prepared from normal mice penile
tissues (500 g of total protein) with antibody to eNOS (1:1000; Becton Dickinson,
Cat. 610297) or diabetic mice penile tissues with antibody to iNOS (1:1000; Becton
Dickinson, Cat. 610332) at 4°C overnight. Then, the protein antibody complex was
incubated with 50 µL Protein G coupled Sepharose beads (Millipore) for 2 hours at Diabetes Page 52 of 84
4°C with gentle rotation. The beads were washed three times with lysis buffer. The pellet was resuspended with 30 µL lysis buffer and the sample was heated at 100°C for 10 minutes and microcentrifuged for 1 minute at 14,000 g. The sample was electrophoresed on 8% SDS PAGE gels and transferred to nitrocellulose membranes.
After blocking with 5% nonfat dry milk for 1 hour at room temperature, the membrane was incubated at room temperature with antibodies to phospho eNOS
(1:1000; Cell Signaling, Cat. 9571), eNOS (1:1000; Becton Dickinson, Cat. 610297), or iNOS (1:1000; Becton Dickinson, Cat. 610332). The signals were visualized by using an ECL detection system (Amersham Pharmacia Biotech.). Page 53 of 84 Diabetes
Supplemental References
1. Krege JH, Hodgin JB, Hagaman JR, Smithies O. A noninvasive computerized tail cuff system for measuring blood pressure in mice. Hypertension 1995;25:1111 1115 2. Bivalacqua TJ, Usta MF, Kendirci M, Pradhan L, Alvarez X, Champion HC, Kadowitz PJ, Hellstrom WJ. Superoxide anion production in the rat penis impairs erectile function in diabetes: influence of in vivo extracellular superoxide dismutase gene therapy. J Sex Med 2005;2:187 197 3. Jin HR, Kim WJ, Song JS, Choi MJ, Piao S, Shin SH, Tumurbaatar M, Tuvshintur B, Nam MS, Ryu JK, Suh JK. Functional and morphologic characterizations of the diabetic mouse corpus cavernosum: comparison of a multiple low dose and a single high dose streptozotocin protocols. J Sex Med 2009;6:3289 3304
Diabetes Page 54 of 84
Supplementary Tables:
Supplementary Table S1. Physiological and metabolic parameters 8 weeks after induction of diabetes with streptozotocin (STZ) in wild type (WT) and DKK2 Tg mice. WT DKK2 Tg WT + STZ DKK2 Tg + STZ Body weight (g) 30.7±1.1 29.7±0.8 25.5±0.9* 25.4±0.5* Fasting glucose (mg/dl) 114.3±6.6 111.3±9.8 393.5±51.1* 376.3±49.0* Postprandial glucose (mg/dl) 157.3±7.1 151.5±5.5 558.8±53.4* 556.5±23.4* MSBP (mmHg) 99.3±5.2 97.1±6.2 100.2±4.8 97.2±7.1
HbA1c (%) 5.0±0.2 4.9±0.3 11.2±0.1* 9.5±1.3*
HbA1c (mmol/mol) 31 30 98* 81* Insulin (µg/l) 2.61±0.21 2.75±0.08 0.18±0.02* 0.20±0.0.03* Values are presented as the mean ± SE for n = 4 5 animals per group. *P < 0.01 vs. WT and DKK2 Tg mice. MSBP, mean systolic blood pressure. Page 55 of 84 Diabetes
Supplementary Table S2. Physiological and metabolic parameters 2 weeks after treatment with DKK2 protein. Control DM+PBS DM+DKK2 (6 µg) Body weight (g) 32.2±2.0 23.7±2.4* 23.6±2.6* Fasting glucose (mg/dl) 105.2±5.0 545.3±36.3* 504.6±54.3* Postprandial glucose (mg/dl) 153.0±13.9 594.5±6.6* 585.8±30.6* MSBP (mmHg) 99.5±4.7 98.6±4.1 91.5±2.9 Values are presented as the mean ± SE for n = 6 animals per group. *P < 0.01 vs. Control. DM, diabetes mellitus; PBS, phosphate buffered saline; MSBP, mean systolic blood pressure.
Diabetes Page 56 of 84
Supplementary Table S3. Physiological and metabolic parameters 8 weeks after induction of diabetes with streptozotocin (STZ) in wild type (WT) and DKK2 Tg mice.
DKK2 Tg WT+STZ DKK2 Tg+STZ+Fc DKK2 Tg + STZ+sTie2 Body weight (g) 33.4±1.4 24.9±2.2* 24.7±1.8* 22.9±1.7* Fasting glucose (mg/dl) 104.5±6.4 483.0±52.5* 466.8±13.3* 499.5±52.6* Postprandial glucose (mg/dl) 159.2±5.3 594.0±6.1* 579.5±25.9* 585.5±25.0* MSBP (mmHg) 98.8±10.6 101.1±13.6 99.8±10.3 103.0±5.5 Values are presented as the mean ± SE for n = 5 animals per group. *P < 0.01 vs. DKK2 Tg mice. Fc, dimeric Fc protein; MSBP, mean systolic blood pressure; sTie2, soluble Tie2 antibody.
