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Excess adenosine A2B receptor signaling contributes to priapism through HIF-1␣ mediated reduction of PDE5 gene expression Chen Ning,*,†,§,1 Jiaming Wen,*,㥋,1 Yujin Zhang,* Yingbo Dai,*,† Wei Wang,*,‡ Weiru Zhang,*,‡ Lin Qi,† Almut Grenz,¶ Holger K. Eltzschig,¶ Michael R. Blackburn,*,# Rodney E. Kellems,*,# and Yang Xia,*,㥋,#,2 *Department of Biochemistry and Molecular Biology, The University of Texas Medical School at Houston, Houston, Texas, USA; †Department of Urology and ‡Department of Nephrology, Xiangya Hospital, Central South University, Changsha, China; §Department of Urology, Beijing Friendship Hospital, Capital Medical University, Beijing, China; 㛳Second Affiliated Hospital, Zhejiang University, Hangzhou, China; ¶Department of Anesthesiology, The University of Colorado School of Medicine, Denver, Colorado, USA; and #Graduate School of Biomedical Science, The University of Texas, Houston, Texas, USA Priapism is featured with prolonged and painful penile erection and is prevalent among males with sickle cell disease (SCD). The disorder is a dangerous urological and hematological emergency since it is associated with ischemic tissue damage and erectile disability. Here we report that phosphodiesterase-5 (PDE5) gene expression and PDE activity is significantly reduced in penile tissues of two independent priapic models: SCD mice and adenosine deaminase (ADA)-deficient mice. Moreover, using ADA enzyme therapy to reduce adenosine or a specific antagonist to block A2B adenosine receptor (ADORA2B) signaling, we successfully attenuated priapism in both ADAⴚ/ⴚ and SCD mice by restoring penile PDE5 gene expression to normal levels. This finding led us to further discover that excess adenosine signaling via ADORA2B activation directly reduces PDE5 gene expression in a hypoxia-inducible factor-1␣ (HIF1␣)-dependent manner. Overall, we reveal that excess adenosine-mediated ADORA2B signaling underlies reduced penile PDE activity by decreasing PDE5 gene expression in a HIF-1␣-dependent manner and provide new insight for the pathogenesis of priapism and novel therapies for the disease.—Ning, C., Wen, J., Zhang, Y., Dai, Y., Wang, W., Zhang, W., Qi, L., Grenz, A., Eltzschig, H. K., Blackburn, M. R., Kellems, R. E., Xia, Y. Excess adenosine A2B receptor signaling contributes to priapism through HIF-1␣ mediated reduction of PDE5 gene expression. FASEB J. 28, 2725–2735 (2014). www.fasebj.org ABSTRACT

Abbreviations: ADA, adenosine deaminase; ADA⫺/⫺, adenosine deaminase-deficient; ADORA2B, adenosine A2B receptor; AUC, area under the curve; CCSMC, corpus cavernosal smooth muscle cell; CNS, cavernous nerve stimulation; ED, erectile dysfunction; eNOS, endothelial nitric oxide synthase; HbS, human sickle hemoglobin; HD, high dose; HIF-1␣, hypoxiainducible factor 1␣; ICP, intracarvenosal pressure; LD, low dose; MAP, mean arterial pressure; nNOS, neuronal nitric oxide synthase; NO, nitric oxide; PDE5, phosphodiesterase-5; PEGADA, polyethylene glycol adenosine deaminase; SCD, sickle cell disease; SCD Tg, sickle cell disease transgenic; WT, wild type 0892-6638/14/0028-2725 © FASEB

Key Words: hypoxia-inducible factor 䡠 sickle cell disease 䡠 phosphodiesterase-5 Priapism is defined as an abnormally prolonged and persistent penile erection in the absence of sexual provocation or desire (1). Although this disorder is rarely seen in the general population (2), it is frequently associated with certain medical conditions, including sickle cell disease (SCD), erectile dysfunction (ED) therapy-related complications, neurological disease, and solid malignancies (3). Of note, ⬃40% of men with SCD experience priapism (4, 5). The condition is dangerous and urgent given its association with erectile tissue damage and ED. Treatment approaches for this disorder include penile blood aspiration, with or without irrigation of the corpora cavernosa, as well as surgical management involving the introduction of a shunt to quickly reduce intracavernosal pressure (ICP) and to relieve symptoms (6, 7). An ␣-adrenergic agonist, such as phenylephrine, is often administered to promote vasoconstriction, thereby promoting detumescence. However, these reactive treatments lack preventive effects and rarely restore normal penile function. Although red blood cell transfusion is a common treatment for the management of many sickle-related complications, including prevention of priapism, evidence supporting its use as a standard care for acute attacks of priapism is lacking (8). A better understanding of the molecular basis of priapism is needed in an effort to develop novel therapeutic approaches. Two potent vasodilators, nitric oxide (NO) and adenosine, contribute to penile erection by promoting cor1

These authors contributed equally to this work. Correspondence: Department of Biochemistry and Molecular Biology, University of Texas Medical School at Houston, 6431 Fannin St., MSB 6.200, Houston, TX 77030, USA. E-mail: [email protected] doi: 10.1096/fj.13-247833 2

