sac-1004, a pseudo-sugar derivative of cholesterol...

Abbreviations

and Acronyms

Ang1 ¼ angiopoietin-1

EC ¼ endothelial cell-cell

ED ¼ erectile dysfunction

eNOS ¼ endothelial NO synthase

ICP ¼ intracavernous pressure

LDL ¼ low density lipoprotein

MCEC ¼ mouse cavernousendothelial cell

MSBP ¼ mean systolic bloodpressure

NO ¼ nitric oxide

PBS ¼ phosphate buffered saline

PDE5 ¼ phosphodiesterase-5

PECAM-1 ¼ platelet/endothelialcell adhesion molecule-1

phospho- ¼ phosphorylated

STZ ¼ streptozotocin

Tie2 ¼ tyrosine kinase with Ig andepidermal growth factor homologydomain-2

VE ¼ vascular endothelial

ZO-1 ¼ zonular occludens-1

1936 j www.jurology.com

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Sac-1004, a Pseudo-Sugar Derivative of Cholesterol,Restores Erectile Function through Reconstructionof Nonleaky and Functional Cavernous Angiogenesisin the Streptozotocin Induced Diabetic Mouse

Dulguun Batbold,* Kang-Moon Song,* Jin-Mi Park, Soo-Hwan Park,

Tack Lee, Dong-Soo Ryu, Young-Ger Suh, Young-Guen Kwon, Ji-Kan Ryu†

and Jun-Kyu Suh†

From the National Research Center for Sexual Medicine, Department of Urology (DB, KMS, JMP, SHP, TL, JKR, JKS) and

Inha Research Institute for Medical Sciences (JKR), Inha University School of Medicine, Incheon, Department of Urology,

Sungkyunkwan University School of Medicine, Samsung Changwon Hospital (DSR), Changwon and Research Institute

of Pharmaceutical Sciences and College of Pharmacy, Seoul National University (YGS) and Department of Biochemistry,

College of Life Science and Biotechnology, Yonsei University (YGK), Seoul, Republic of Korea

Purpose: We examined whether and how Sac-1004, a vascular leakage blocker,would restore erectile function in an animal model of diabetic erectiledysfunction.

Materials and Methods: Eight-week-old C57BL/6J mice were used. Diabetes wasinduced by intraperitoneal injection of streptozotocin. Eight weeks after diabetesinduction the animals were divided into 6 groups, including controls, diabeticmice that received repeat intracavernous injections of phosphate buffered saline(20 ml) on days e3 and 0, and diabetic mice that received repeat intracavernousinjections of Sac-1004 on days e3 and 0 (1, 2, 5 and 10 mg, respectively, in 20 mlphosphate buffered saline). One week after injection erectile function wasmeasured by cavernous nerve stimulation. The penis was then harvested forhistological examinations and Western blot analysis.

Results: Local delivery of Sac-1004 in the corpus cavernosum restored erectilefunction in diabetic mice. The highest erectile response was noted at a dose of5 mg with a response comparable to that in the control group. Sac-1004 signifi-cantly increased cavernous endothelial and smooth muscle contents, and inducedendothelial nitric oxide synthase phosphorylation (Ser1177). Sac-1004 decreasedextravasation of oxidized low density lipoprotein by restoring endothelial cell-celljunction proteins and pericyte content. Sac-1004 also promoted tube formation inprimary cultured mouse cavernous endothelial cells in vitro. Sac-1004 mediatedcavernous angiogenesis and erectile function recovery was abolished by inhib-iting angiopoietin-1-Tie2 signaling with soluble Tie2 antibody.

Accepted for publication December 30, 2015.No direct or indirect commercial incentive associated with publishing this article.The corresponding author certifies that, when applicable, a statement(s) has been included in the manuscript documenting institutional

review board, ethics committee or ethical review board study approval; principles of Helsinki Declaration were followed in lieu of formal ethicscommittee approval; institutional animal care and use committee approval; all human subjects provided written informed consent with gua-rantees of confidentiality; IRB approved protocol number; animal approved project number.

Supported by Korea Healthcare Technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea Grant A110076 (JKSand JKR) and Medical Research Center Grant 2014R1A5A2009392 funded by the Korean Ministry of Science, ICT and Future Planning (JKR).