Page 57 of 84 Diabetes
Supplementary Table S4. Physiological and metabolic parameters 2 weeks after treatment with DKK2 protein or soluble Tie2 antibody (sTie2). Control DM+PBS DM+DKK2+Fc DM+DKK2+sTie2 Body weight (g) 32.4±1.9 22.2±1.2* 22.0±1.3* 22.8±0.6*
Fasting glucose (mg/dl) 104.3±5.2 557.8±17.4* 508.5±61.9* 497.5±42.0*
Postprandial glucose (mg/dl) 155.5±14.7 597.8±4.5* 582.3±34.2* 565.5±33.7* MSBP (mmHg) 100.0±5.7 98.4±3.8 100.8±6.9 99.1±1.8 Values are the mean ± SE for n = 5 animals per group. *P < 0.01 vs. Control. DM, diabete mellitus; Fc, dimeric Fc protein; MSBP, mean systolic blood pressure; PBS, phosphate buffered saline.
Diabetes Page 58 of 84
Supplementary Table S5. Summary of selected genes decreased in the HG + PBS group compared to the NG group and restored by DKK2 treatment. HG + PBS/ HG + DKK2/ Gene Product GenBank NG* HG + PBS* Angpt1 Angiopoietin 1 0.2 2.0 NM_009640 Sh3bgrl2 SH3 domain binding glutamic acid rich protein like 0.4 3.9 NM_172507 Chmp4b Chromatin modifying protein 4B 0.4 2.2 NM_029362 * MCECs were exposed to normal glucose (NG) or high glucose (HG) conditions and treated with phosphate buffered saline (PBS) or DKK2 protein. We selected genes that changed more than 2 fold for both HG + PBS/NG and HG + DKK2/HG + PBS.
Page 59 of 84 Diabetes
Supplementary Table S6. Summary of selected genes that increased in the HG + PBS group compared to the NG group and restored by DKK2 treatment. HG + HG + DKK2/ Gene Product GenBank PBS/ NG* HG + PBS* Ptpn13 Protein tyrosine phosphatase, non receptor type 13 2.0 0.5 NM_011204 Sema7a Sema domain, immunoglobulin domain (Ig), and GPI membrane anchor 2.0 0.5 NM_011352 Lxn Latexin 2.0 0.5 NM_016753 Nhsl2 NHS like 2 2.0 0.5 NM_001163610 Fabp4 Fatty acid binding protein 4 2.0 0.4 NM_024406 Tsr2 TSR2, 20S rRNA accumulation, homolog 2.0 0.4 NM_001164578 Odz4 Odd Oz/ten m homolog 4 2.0 0.4 NM_011858 Lhx8 LIM homeobox protein 8 2.0 0.4 NM_010713 Fam164a Family with sequence similarity 164, member A 2.0 0.4 NM_173181 Zfr2 Zinc finger RNA binding protein 2 2.0 0.5 NM_001034895 Kcnip3 Kv channel interacting protein 3, calsenilin 2.0 0.5 NM_001111331 Fzd4 Frizzled homolog 4 (Drosophila) 2.0 0.2 NM_008055 C1rb Complement component 1, r subcomponent B 2.0 0.3 NM_001113356 Klf15 Kruppel like factor 15 2.0 0.4 NM_023184 Arl4d ADP ribosylation factor like 4D 2.0 0.4 NM_025404 Nkd1 Naked cuticle 1 homolog (Drosophila) 2.0 0.2 NM_027280 Diabetes Page 60 of 84
Synm Synemin, intermediate filament protein 2.0 0.4 NM_201639 Fam149a Family with sequence similarity 149, member A 2.0 0.5 NM_153535 Zfp82 Zinc finger protein 82 2.0 0.4 NM_177889 Fign Mus musculus fidgetin (Fign), mRNA [NM_021716] 2.0 0.4 NM_021716 Rnf125 Ring finger protein 125 2.1 0.5 NM_026301 Xrcc6bp1 XRCC6 binding protein 1 2.1 0.4 NM_026858 Lpl Lipoprotein lipase 2.1 0.3 NM_008509 Entpd2 Ectonucleoside triphosphate diphosphohydrolase 2 2.1 0.4 NM_009849 Amz2 Archaelysin family metallopeptidase 2 2.1 0.5 NM_025275 Jag1 Jagged 1 2.1 0.3 NM_013822 Aspn Asporin 2.1 0.3 NM_025711 Dnahc7b Dynein, axonemal, heavy chain 7B 2.1 0.5 NM_001160386 Gm1661 Predicted gene 1661 2.1 0.2 NM_001145637 Ankrd29 Ankyrin repeat domain 29 2.1 0.5 NM_001190371 Susd2 Sushi domain containing 2 2.1 0.2 NM_027890 Scara5 Scavenger receptor class A, member 5 2.1 0.2 NM_028903 Plscr2 Phospholipid scramblase 2 2.1 0.3 NM_008880 Tfpi Tissue factor pathway inhibitor 2.1 0.4 NM_011576 Tspyl3 TSPY like 3 2.1 0.4 NM_198617 Zmat1 Zinc finger, matrin type 1 2.1 0.4 NM_175446 Gulp1 GULP, engulfment adaptor PTB domain containing 1 2.1 0.4 NM_027506 Page 61 of 84 Diabetes