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pus cavernosal smooth muscle cell (CCSMC) relaxation, which thereby allows the cavernosa to be engorged by incoming blood. NO is initially derived from cavernosal nerves [neuronal NO synthase (nNOS)] and subsequently from the endothelium [endothelial NO synthase (eNOS)] and acts directly on cavernosal smooth muscle guanylate cyclase to stimulate cGMP synthesis and CCSMC relaxation. This process is counterbalanced by phosphodiesaterase 5 (PDE5), the predominant cGMP-specific PDE isozyme in penile tissues that plays a key role in erectile function by eliminating cGMP (9). Similar to NO, adenosine plays a multifaceted role during penile erection involving the activation of two adenosine receptors on multiple cell types in the corpora cavernosa. Activation of adenosine A2B receptors (ADORA2Bs) on CCSMCs results in the activation of adenylyl cyclase, increased synthesis of cAMP, and CCSMC relaxation. Activation of ADORA2Bs on cavernosal endothelial cells results in increased NO production by PI3K/AKT mediated posttranslational activation of eNOS (10). More recent studies showed (11) that adenosine signaling through ADORA1 adenosine receptors reduces norepinephrine release from cavernosal neurons and results in reduced cavernosal smooth muscle contraction, which thereby facilitates cavernosal smooth muscle relaxation and promotes erection. Thus, both NO and adenosine signaling orchestrate a coordinated signaling program to promote penile erection. Impaired NO and adenosine signaling are reported in erectile disorders. For example, early studies showed that endothelial ADORA2B dysfunction may contribute to ED in men (12). Recently, an unexpected priapism phenotype in adenosine deaminase (ADA)-deficient (ADA⫺/⫺) mice revealed a previously unrecognized role of excess adenosine in priapism (13). ADA is a purine metabolic enzyme converting adenosine to inosine. Mice deficient in this enzyme exhibit a marked increase in systemic adenosine concentrations, including penile tissues. These mice display features of priapism seen in humans, including spontaneous prolonged penile erection, increased vascular relaxation in response to neurostimulation, and penile fibrosis. Thus, ADA⫺/⫺ mice represent a novel and important animal model to study the role of adenosine signaling in priapism. Subsequent in vitro studies showed that excess adenosine signaling via ADORA2B activation contributes to increased corpus cavernosal relaxation in both ADA⫺/⫺ and SCD transgenic (SCD Tg) mice, a well-accepted animal model of priapism (13). Thus, these findings revealed that elevated adenosine contributes to priapism via ADORA2B signaling. Early studies showed that reduced PDE5 activity in SCD mouse model contributes to priapism (14). However, the specific factors and signaling pathways responsible for reduced PDE5 activity in priapism remain largely unidentified, and treatment options remain limited. Here, we provide the first in vivo evidence that reduced penile PDE5 gene expression underlies decreased PDE activity in two independent animal models, ADA⫺/⫺ and SCD Tg mice. These results indicate that reduced PED5 gene expression is a general mechanism underlying decreased PDE activity in priapism. 2726

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We further revealed that lowering elevated adenosine by ADA enzyme therapy or interference with ADORA2B signaling using a specific receptor antagonist restored penile PDE5 gene expression and PDE activity to normal levels and thereby successfully reduced priapism in both animal models. Mechanistically, we revealed that hypoxia-inducible factor 1␣ (HIF-1␣)-mediated reduction in PDE5 gene expression is a previously unrecognized signaling pathway functioning downstream of ADORA2B underlying priapism in both animal models of priapism. Altogether, using genetic, pharmacological, physiological, biochemical, and cellular approaches we have revealed that ADORA2B-mediated HIF-1␣ induction contributes to reduced PDE5 gene expression, PDE activity, and the pathogenesis of priapism. Preclinical studies presented here reveal novel and potentially effective therapeutic approaches that may be useful for disease management.

MATERIALS AND METHODS Mice ADA⫺/⫺ mice were generated and genotyped as previously described (15–17). These mice were backcrossed ⱖ10 generations onto the C57BL/6 background and were genotyped according to established protocols (15–17). SCD Tg mice, expressing exclusively human sickle hemoglobin (HbS), were purchased from The Jackson Laboratory (Bar Harbor, ME, USA; refs. 18, 19). ADORA2B-deficient mice congenic on a C57BL/6 background were generated and genotyped as described (10). For all studies, age-matched wild-type (WT) C57BL/6 mice were used as controls. All mice were maintained and housed in accordance with U.S. National Institutes of Health guidelines and with approval of the Animal Care and Use Committee at the University of Texas Health Science Center at Houston. Polyethylene glycol ADA (PEG-ADA) and PSB1115 therapy PEG-ADA was generated by the covalent modification of purified bovine ADA with activated PEG as described previously (20 –22). Specifically, the ADA⫺/⫺ mice were maintained on high-dose (HD) enzyme therapy at 5 U/wk until 12 wk to keep the mice alive and to allow for normal penile development. At 12 wk, ADA⫺/⫺ mice were divided into 3 groups. The first group continued to be treated with a high dosage of PEG-ADA (5 U/wk) and was termed the PEG-ADA treatment group. The other two groups received a low dosage of PEG-ADA (0.625 U/wk) to allow for an increase in circulating adenosine levels. One of the low-dose (LD) PEGADA groups was injected with PSB1115 (200 ␮g/d; Tocris Bioscience, Ellisville, MO, USA), and the other, serving as a control, was injected with normal saline. SCD Tg mice, at 12–14 wk of age, were also divided into 3 groups: one group received PEG-ADA (2.5 U/wk), the others received PSB1115 (200 ␮g/d) or normal saline for 2 wk. WT mice (C57BL/6) were used as controls and were injected with saline, PEG-ADA (2.5 U/wk) or PSB1115 (200 ␮g/d) for 2 wk. An article describing the use of PEG-ADA enzyme therapy to regulate the metabolic and phenotypic consequences of ADA deficiency in these mice was first published many years ago (23). This article describes the use of different doses of PEG-ADA enzyme replacement therapy to achieve different levels of

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adenosine in the ADA⫺/⫺ mice with different phenotypic responses. Data appearing therein guided us to choose 5 U/wk for treating ADA⫺/⫺ mice. A dose of 2.5 U/wk was chosen for SCD mice because these mice are ADA positive and the elevated levels of adenosine in SCD mice are not as high as in ADA⫺/⫺ mice. The use of 2.5 U/wk for treatment of SCD mice is described in a recent article from our laboratory (24).

activated PDE and was further cleaved to guanosine and phosphate by purine nucleoside phosphorylase present in the assay cocktail. The phosphate generated is quantified using a colorimetric reaction using Biomol Green reagent (Biomol International, Plymouth Meeting, PA, USA) in a modified Malachite Green assay (26). Isolation of CCSMCs from WT and ADORA2B-deficient mice and in vitro culture conditions

Quantification of penile adenosine levels Mice were anesthetized, and the penises were rapidly removed and frozen in liquid nitrogen. Adenine nucleosides were extracted from frozen penises using 0.4 N perchloric acid, and adenosine was separated and quantified using reverse-phase HPLC as described previously (15, 16). Cavernous nerve stimulation (CNS) and ICP measurement In vivo erectile function in response to CNS was studied in ADA⫺/⫺, SCD Tg, and WT anesthetized mice. Mice were anesthetized by intraperitoneal injection with 250 mg/kg avertin (made by mixing 10 g of 2,2,2-tribromoethyl alcohol with 10 ml of tert-amyl alcohol). A heating pad at 38°C was used to maintain body temperature during the procedure. The shaft of the penis was freed of skin and fascia; by dissecting bilaterally overlying ischiocavernous muscle, exposure of the penile crus was performed. The bladder and prostate gland were exposed through a lower midline abdominal incision. The right major pelvic ganglion and cavernous nerve, located posterolateral to the prostate on one side, were identified, and an electrical stimulator with a bipolar silver electrode was placed around the cavernous nerve. Electric stimulation was carried out by a Grass stimulator to induce penile erection (AD Instruments Inc., Colorado Springs, CO, USA) at 1, 2, and 4 V with 16 Hz and 5 ␮s duration for 1 min (14, 25). To monitor the ICP, the right corpus cavernosum was penetrated by a 25-gauge needle, and a heparinized (250 U/ml) mouse jugular catheter (Alzet Osmotic Pumps, Cupertino, CA, USA) was inserted into the right corpus cavernosum. The cannula was connected to a pressure transducer and an amplifier unit, which was connected to a data acquisition module. ICP before and after CNS was recorded on a personal computer by Chart 5 software (AD Instruments). Mean arterial pressure (MAP) measurement ICP data were normalized to MAP. MAP was monitored by cannulating the right carotid artery with a mouse jugular catheter connected to a pressure transducer and an amplifier unit. The amplifier was linked to a data acquisition module, and MAP was recorded simultaneously with ICP monitoring on a personal computer by Chart 5 Software (AD Instruments). PDE activity measurement in mouse penile tissues PDE activity was determined by using a colorimetric cyclic nucleotide PDE assay kit (cat. no. ab139460; Abcom, Inc., Atlanta, GA, USA). Briefly, penile tissues were sonicated in 0.3 ml lysis buffer containing complete protease inhibitors and phosphatase inhibitors (Cell Signaling Technology, Danvers, MA, USA) for 2 min at 25 Hz. The lysate was centrifuged at 20,000 g for 10 min at 4°C. The supernatant was transferred to a new tube for the assay. The cell lysates (⬃100 ␮g) were incubated with the substrate cGMP at 37°C for 10 min. At the end of the experiments, cGMP was cleaved to 5=GMP by HIF1-␣ AND PDE5 IN PRIAPISM

CCSMCs were isolated from WT mice and ADORA2B-deficient mice as described previously (13). Briefly, the isolated CCSMCs were cultured in DMEM containing 10% FBS in a humidified atmosphere of 5% CO2 at 37°C. After overnight culture, cells were serum starved in DMEM without FBS and treated with various drugs for 24 h. Specifically, NECA (10 ␮M), a potent nonmetabolized adenosine analog; PSB1115 (10 ␮M), a specific ADORA2B antagonist; DMOG (10 ␮M), a potent HIF-1␣ stabilizer; and Chrysin (10 ␮M), a specific HIF-1␣ destabilizer, were used. After 24 h treatment, cell lysates were isolated, and HIF-1a and PDE5 mRNA were measured by RT-PCR as described below. Real-time RT-PCR analysis Total RNA was isolated from mouse penile tissues and from wild-type and ADORA2B-deficient mouse CCSMCs by using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNase-free DNase (Invitrogen) was used to eliminate genomic DNA contamination. Transcript levels were quantified using real-time quantitative RT-PCR. SYBER green was used for analysis of HIF-1␣, PDE5, and ␤-actin using the following primers: mouse HIF-1␣, forward 5=-CCTTCATCGGAAACTCCAAA-3= and reverse 5=-TGGGGCATGGTAAAAGAAAG-3=; mouse PDE5: forward 5=-CGGCCTACCTGGCATTCTG-3= and reverse 5=GCAAGGTCAAGTAACACCTGATT-3=; mouse ␤-actin, forward 5=-GGGAATGGGTCAAAACT-3= and reverse 5=-CTTCTCCATGTCGTCCCAGT-3=. Statistical analysis All data are expressed as means ⫾ sem. Data were analyzed for statistical significance using statistical programs run by GraphPad Prism 5 software (GraphPad Software, San Diego, CA, USA). Student’s t test (paired or unpaired as appropriate) was applied in 2-group analysis. Differences between the means of multiple groups were compared by 1-way analysis of variance (ANOVA), followed by Tukey’s multiple comparisons test. A value of P ⬍ 0.05 was considered significant and was the threshold to reject the null hypothesis.