Ccdc80 Coiled coil domain containing 80 2.1 0.5 NM_026439 Adh7 Alcohol dehydrogenase 7 (class IV), mu or sigma polypeptide 2.1 0.2 NM_009626 Pcdh10 Protocadherin 10 2.1 0.4 NM_001098172 Igdcc4 Immunoglobulin superfamily, DCC subclass, member 4 2.1 0.3 NM_020043 Reps2 RALBP1 associated Eps domain containing protein 2 2.1 0.3 NM_178256 Phxr4 Per hexamer repeat gene 4 2.1 0.4 NM_008835 Mcc Mutated in colorectal cancers 2.1 0.3 NM_001085373 Cxcl14 Chemokine (C X C motif) ligand 14 2.1 0.3 NM_019568 Zfp467 Zinc finger protein 467 2.1 0.3 NM_001085417 Prr24 Proline rich 24 2.1 0.5 NM_001136270 N6amt1 N 6 adenine specific DNA methyltransferase 1 2.1 0.4 NM_026366 Tfpi Tissue factor pathway inhibitor 2.1 0.5 NM_011576 Mrvi1 MRV integration site 1 2.1 0.3 NM_194464 Dpep1 Dipeptidase 1 (renal) 2.1 0.2 NM_007876 Rab3d RAB3D, member RAS oncogene family 2.1 0.4 NM_031874 Pappa Pregnancy associated plasma protein A 2.1 0.3 NM_021362 Smad9 MAD homolog 9 (Drosophila) 2.1 0.5 NM_019483 Rab6b RAB6B, member RAS oncogene family 2.2 0.4 NM_173781 Lifr Leukemia inhibitory factor receptor 2.2 0.2 NM_013584 C1ra Complement component 1, r subcomponent A 2.2 0.2 NM_023143 H19 H19 fetal liver mRNA 2.2 0.3 NR_001592 Diabetes Page 62 of 84
BC026782 cDNA sequence BC026782 2.2 0.3 NM_001025575 Vcam1 Vascular cell adhesion molecule 1 2.2 0.5 NM_011693 Rbms3 RNA binding motif, single stranded interacting protein 2.2 0.4 NM_001172123 Arhgef17 Rho guanine nucleotide exchange factor (GEF) 17 2.2 0.4 XM_133692 Bace2 Beta site APP cleaving enzyme 2 2.2 0.4 NM_019517 Rnd3 Rho family GTPase 3 2.2 0.4 NM_028810 Gabra1 Gamma aminobutyric acid (GABA) A receptor, subunit alpha 1 2.2 0.2 NM_010250 Sspn Sarcospan 2.2 0.4 NM_010656 Clca2 Chloride channel calcium activated 2 2.2 0.4 NM_030601 Ap2b1 Adaptor related protein complex 2, beta 1 subunit 2.2 0.5 NM_027915 Msmp Microseminoprotein, prostate associated 2.2 0.2 NM_001099314 Ccl27a Chemokine (C C motif) ligand 27A 2.2 0.5 NM_001048179 Fh1 Fumarate hydratase 1 2.2 0.5 NM_010209 Abca5 ATP binding cassette, sub family A (ABC1), member 5 2.2 0.5 NM_147219 Myh8 Myosin, heavy polypeptide 8, skeletal muscle, perinatal 2.2 0.1 NM_177369 Epyc Epiphycan 2.2 0.3 NM_007884 Tnfrsf25 Tumor necrosis factor receptor superfamily, member 25 2.2 0.3 NM_033042 Kctd12 Potassium channel tetramerization domain containing 12 2.2 0.3 NM_177715
Kcnab1 Potassium voltage gated channel, shaker related subfamily, beta member 1 2.2 0.5 NM_010597
Crispld2 Cysteine rich secretory protein LCCL domain containing 2 2.2 0.3 NM_030209 C1qtnf7 C1q and tumor necrosis factor related protein 7 2.2 0.4 NM_001135172 Page 63 of 84 Diabetes
Art3 ADP ribosyltransferase 3 2.2 0.2 NM_181728 Ebf2 Early B cell factor 2 2.2 0.2 NM_010095 Ctla2a Cytotoxic T lymphocyte associated protein 2 alpha 2.2 0.4 NM_007796 Marcks Myristoylated alanine rich protein kinase C substrate 2.2 0.5 NM_008538 Ctps2 Cytidine 5' triphosphate synthase 2 2.2 0.3 NM_018737 Art3 ADP ribosyltransferase 3 2.2 0.4 NM_181728 Sgce Sarcoglycan, epsilon 2.2 0.5 NM_011360 Zscan18 Zinc finger and SCAN domain containing 18 2.2 0.5 NM_001017955 Eif2c4 Eukaryotic translation initiation factor 2C, 4 2.2 0.4 NM_153177 Col5a2 Collagen, type V, alpha 2 2.3 0.3 NM_007737 Raver2 Ribonucleoprotein, PTB binding 2 2.3 0.2 NM_183024 Gabrd Gamma aminobutyric acid (GABA) A receptor, subunit delta 2.3 0.3 NM_008072 Radil Ras association and DIL domains 2.3 0.5 NM_178702 Ptpru Protein tyrosine phosphatase, receptor type, U 2.3 0.3 NM_001083119 Lgi4 Leucine rich repeat LGI family, member 4 2.3 0.2 NM_144556 Mum1l1 Melanoma associated antigen (mutated) 1 like 1 2.3 0.4 NM_175541 Postn Periostin, osteoblast specific factor 2.3 0.2 NM_015784 Nsun7 NOL1/NOP2/Sun domain family, member 7 2.3 0.5 NM_027602 Me3 Malic enzyme 3, NADP(+) dependent, mitochondrial 2.3 0.2 NM_181407 Calml4 Calmodulin like 4 2.3 0.3 NM_138304 Bicc1 Bicaudal C homolog 1 (Drosophila) 2.3 0.3 NM_031397 Diabetes Page 64 of 84
Fhl2 Four and a half LIM domains 2 2.3 0.3 NM_010212 Caprin2 Caprin family member 2 2.3 0.5 NM_181541 Rspo2 R spondin 2 homolog 2.3 0.3 NM_172815 Rufy4 RUN and FYVE domain containing 4 2.3 0.4 NM_001034060 Sipa1l1 Signal induced proliferation associated 1 like 1 2.3 0.4 NM_001167983 Flrt3 Fibronectin leucine rich transmembrane protein 3 2.3 0.4 NM_001172160 Ddx4 EEAD (Asp Glu Ala Asp) box polypeptide 4 2.3 0.1 NM_010029 Sparcl1 SPARC like 1 2.3 0.2 NM_010097 Gstm1 Glutathione S transferase, mu 1 2.3 0.5 NM_010358 Acrbp Proacrosin binding protein 2.3 0.4 NM_016845 Myl1 Myosin, light polypeptide 1 2.3 0.0 NM_021285 Man1a2 Mannosidase, alpha, class 1A, member 2 2.3 0.5 NM_010763 Nrbp2 Nuclear receptor binding protein 2 2.4 0.3 NM_144847 Aqp11 Aquaporin 11 2.4 0.5 NM_175105 Rasgrp2 RAS, guanyl releasing protein 2 2.4 0.4 NM_011242 Ccl27a Chemokine (C C motif) ligand 27A 2.4 0.5 NM_011336 Ppara Peroxisome proliferator activated receptor alpha 2.4 0.5 NM_011144 Abi3bp ABI gene family, member 3 (NESH) binding protein 2.4 0.3 NM_001014423 Dyx1c1 Dyslexia susceptibility 1 candidate 1 homolog (human) 2.4 0.4 NM_026314 Pde2a Phosphodiesterase 2A, cGMP stimulated 2.4 0.4 NM_001008548 Mosc1 MOCO sulfurase C terminal domain containing 1 2.4 0.3 NM_001081361 Page 65 of 84 Diabetes
Lrrn3 Leucine rich repeat protein 3, neuronal 2.4 0.4 NM_010733 Cxcl12 Chemokine (C X C motif) ligand 12 2.4 0.5 NM_021704 Il17rd Interleukin 17 receptor D 2.5 0.4 NM_13443 Dlc1 Deleted in liver cancer 1 2.5 0.4 XM_486096 Mtap7d3 MAP7 domain containing 3 2.5 0.4 NM_177293 Nxnl2 Nucleoredoxin like 2 2.5 0.3 NM_029173 Chadl Chondroadherin like 2.5 0.4 NM_001164320 Spry1 Sprouty homolog 1 (Drosophila) 2.5 0.2 NM_011896 Ogn Osteoglycin 2.5 0.1 NM_008760 Clca1 Chloride channel calcium activated 1 2.5 0.4 NM_009899 Amy1 Amylase 1, salivary 2.5 0.2 NM_007446 Prss12 Protease, serine, 12 neurotrypsin 2.5 0.4 NM_008939 Fam71e1 Family with sequence similarity 71, member E1 2.5 0.5 NM_028169 Uba7 Ubiquitin like modifier activating enzyme 7 2.5 0.5 NM_023738 Ctla2b Cytotoxic T lymphocyte associated protein 2 beta 2.5 0.5 NM_007797 Dnahc2 Dynein, axonemal, heavy chain 2 2.5 0.2 NM_001081330 Adig Adipogenin 2.6 0.2 NM_145635 Zfpm1 Zinc finger protein, multitype 1 2.6 0.5 NM_009569 Thbs4 Thrombospondin 4 2.6 0.3 NM_011582 Ccdc46 Coiled coil domain containing 46 2.6 0.5 NM_029606 Sfrs18 Splicing factor, arginine/serine rich 18 2.6 0.4 NM_025669 Diabetes Page 66 of 84