RESULTS Reduced penile PDE5 mRNA levels underlie decreased PDE activity in both SCD Tg and ADAⴚ/ⴚ mice Earlier studies showed that reduced penile PDE5 activity contributes to priapism in SCD Tg mice (14). However, the molecular basis responsible for reduced PDE5 activity in priapism remains unidentified. In an effort to identify molecular mechanisms underlying reduced PDE5 activity in priapism in SCD Tg mice, we first measured PDE5 gene expression in the penile tissues of SCD Tg mice. We found that the PDE5 mRNA 2727

Figure 1. Excess adenosine contributes to reduced PDE5 mRNA levels and PDE activity in two independent priapic animal models. A, B) PDE5 mRNA levels (A) and PDE activity (B) were significantly reduced to a similar extent in penile tissues of SCD Tg and ADA⫺/⫺ mice. C, D) PDE5 mRNA levels (C) and PDE activity (D) were restored to normal levels by chronic PEG-ADA treatment. Data are expressed as means ⫾ sem; n ⫽ 6. *P ⬍ 0.05 vs. control mice; **P ⬍ 0.05 vs. untreated ADA⫺/⫺ or SCD mice.

levels were significantly reduced in penile tissue of SCD Tg mice (Fig. 1A). Furthermore, we confirmed that cGMP-specific PDE activity was also significantly reduced in penile tissue of SCD Tg mice (Fig. 1B). Of note, the fold reduction of PDE activity was similar to the reduced PDE5 gene expression, indicating that reduced PDE5 gene expression is likely responsible for the reduced cGMP-specific PDE activity in the penile tissues of SCD Tg mice. To further determine whether reduced PDE5 gene expression and PDE activity are general features seen in priapism, we assessed their levels in penile tissue of ADA⫺/⫺ mice, another priapic animal model due to excess accumulated adenosine (13). Similarly, we found that both PDE5 mRNA levels and PDE activity in penile tissues were reduced to a similar extent compared to normal controls (Fig. 1A, B). Thus, these findings provide the first evidence that reduced PDE5 gene expression in penile tissue is a general molecular mechanism underlying reduced PDE activity seen in priapism. Elevated adenosine is a common signaling molecule contributing to reduced penile PDE5 gene expression in both SCD Tg and ADAⴚ/ⴚ mice Although early studies showed that elevated penile adenosine contributes to priapism in both ADA⫺/⫺ and 2728

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SCD Tg mice (13), nothing is known about the role of excess adenosine in reduced PDE5 gene expression in priapism. In view of our observation that PDE5 mRNA levels and cGMP-specific PDE activity were reduced in penile tissues of these two independent animal models of priapism, we hypothesized that elevated adenosine is the signaling molecule involved in PDE5 gene expression. To test this hypothesis, we took a pharmacological approach by using ADA enzyme therapy to lower elevated adenosine in both ADA⫺/⫺ and SCD Tg mice. Briefly, we treated ADA⫺/⫺ mice with an HD regimen of PEG-ADA (5 U/wk) from birth to 12 wk of age in an effort to maintain low adenosine levels and allow normal growth and penile system development. At 12 wk of age a group of ADA⫺/⫺ mice, termed the PEG-ADA treatment group, continued to receive HD PEG-ADA therapy. Another group, termed ADA⫺/⫺, were switched to an LD regimen of PEG-ADA (0.625 U/wk) to allow adenosine to accumulate in penile tissues and to generate a priapic phenotype. We found that adenosine levels in the penile tissues of ADA⫺/⫺ mice maintained on LD PEG-ADA were remarkably higher than in control mice. However, HD PEG-ADA treatment significantly reduced adenosine levels in the penises of ADA⫺/⫺ mice (Table 1). PEG-ADA (2.5 U/wk for 2 wk) was also administrated to one group of SCD Tg mice from 12–14 wk of age, while another group of SCD Tg mice received saline injections and served as controls.

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TABLE 1. Adenosine levels in penile tissues of ADA-deficient mice and SCD Tg mice

Group

n

Penile adenosine level (nmol/mg protein)

Control Control ⫹ PEG-ADA ADA⫺/⫺ ADA⫺/⫺ ⫹ PEG-ADA SCD SCD⫹ PEG-ADA

9 7 7 6 8 6

9.91 ⫾ 1.66 7.47 ⫾ 1.20 68.47 ⫾ 5.59* 22.15 ⫾ 2.45# 19.52 ⫾ 3.32* 10.90 ⫾ 1.67$

*P ⬍ 0.05 vs. control mice; #P ⬍ 0.05 vs. ADA⫺/⫺ mice; $P ⬍ 0.05 vs. SCD Tg mice

(P⬍0.05; Fig. 2C). Overall, these results provide in vivo evidence that ADA⫺/⫺ and SCD Tg mice display priapism features like humans with potent and prolonged penile erection. Next, we treated both ADA⫺/⫺ and SCD Tg mice with PEG-ADA as described above. At the end of 2-wk treatment, we measured the erectile response to CNS to assess in vivo erectile function. Because the most significant differences in erectile responses between mutant and control groups were shown at 2 V CNS for 1 min (Fig. 2B), we chose these parameters for our experiments. The results (Fig. 3) show that ADA⫺/⫺ mice with elevated adenosine levels exhibited an increased erectile response (maximal ICP, 0.89⫾0.05) and a pro-