Ggt5 Gamma glutamyltransferase 5 2.6 0.5 NM_011820 Scai Suppressor of cancer cell invasion 2.6 0.5 NM_178778 Lpl Lipoprotein lipase 2.6 0.3 NM_008509 Zfpm1 Zinc finger protein, multitype 1 2.6 0.5 NM_009569 Ccdc106 Coiled coil domain containing 106 2.6 0.4 NM_146178 Avpr1a Arginine vasopressin receptor 1A 2.6 0.2 NM_016847 Ccdc80 Coiled coil domain containing 80 2.7 0.3 NM_026439 Igf1 Insulin like growth factor 1 2.7 0.3 NM_010512 Ankrd29 Ankyrin repeat domain 29 2.7 0.4 NM_001190371 Gnb1l Guanine nucleotide binding protein (G protein), beta polypeptide 1 like 2.7 0.5 NM_001081682 EG665756 Predicted gene, EG665756 2.7 0.5 XM_979235 Agtr1a Angiotensin II receptor, type 1a 2.7 0.2 NM_177322 Cd200 CD200 antigen 2.7 0.3 NM_010818 Txnrd3 Thioredoxin reductase 3 2.7 0.4 NM_153162 Epas1 Endothelial PAS domain protein 1 2.8 0.4 NM_010137 Cacna1g Calcium channel, voltage dependent, T type, alpha 1G subunit 2.8 0.3 NM_009783 Gprasp2 G protein coupled receptor associated sorting protein 2 2.8 0.3 XM_142154 Gpihbp1 GPI anchored HDL binding protein 1 2.8 0.5 NM_026730 Lrrtm1 Leucine rich repeat transmembrane neuronal 1 2.8 0.3 NM_028880 Pcsk6 Proprotein convertase subtilisin/kexin type 6 2.8 0.5 XM_355911 Id3 Inhibitor of DNA binding 3 2.8 0.5 NM_008321 Page 67 of 84 Diabetes
Cfh Complement component factor h 2.8 0.3 NM_009888 Wisp2 WNT1 inducible signaling pathway protein 2 2.8 0.1 NM_016873 Plekha4 Pleckstrin homology domain containing, family A member 4 2.8 0.3 NM_148927 Clstn2 Calsyntenin 2 2.8 0.4 NM_022319 Aldh1a3 Aldehyde dehydrogenase family 1, subfamily A3 2.8 0.2 NM_022319 Apoc1 Apolipoprotein C I 2.8 0.3 NM_007469 Myl9 Myosin, light polypeptide 9, regulatory 2.9 0.3 XM_485171 Cdh6 Cadherin 6 2.9 0.3 NM_007666 Kif5a Kinesin family member 5A 2.9 0.4 NM_001039000 Crygs Crystallin, gamma S 3.0 0.1 NM_009967 Cyp1a1 Cytochrome P450, family 1, subfamily a, polypeptide 1 3.0 0.4 NM_009992 Lgr5 Leucine rich repeat containing G protein coupled receptor 5 3.0 0.1 NM_010195 Crispld2 Cysteine rich secretory protein LCCL domain containing 2 3.0 0.3 NM_030209 Kcnip4 Kv channel interacting protein 4 3.1 0.4 NM_030265 Fat4 FAT tumor suppressor homolog 4 (Drosophila) 3.1 0.4 XM_619892 Gpd1 Glycerol 3 phosphate dehydrogenase 1 3.1 0.2 NM_010271 Kera Keratocan 3.2 0.5 NM_008438 Scube1 Signal peptide, CUB domain, EGF like 1 3.2 0.4 NM_022723 Sh3d19 SH3 domain protein D19 3.2 0.4 NM_001082414 Ptpn13 Protein tyrosine phosphatase, non receptor type 13 3.3 0.4 NM_011204 Omd Osteomodulin 3.4 0.2 NM_012050 Diabetes Page 68 of 84
Adrb3 Adrenergic receptor, beta 3 3.6 0.2 NM_013462 Itga11 Integrin alpha 11 3.7 0.2 NM_176922 G0s2 G0/G1 switch gene 2 3.9 0.2 NM_008059 Tspan18 Tetraspanin 18 3.9 0.1 NM_183180 Aoc3 Amine oxidase, copper containing 3 4.1 0.1 NM_009675 Ccny Cyclin Y 4.2 0.5 NM_026484 Cfd Complement factor D (adipsin) 4.3 0.1 NM_013459 Cdcp1 CUB domain containing protein 1 4.4 0.4 NM_133974 Tmed9 Transmembrane emp24 protein transport domain containing 9 4.8 0.4 NM_026211 Ptx3 Pentraxin related gene 5.3 0.2 NM_008987 Trio Triple functional domain (PTPRF interacting) 5.4 0.4 NM_001081302 Angpt2 Angiopoietin 2 5.5 0.5 NM_007426 Saa3 Serum amyloid A 3 5.8 0.3 NM_011315 Hp Haptoglobin 9.7 0.1 NM_017370
* MCECs were exposed to normal glucose (NG) or high glucose (HG) conditions and treated with phosphate buffered saline (PBS) or DKK2 protein. We selected genes that changed more than 2 fold for both HG + PBS/NG and HG + DKK2/HG + PBS.
Page 69 of 84 Diabetes
Supplementary Table S7. Physiological and metabolic parameters 10 weeks after induction of diabetes with streptozotocin (STZ) with or without treatment of insulin. Control STZ+PBS STZ+Insulin Body weight (g) 31.6±0.8 23.3±0.5* 26.9±1.1*
Fasting glucose (mg/dl) 104.4±3.2 492.8±12.6* 122.6±13.2#
Postprandial glucose (mg/dl) 160.6±3.5 591.4±5.3* 190.0±20.0# MSBP (mmHg) 106.1±2.2 102.6±1.7 104.2±2.2 Values are the mean ± SE for n = 4 5 animals per group. *P < 0.01 vs. Control. #P < 0.01 vs. STZ+PBS. MSBP, mean systolic blood pressure; PBS, phosphate buffered saline.
Diabetes Page 70 of 84
Supplemental Figures:
Supplementary Figure 1. DKK2 is mainly expressed in pericytes.
A and B: Representative RT PCR and Western blots for DKK2 in human umbilical vein endothelial cells (HUVECs) and human brain microvascular pericytes (HBMPs).
C and D: Normalized band intensity values (n = 4). *P < 0.05 vs. HUVECs, Mann
Whitney U test. Data are presented as mean ± SE. The relative ratio in the HUVEC group was arbitrarily set to 1.
Page 71 of 84 Diabetes
Supplementary Figure 2. DKK2 overexpression enhances proliferation of
endothelial cells and pericytes. A: PDGFRβ (red) and BrdU (green) staining in penis
tissue from wild type (WT) and DKK2 Tg mice, WT mice receiving streptozotocin
(STZ), and DKK2 Tg mice receiving STZ. Nuclei were labeled with DAPI (blue).
Scale bars = 50 µm. B: BrdU (green) staining in mouse cavernous endothelial cells
(MCECs), mouse cavernous pericytes (MCPs), human umbilical vein endothelial
cells (HUVECs), and human brain microvascular pericytes (HBMPs). The cells were
exposed to normal glucose (NG) or high glucose (HG) conditions for 48 hours and
treated with PBS or DKK2 protein (200 ng/ml). C: Number of BrdU immunopositive
pericytes per high power field (n = 4). *P < 0.05 vs. WT and DKK2 Tg groups. #P <
0.001 vs. WT + STZ group. D to G: Number of BrdU immunopositive endothelial
cells or pericytes per high power field (n = 4). *P < 0.05 vs. NG group. #P < 0.01 vs. Diabetes Page 72 of 84
HG group. P values were determined by Kruskal Wallis test. Data in graphs are presented as mean ± SE. PDGFRβ, platelet derived growth factor receptor beta.
Supplementary Figure 3. Preserved eNOS phosphorylation in DKK2 Tg mice under diabetic conditions. A: CD31 (red) and phospho eNOS (Ser1177; green) staining of cavernous tissue from wild type (WT) and DKK2 Tg mice, WT mice receiving streptozotocin (STZ), and DKK2 Tg mice receiving STZ. Scale bar = 100 Page 73 of 84 Diabetes
µm. B: Quantification of the phospho eNOS immunopositive area in cavernous
tissue by Image J (n = 4). *P < 0.001 vs. WT and DKK2 Tg groups. #P < 0.001 vs.