Consistently, we found that following 2 wk of PEG-ADA enzyme therapy, the elevated adenosine concentrations in penises of SCD Tg mice were significantly reduced (Table 1). Thus, PEG-ADA enzyme therapy effectively lowered adenosine levels in penile tissues of both ADA⫺/⫺ and SCD Tg mice. Next, we found that PEG-ADA enzyme therapy restored penile PDE5 mRNA levels and PDE activity to wild-type levels in both ADA⫺/⫺ and SCD Tg mice (Fig. 1C, D). Thus, our findings revealed that elevated penile adenosine is a common factor that contributes to reduced penile PDE5 mRNA levels and PDE enzyme activity in ADA⫺/⫺ and SCD Tg mice. PEG-ADA enzyme therapy reduced priapism in ADAⴚ/ⴚ mice and SCD Tg mice In a view of our finding that elevated adenosine is a common molecule involved in reduced PDE5 mRNA and cGMP-specific PDE activity in the penile tissues of two independent models of priapism, we hypothesized that PEG-ADA would be an effective chronic treatment for priapism by reversing the reduction of PDE5 mRNA levels. To test this hypothesis, we first precisely characterized the priapic features of ADA⫺/⫺ and SCD Tg mice without treatment. We measured basal ICP of both mice at resting state without CNS. We found that both ADA⫺/⫺ and SCD Tg mice showed an increased resting ICP (P⬍0.05) compared to the control mice (Fig. 2A). These findings provide in vivo evidence that both ADA⫺/⫺ and SCD Tg mice display increased basal penile cavernosal pressure in the absence of sexual stimulation, a hallmark feature of priapism. Next, to precisely assess the erectile function, including potency and length of erection, we took a well-established experimental approach to induce penile erection by CNS. We found that both ADA⫺/⫺ and SCD Tg mice had a significant increase in maximal ICP at all voltage settings tested (Fig. 2B) compared to the basal ICP in the absence of CNS (Fig. 2A). We also found that in control mice, ICP gradually increased at a standard voltage range, with a maximal response at 4 V. However, ADA⫺/⫺ and SCD Tg mice displayed highly responsive reactions to CNS even at a low voltage, reaching a maximal response at 2 V (Fig. 2B). Moreover, we found that during the detumescence phase, the erectile responses after terminating CNS (2 V) were also remarkably prolonged in ADA⫺/⫺ and SCD Tg mice HIF1-␣ AND PDE5 IN PRIAPISM

Figure 2. Elevated erectile response and prolonged detumescence phase in ADA⫺/⫺ and SCD Tg mice. A) Baseline resting ICP. B) Voltage-dependent erectile responses to CNS. C) Area under curve (mmHg·s) post-CNS (2 V). *P ⬍ 0.05 vs. control mice; n ⫽ 4 – 6. 2729

Figure 3. PEG-ADA enzyme therapy reduces priapic features seen in ADA⫺/⫺ and SCD Tg mice. A) Representative recordings of ICP induced by CNS at 2 V for 1 min in control, ADA⫺/⫺, and SCD Tg mice in the absence of treatment or following 2 wk treatment with PEG-ADA. B) Maximum ICP normalized to MAP. C) Area under curve (mmHg·s) post-CNS (2 V). See Materials and Methods for details regarding PEG-ADA treatments. *P ⬍ 0.05 vs. control mice; **P ⬍ 0.05 vs. ADA⫺/⫺ mice; ## P ⬍ 0.05 vs. SCD Tg mice; n ⫽ 4 – 6.

longed detumescence phase [area under the curve (AUC) poststimulation, 5110⫾1083 mmHg·s] compared with control mice (0.66⫾0.09, 627⫾60 mmHg·s, P⬍0.05) (Fig. 3). However, HD PEG-ADA treatment reduced the heightened erectile response (ICP/MAP, 0.61⫾0.09, P⬍0.05; Fig. 3B) and largely eliminated the extended detumescence phase (AUC, 878⫾218 mmHg·s) in the HD-PEG-ADA treated group (P⬍0.05, Fig. 3C). Features of priapism observed in SCD Tg mice were also reduced following 2 wk of PEG-ADA treatment. Maximal ICP was reduced to 0.68 ⫾ 0.11 from 0.88 ⫾ 0.04, and AUC poststimulation was reduced from 3936 ⫾ 781 to 1015 ⫾ 514 mmHg · s (Fig. 3B, C). Overall, in two lines of mice with priapism, we showed that PEG-ADA enzyme therapy lowered penile adenosine levels, restored PDE5 mRNA levels and PDE activity, and thereby successfully reduced features associated with priapism. These results indicate that PEG-ADA therapy may have general utility for the treatment of priapism. ADORA2B is responsible for excess adenosinemediated reduction of PDE5 gene expression in the penile tissues of both ADAⴚ/ⴚ and SCD Tg mice Early in vitro studies showed that elevated adenosine signaling via ADORA2B contributes to increased corporal cavernosal relaxation (13). However, whether 2730

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excessive ADORA2B signaling is responsible for reduced penile PDE5 gene expression is unknown. To address this possibility, we treated both ADA⫺/⫺ and SCD Tg mice with the ADORA2B-specific antagonist, PSB1115. Briefly, ADA⫺/⫺ and SCD Tg mice were treated with PSB1115 for 2 wk. At the end of the protocol, PDE5 mRNA levels and PDE activity were measured in the penile tissues of these mice. Similar to PEG-ADA enzyme therapy, we found that PSB1115 significantly reversed the reduction in PDE5 gene expression and restored PDE activity in penile tissues of both priapism models (Fig. 4A, B). Taken together, these studies provide in vivo evidence that excessive ADOA2B signaling contributes to reduced PDE5 mRNA levels and PDE enzyme activity in priapism. PSB1115 treatment ameliorates the priapic feature in ADAⴚ/ⴚ and SCD Tg mice Because we found that PSB1115 treatment restores PDE5 mRNA levels and PDE activity to normal levels in penile tissues of both mouse models of priapism, we hypothesized that PSB1115 would be effective to treat priapism. To test this hypothesis, ADA⫺/⫺ and SCD Tg mice were treated with PSB1115 for 2 wk prior to in vivo assessment of erectile function following CNS. For ADA⫺/⫺ mice, the maximum ICP and AUC poststimulation were significantly reduced to 0.61 ⫾ 0.05 and 916 ⫾

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ADA⫺/⫺ and SCD mice (Fig. 6A). These findings provide in vivo evidence that excess adenosine signaling via ADORA2B activation underlies increased HIF-1␣ mRNA production in both ADA⫺/⫺ and SCD Tg mice. Genetic deletion of ADOAR2B or treatment with an ADORA2B antagonist abolishes adenosine-mediated changes in HIF-1␣ and PDE5 gene expression in primary CCSMCs

Figure 4. ADORA2B activation contributes to excess adenosine-mediated reduced PDE5 mRNA levels in both ADA⫺/⫺ and SCD Tg mice. PSB1115 treatment significantly reduced PDE5 mRNA levels (A) and PDE activity (B) in both ADA⫺/⫺ and SCD Tg mice. Data are expressed as means ⫾ sem; n ⫽ 6. *P ⬍ 0.05 vs. control mice; **P ⬍ 0.05 vs. untreated ADA⫺/⫺ or SCD mice.

174 mmHg · s, respectively (P⬍0.05; Fig. 5). Similarly, for SCD Tg mice, priapic features were significantly ameliorated following a 2-wk PSB1115 treatment protocol. Maximum ICP and AUC poststimulation were reduced to 0.68 ⫾ 0.09 and 745 ⫾ 105 mmHg · s, respectively (Fig. 5). Overall, these results demonstrated that ADORA2B antagonism was an effective therapeutic treatment for priapism in these two mouse models. ADORA2B activation leads to increased HIF-1␣ gene expression in the penile tissues of both ADAⴚ/ⴚ and SCD Tg mice Priapism is characterized by ischemia leading to hypoxia (7). Both adenosine (27) and HIF-1␣ (28 –30) are induced under hypoxic conditions. Here we report that ADORA2B activation is responsible for reduced PDE5 gene expression in priapic mouse models. Thus, in an effort to identify the intracellular molecules responsible for ADORA2B-mediated reduced PDE gene expression, we chose to focus on HIF-1␣ gene expression in these two priapic mouse models. Intriguingly, we found that penile HIF-1␣ mRNA levels were significantly elevated in ADA⫺/⫺ and SCD Tg mice relative to control mice (Fig. 6A). Moreover, elevated HIF-1␣ mRNA levels were significantly reduced following PSB1115 treatment in the penile tissues of both HIF1-␣ AND PDE5 IN PRIAPISM

In view of our findings that HIF-1␣ is a common transcription factor induced by ADORA2B activation and that ADORA2B signaling underlies reduced penile PDE5 gene expression in two different animal models of priapism, we hypothesize that HIF-1␣ is likely an intracellular molecule responsible for ADORA2B-mediated reduced PDE5 gene expression. It is difficult to decipher the molecular basis for ADORA2B-mediated regulation of HIF-1␣ and PDE5 gene expression in intact animals. We therefore isolated primary CCSMCs, a recognized source of PDE5 production, as a cellular model system to determine whether elevated adenosine can directly induce HIF-1␣ and reduce PDE5 gene expression. Similar to our in vivo observations, we found that NECA, a potent nonmetabolized adenosine analog, significantly induced HIF-1␣ and reduced PDE5 gene expression in cultured primary CCSMCs isolated from WT mice (Fig. 6B, C). Treatment of PSB1115 significantly reduced NECA-mediated altered gene expression of HIF-1␣ and PDE5 in WT CCSMCs (Fig. 6B C). To confirm our pharmacological studies, we further isolated CCSMCs from ADORA2B-defiicent mice. Similar to PSB1115 treatment, genetic deletion of ADOAR2B significantly abolished induction of HIF-1␣ and reduction of PDE5 gene expression by NECA treatment in CCSMCs(Fig. 6B, C). Thus, we provide both pharmacologic and genetic evidence that excess adenosine signaling via ADORA2B regulates both HIF-1␣ and PDE5 gene expression in primary cultured mouse CCSMCs. ADORA2B-mediated HIF-1␣ induction is essential for reduced PDE5 gene expression in primary CCSMCs To further delineate whether HIF-1␣ is a key transcription factor responsible for ADORA2B-mediated reduction in PDE5 gene expression, we treated WT CCSMCs with or without DOMG, a potent HIF-1␣ stabilizer, or Chrysin, a HIF-1␣ destabilizer. We found that NECAmediated reduction of PDE5 mRNA was further enhanced by DMOG treatment to stabilize HIF-1␣ (Fig. 6D). In contrast, Chrysin treatment significantly inhibited the NECA-mediated reduction of PDE5 gene expression (Fig. 6D). Overall, our findings show that HIF-1␣ is essential for adenosine signaling-mediated reduction of PDE5 gene expression in mouse CCSMCs.