WT + STZ group. C: Representative Western blots for phospho Akt and Akt or
phospho eNOS and eNOS in age matched control and diabetic mice 2 weeks after
repeated intracavernous injections of PBS (20 µl) or DKK2 protein (days 3 and 0; 6
µg/20 µl). Arrows represent target bands. D and E: Normalized band intensity values
(n = 4). *P < 0.05 vs. control (C) group. #P < 0.05 vs. PBS treated group. F and G:
Immunoprecipitation (IP) to confirm the phospho eNOS and eNOS Western blot
data. P values were determined by Kruskal Wallis test. Data in graphs are presented
as mean ± SE. The relative ratio in the WT or C group was arbitrarily set to 1.
Diabetes Page 74 of 84
Supplementary Figure 4. Preserved endothelial cell cell junction proteins in
DKK2 Tg mice under diabetic conditions. A and B: CD31 (red) and claudin 5 (green) staining of cavernous tissue from wild type (WT) and DKK2 Tg mice, WT mice receiving streptozotocin (STZ), and DKK2 Tg mice receiving STZ. Scale bar = 100
µm. Quantification of the claudin 5 immunopositive area in cavernous tissue by
Image J (n = 4). *P < 0.001 vs. WT and DKK2 Tg groups. #P < 0.001 vs. WT + STZ group. C and D: CD31 (red) and VE cadherin (green) staining of cavernous tissue.
Scale bar = 100 µm. Quantification of the VE cadherin immunopositive area (n = 4). Page 75 of 84 Diabetes
*P < 0.01 vs. WT and DKK2 Tg groups. #P < 0.001 vs. WT + STZ group. P values
were determined by Kruskal Wallis test. Data in graphs are presented as mean ± SE.
The relative ratio in the WT group was arbitrarily set to 1. VE, vascular endothelial.
Supplementary Figure 5. DKK2 Tg mice decrease the extravasation of oxidized
LDL under diabetic conditions. A and B: CD31 (red) and oxidized LDL (green)
staining of cavernous tissue from wild type (WT) and DKK2 Tg mice, WT mice
receiving streptozotocin (STZ), and DKK2 Tg mice receiving STZ. Scale bar = 100
µm. Quantification of the oxidized LDL immunopositive area in cavernous tissue by
Image J (n = 4). *P < 0.05 vs. WT and DKK2 Tg groups. #P < 0.05 vs. WT + STZ
group. P values were determined by Kruskal Wallis test. Data are presented as mean
± SE. The relative ratio in the WT group was arbitrarily set to 1. LDL, low density
lipoprotein.
Diabetes Page 76 of 84
Supplementary Figure 6. Establishment of a direct mixed MCEC MCP co culture system. To determine the optimal ratio of MCECs and MCPs for direct mixed cell culture, the cells were cultivated at different ratios (10:1, 5:1, 3:1, 2:1, 1:1). Tube formation assays were performed on matrigel in 96 well plates and images taken at
24 and 48 hours. 40× magnification. MCEC, mouse cavernous endothelial cell; MCP, mouse cavernous pericyte.
Page 77 of 84 Diabetes
Supplementary Figure 7. DKK2 overexpression promotes migration of endothelial
cells and pericytes. A to C: Scratch wound healing assay in mouse cavernous
pericytes (MCPs), human umbilical vein endothelial cells (HUVECs), and human
brain microvascular pericytes (HBMPs) exposed to normal glucose (NG) or high
glucose (HG) conditions for 48 hours and treated with PBS or DKK2 protein (200
ng/ml). The images were taken 24 hours after scratching. 40× magnification. D to F:
Number of migrated endothelial cells and pericytes (n = 4). *P < 0.001 vs. NG group.
#P < 0.001 vs. PBS treated group. P values were determined by Kruskal Wallis test.
Data in graphs are presented as mean ± SE. Diabetes Page 78 of 84
Supplementary Figure 8. DKK2 overexpression decreases cavernous reactive oxygen species (ROS) production under diabetic conditions. A: Representative
Western blots for iNOS in age matched control and diabetic mice 2 weeks after repeated intracavernous injections of PBS (20 µl) or DKK2 protein (days 3 and 0; 6
µg/20 µl). B: Normalized band intensity values (n = 4). *P < 0.01 vs. control (C) group. #P < 0.01 vs. PBS treated group. C: Immunoprecipitation (IP) to confirm the iNOS Western blot data. D: In situ detection of superoxide anion in cavernous endothelial cells of each group of animals. Corpus cavernosum tissue was incubated with hydroethidine (red), an oxidative fluorescent dye used to detect superoxide anion, and antibody to CD31 (green). E and F: Number of ethidium bromide Page 79 of 84 Diabetes
fluorescence positive endothelial cells (ECs) per high power field (screen
magnification ×400) (n = 4). (E) *P < 0.05 vs. WT and DKK2 Tg groups. #P < 0.05
vs. WT + STZ group. (F) *P < 0.05 vs. C group. #P < 0.05 vs. PBS treated group. G:
Nitrotyrosine (red) and CD31 (green) staining in cavernous endothelial cell of each
group of animals (n = 4). H and I: The nitrotyrosine immunopositive area quantified
in cavernous endothelial cells by Image J. (H) *P < 0.05 vs. WT and DKK2 Tg
groups. #P < 0.05 vs. WT + STZ group. (I) *P < 0.05 vs. C group. #P < 0.05 vs. PBS
treated group. P values were determined by Kruskal Wallis test. Data in graphs are
presented as mean ± SE. The relative ratio in the C group was arbitrarily set to 1.