DISCUSSION The work reported here is the first to show that elevated adenosine, coupled with excessive ADORA2B activa2731

Figure 5. ADORA2B antagonist (PSB1115) treatment has a therapeutic effect on priapic feature in ADA⫺/⫺ and SCD Tg mice. A) Representative recordings of ICP induced by CNS at 2 V for 1 min in control, ADA⫺/⫺, and SCD Tg mice in the absence of treatment or following 2 wk of PSB1115 infusion. B) Maximum ICP normalized to MAP. C) Area under curve (mmHg·s) post-CNS (2 V). *P ⬍ 0.05 vs. control mice; **P ⬍ 0.05 vs. ADA⫺/⫺ mice; #P ⬍ 0.05 vs. control mice; ##P ⬍ 0.05 vs. SCD Tg mice; n ⫽ 4 – 6.

tion, underlies reduced PDE5 gene expression in a HIF-1␣-dependent manner in priapism. Therefore, the use of PEG-ADA to lower adenosine or PSB1115 to interfere with ADORA2B activation may represent novel treatments for priapism by reducing HIF-1␣mediated reduction of PDE5 gene expression, reduced PDE activity, and subsequent elevations of cGMP (Fig. 6E). Our current findings have significantly advanced our understanding of the pathogenesis of priapism by revealing the pathogenic role of ADORA2B-mediated HIF-1␣ signaling in reduced PDE5 gene expression and also provide a strong foundation for therapeutic approaches based on interfering with adenosineADORA2B signaling. Priapism is considered a dangerous disorder that results in penile structural damage and progressive ED if intervention is not prompt. It is generally accepted that the longer priapism continues, the greater the risk of ED. Pryor et al. (31) documented that 90% of patients develop ED if priapism ⬎24 h. It has also been reported that if the priapic episode lasts ⬎36 h, no patients were able to generate a functional erection (32). Although treatment options of priapism have improved in recent years, most are based on surgical procedures to relieve symptoms during a single priapic episode. In addition, preventive approaches are lacking for abnormal erection tendencies associated with priapism. Among SCD patients with priapism, ⬃75% had their first occurrence before the age of 20 with the 2732

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mean age being 12 to 15 yr (4, 5, 33). Without intervention or prevention, these boys will not only suffer painful erections but also incur ED at an early age. Thus, not surprisingly, a high prevalence of ED is found among patients with SCD with recurrent priapism (34). Therefore, it is urgent to identify the molecular basis for pathogenesis and in turn find a way to prevent priapism and avoid the erectile tissue damage, fibrosis, and loss of erectile function for both young and old male patients with SCD. Of note, early studies showed that PDE activities are significantly reduced in the penile tissues of SCD Tg mice and underlie the pathogenesis of priapism associated with SCD (14). This same study also reported that reduced PDE5 activity is observed in eNOS and nNOS double-deficient (NOS⫺/⫺) mice, a mouse model of priapism (14). This study reveals that NOS deficiency leads to impaired NO signaling and reduced cGMP levels in cavernosal smooth muscle. Because cGMP is a positive regulator of PDE5 gene expression in cavernosal smooth muscle (35), the chronically low cGMP levels (resulting from low endothelial and neuronal derived NO production) results in low levels of PDE5 in cavernosal smooth muscle. More recent studies demonstrate that continuous treatment with sildenafil citrate (a PDE5 inhibitor) restores PDE5 levels in SCD Tg mouse penile tissues and prevents priapism (36). Presumably the use of a PDE5 inhibitor allows cGMP levels to accumulate and exert a positive feedback stimulation

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Figure 6. Elevated penile HIF-1␣ is required for ADORA2Bmediated reduced PDE5 mRNA levels associated with priapism. A) Increased HIF-1␣ mRNA levels in the penile tissues of ADA⫺/⫺ and SCD Tg mice were reduced by PSB1115 treatment. Data are expressed as means ⫾ sem. *P ⬍ 0.05 vs. control mice; **P ⬍ 0.05 vs. untreated ADA⫺/⫺ or SCD Tg mice. B, C) NECA stimulated HIF-1␣ gene expression (B) and reduced PDE5 gene expression (C) in CCSMCs of WT mice. NECA-mediated induction of HIF-1␣ mRNA and reduction of PDE5 mRNA were inhibited by PSB1115 in CCSMCs from WT mice or in CCSMCs from ADORA2B-deficient mice. D) Treatment of WT CCSMCs with DOMG (stabilizes HIF-1␣) further enhanced the NECA-mediated decrease in PDE5 gene expression. Treatment of CCSMCS with Chrysin (destabilizes HIF-1␣) significantly attenuated the NECA-mediated decrease in PDE5 gene expression. Data are expressed as means ⫾ sem; n ⫽ 5 or 6. *P ⬍ 0.05 vs. untreated CCSMCs; **P ⬍ 0.05 vs. NECA-treated WT CCSMCs. E) Working model for excess penile adenosine signaling via ADORA2B in the induction of HIF-1␣ and subsequent reduction of PDE5 in priapism and novel therapies. Excess adenosine induces priapism via ADORA2B-mediated induction of HIF-1␣ gene expression. Increased HIF-1␣ serves a negative transcriptional regulator of PDE5 gene expression, resulting in decreased PDE5 levels and increased cavernosal smooth muscle relaxation. HIF-1␣ can also induce ADORA2B gene expression (41), thereby enhancing this signaling pathway responsible for decreased expression of PDE5. Moreover, ADORA2B activation in cavernosal smooth muscle cells directly leads to elevated cAMP production (13). Thus, adenosine signaling via ADORA2B promotes CCSMC relaxation and priapism by two mechanisms: decreased PDE5 gene expression, resulting in elevated cGMP production; and increased cAMP production via PKA-mediated activation of adenylyl cyclase. Lowering chronically elevated adenosine or interfering with ADORA2B activation are novel therapeutic possibilities to treat priapism.

of PDE5 gene expression (35). Here we report that reduced penile PDE5 gene expression is a general mechanism underlying reduced PDE activity seen in two different models of priapism: SCD Tg mice and ADA⫺/⫺ mice. We show that excess adenosine activates HIF1-␣ AND PDE5 IN PRIAPISM

ADORA2B, resulting in reduced PDE5 gene expression in a HIF-1␣-dependent manner in the penile tissues of these two independent priapic models. Thus, our studies reveal that adenosine signaling is a previously unrecognized common signaling pathway underlying re2733

duced PDE activity by reduction of PDE5 gene expression in priapism. It will be interesting to investigate whether excess adenosine signaling via ADORA2B activation also contributes to reduced PDE5 gene expression and subsequent decreased PDE5 activities in NOS⫺/⫺ mice. Ischemic priapism, also known as vasoocclusive or low-flow priapism, is the most prevalent and worst type of priapism in patients with SCD (6, 7). In the penis of patients with ischemic priapism, the corpora cavernosa are exposed to intermittent hypoxic episodes. Thus, we are not surprised to see that penile HIF-1␣ gene expression is significantly induced in both ADA⫺/⫺ and SCD Tg mice. HIF-1␣ is a transcription factor that regulates the expression of numerous genes under hypoxic conditions (37). Some genes are activated, whereas others are repressed. Although adenosine is highly induced under ischemic and hypoxic conditions and multiple studies also demonstrated that adenosine signaling stimulates HIF-1␣ accumulation in hypoxic cells (28 –30), whether adenosine contributes to reduction of PDE5 gene expression via HIF-1␣-dependent manner was unknown. Here we provide the first in vivo evidence that ADORA2B signaling is essential for adenosine-mediated induction of HIF-1␣ and reduced PDE5 in two priapic animal models. Second, using primary CCSMCs from wild-type mice and ADORA2Bdeficient mice, we demonstrate that adenosine signaling via ADORA2B activation directly contributes to elevated HIF-1␣ and decreased PDE5 gene expression. Finally, we provide direct evidence that HIF-1␣ is a key factor responsible for ADORA2B-mediated down-regulation of PDE5 gene expression in CCSMCs. Thus, both in vivo and in vitro studies, coupled with genetic and pharmacologic evidence, indicate that the HIF-1␣-mediated reduced PDF5 gene expression contributes to adenosine-induced priapism. As long as an ischemic episode persists, adenosine levels will increase as an extracellular breakdown product of ATP under hypoxic conditions. The excessive adenosine activates ADORA2B, leading to increased HIF-1␣ gene expression. Increased HIF-1␣ production not only reduces PDE5 gene transcription but also stimulated ADORA2B gene expression forming a positive signaling loop (ref. 38 and Fig. 6E). Thus, these studies immediately suggest that lowering adenosine and interfering with ADORA2B activation are potential therapeutic options for priapism in men with SCD. Our studies reported here show that treatment of ADA⫺/⫺ mice or SCD Tg mice with PEG-ADA or PSB1115 for 2 wk significantly reduced priapic features (Fig. 6E). Of note, recent studies showed that excessive ADORA2B signaling on red blood cells promoted sickling by stimulating an increase in 2,3-bisphosphoglycerate production, HbS deoxygenation, and erythrocyte sickling (24). These studies highlight the possibility that lowering adenosine by PEG-ADA or interfering with ADOARA2B activation by a specific antagonist may lead to reduced priapism in SCD mice because of reduced sickling and vasoocclusion, central pathogenic features of the disease. However, we observed the similar therapeutic benefit of PEG-ADA and PSB1115 on priapism in ADA⫺/⫺ mice as in SCD mice. These 2734

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studies provide strong genetic evidence indicating the direct pathological role of excessive adenosine signaling in promoting priapism through the activation of ADORA2B in ADA⫺/⫺ mice. Overall, our preclinical studies in both ADA⫺/⫺ and SCD Tg mice have revealed the beneficial effects of PEG-ADA and PSB1115 (an ADORA2B antagonist) for the prevention and treatment of priapism (Fig. 6E). With regard to adenosine signaling, it is important to note that early studies showed that activation of ADORA2A on iNKT cells reduces pulmonary inflammation and injury in mice with SCD (39). Recent promising phase I trials indicate that activation of iNKT cells in SCD is decreased by the ADORA2A agonist regadenoson (40). Because regadenoson acts on ADORA2A, and presumably not on ADORA2B, we would not expect regadenoson to cause priapism or promote sickling. A potentially promising therapeutic approach for SCD may be to reduce inflammation with regadenoson and prevent the adverse effects of A2B receptor activation (sickling and priapism) with an A2B receptor antagonist. The significance of our findings extends well beyond priapism. Our findings reveal a previously unrecognized important role for excess adenosine signaling in PDE5 gene expression and indicate a new concept for the regulation of penile erection under normal and abnormal conditions. Interfering with excess adenosine-mediated ADORA2B activation is a potential therapeutic strategy for the prevention and treatment of priapism. For ED, enhancing adenosine signaling with an ADORA2B agonist is likely beneficial by increasing CCSMC relaxation and penile erection. It is worth noting that our findings have implications regarding caffeine, a widely used adenosine receptor antagonist consumed in rather high doses. Our findings raise the possibility that high doses of caffeine may be useful to prevent priapism. Conversely, a high intake of caffeine could contribute to ED. Taken together, our findings reveal important novel opportunities for the treatment and or prevention of priapism and ED. This work was supported by U.S. National Institutes of Health grants DK083559 (to Y.X.), HL119549 (to Y.X.), HL114457 (M.R.B., Y.X. and H.K.E.) HL070952 (to M.R.B.), and HL092188 (to H.K.E.), China Scholarship Council grant 2009637520 (to W.R.Z.), and Beijing Natural Science Foundation grant 7144203 (to C.N.). The authors declare no conflicts of interest.

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