Supplementary Figure 9. DKK2 overexpression is resistant to diabetes induced Diabetes Page 80 of 84
erectile dysfunction. A: Representative intracavernous pressure (ICP) responses for wild type (WT) and DKK2 Tg mice, WT mice receiving streptozotocin (STZ), and
DKK2 Tg mice receiving STZ. The cavernous nerve was stimulated at 5 V. The stimulus interval is indicated by a solid bar. The ICP was measured 8 weeks after
STZ injection. B Representative ICP responses for age matched control and diabetic mice stimulated 2 weeks after repeated intracavernous injections of PBS (20 µl) or
DKK2 protein (days 3 and 0; 6 µg/20 µl). C to F: Ratios of mean maximal ICP and total ICP (area under the curve) to mean systolic blood pressure (MSBP) were calculated for each group (n = 5 8). (C and D) *P < 0.001 vs. WT and DKK2 Tg groups. #P < 0.001 vs. WT + STZ group. (E and F) *P < 0.001 vs. control (C) group.
#P < 0.001 vs. PBS treated group. P values were determined by Kruskal Wallis test.
Data are presented as mean ± SE.
Page 81 of 84 Diabetes
Supplementary Figure 10. Determination of optimal dosage of DKK2 protein to
induce maximal erectile function recovery under diabetic conditions. A:
Representative ICP responses for age matched control and diabetic mice stimulated
2 weeks after repeated intracavernous injections of PBS (20 µl) or DKK2 protein
(days 3 and 0; 1 µg/20 µl, 6 µg/20 µl, and 10 µg/20 µl, respectively). The cavernous
nerve was stimulated at 5 V. The stimulus interval is indicated by a solid bar. B and
C: Ratios of mean maximal ICP and total ICP (area under the curve) to mean systolic
blood pressure (MSBP) were calculated for each group (n = 5 10). *P < 0.05 vs.
control (C) group. #P < 0.05 vs. PBS treated group. P values were determined by
Kruskal Wallis test. Data are presented as mean ± SE.
Diabetes Page 82 of 84
Supplementary Figure 11. Angiopoietin 1 (Ang1) derived from mouse cavernous pericytes (MCPs) rather than mouse cavernous endothelial cells (MCECs) plays a crucial role in DKK2 mediated angiogenesis and neural regeneration. A and B: Tube formation assay in MCEC MCP mixed co culture exposed to normal glucose (NG) or high glucose (HG) conditions for 48 hours and treated with DKK2 protein (200 ng/ml). MCECs and MCPs were transfected with scrambled or Ang1 siRNA (200 pmol/l). (A) 40× magnification. (B) Number of tubes per high power field (n = 4).
*P < 0.05 vs. NG group; #P < 0.05 vs. HG group. †P < 0.05 vs. HG + DKK2 group. Page 83 of 84 Diabetes
C and D: Conditioned medium derived from MCEC MCP mixed co culture regulates
neurite sprouting in mouse pelvic ganglion (MPG) tissue culture. (C) βIII tubulin
(red) staining in MPG tissue culture exposed to NG or HG conditions for 72 hours
and treated with conditioned medium from each group. (D) Quantification of neurite
length by Image J (n = 4). *P < 0.001 vs. NG group; #P < 0.001 vs. HG group. †P <
0.001 vs. HG + DKK2 group. P values were determined by Kruskal Wallis test. Data
in graphs are presented as mean ± SE. The relative ratio in the NG group was
arbitrarily set to 1.
Supplementary Figure 12. Insulin treatment does not rescue diabetes induced
erectile dysfunction. A: Representative intracavernous pressure (ICP) responses for Diabetes Page 84 of 84
age matched control and diabetic mice treated with PBS or insulin (4 IU/day).
Insulin treatment started 1 week after STZ injection (9 weeks of age) and continued for 9 weeks (18 weeks of age). The cavernous nerve was stimulated at 5 V. The stimulus interval is indicated by a solid bar. B and C: Ratios of mean maximal ICP and total ICP (area under the curve) to mean systolic blood pressure (MSBP) were calculated for each group (n = 5). *P < 0.001 vs. control (C) group. D:
Representative Western blots for DKK2 in each group of animals. E: Normalized band intensity values (n = 4). *P < 0.01 vs. C group. P values were determined by
Kruskal Wallis test. Data in graphs are presented as mean ± SE. The relative ratio in the C group was arbitrarily set to 1.