* Equal study contribution.† Correspondence: National Research Center for Sexual Medicine, Department of Urology, Inha University School of Medicine, 7-206, 3rd St.,

Shinheung-Dong, Jung-Gu, Incheon 400-711, Republic of Korea (telephone: 82-32-890-3505; FAX: 82-32-890-3099; e-mail: [email protected];and telephone: 82-32-890-3441; FAX: 82-32-890-3097; e-mail: [email protected]).

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2016 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION AND RESEARCH, INC.

http://dx.doi.org/10.1016/j.juro.2015.12.103

Vol. 195, 1936-1946, June 2016

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http://dx.doi.org/10.1016/j.juro.2015.12.103

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PSEUDO-SUGAR DERIVATIVE OF CHOLESTEROL RESTORES ERECTILE FUNCTION 1937

Conclusions: With the effects of angiogenesis and antipermeability Sac-1004 reestablishes structural andfunctional cavernous sinusoids. This is highly promising for future treatment of erectile dysfunction fromvascular causes.

Key Words: penis, erectile dysfunction, diabetes mellitus, Sac-1004, angiogenesis inducing agents

Figure 1. Sac-1004 structure

A variety of pathological conditions, includingvascular risk factors or diseases, neurological ab-normalities and hormonal disturbances, are knownto be involved in the pathogenesis of ED.1 Diabetesmellitus is strongly associated with an increasedrisk of ED and about 50% to 75% of diabetic menexperience ED.2,3 Although oral PDE5 inhibitorsare generally effective and considered a first linetreatment modality for ED,4 men with diabetic EDrespond more poorly to these agents than nondia-betic ED patients.5 Diabetes induced severe endo-thelial dysfunction is responsible for poorresponsiveness to PDE5 inhibitors.6,7 Because theaction of PDE5 inhibitors relies on endogenous NOproduction, these agents may fail to increase thecGMP concentration if bioavailable NO does notreach the necessary threshold in cases of severeendothelial dysfunction from diabetes. MoreoverPDE5 inhibitors are not cures for ED and they areused on demand prior to sexual intercourse, whichgreatly limits the spontaneity of the sexual act.Therefore new therapeutic approaches are neces-sary for diabetes associated ED.

Previous groups have reported functional andstructural derangements in cavernous endotheliumin animal models of diabetic ED.8e10 The generationof reactive oxygen species is regarded as an impor-tant cause of endothelial cell damage and reducedNO bioavailability in the diabetic condition.8,11,12

The EC junction, which serves as a barrier byregulating paracellular permeability, has a key rolein vascular formation, networking and remodel-ing.13,14 Recently we reported an increase incavernous endothelial permeability with a concur-rent decrease in cavernous EC junction proteins inanimal models of diabetes.15,16 These findings sug-gest that derangements in both cavernous endo-thelial cells and EC junctions are majorpathophysiological mechanism involved in diabeticED. Therefore restoration of the functional andstructural integrity of the cavernous endotheliummay be a promising therapeutic strategy forcuring ED.

Sac-1004, a pseudo-sugar derivative of choles-terol known to block vascular leakage by enhancingendothelial integrity, has a protective effect onendothelial cells.17 Moreover Sac-1004 was reportedto induce normalization of tumor blood vessels andincrease perfusion.18

In the current study we determined the effec-tiveness of Sac-1004 in promoting nonleaky andfunctional cavernous angiogenesis, and restoringerectile function in STZ induced diabetic micein vivo. We also determined the angiogenic poten-tial of Sac-1004 in primary cultured MCECsin vitro.

METHODS

Sac-1004 Compound FormulationSac-1004 was synthesized as previously described.17

Briefly pregnenolone was reacted with dihydropyran inthe presence of p-toluenesulfonic acid to prepare thetetrahydropyran analogue. After Wittig olefination with4-(carboxybutyl) triphenyl phosphonium bromide theacid moiety was methylated by trimethylsilyl-diazomethane. Sac-1004 was synthesized via tetrahy-dropyran deprotection and subsequent glycosidationwith 4,6-di-O-acetyl-2,3-dideoxyhex-2-enopyran in thepresence of acid (fig. 1). Stock solution of Sac-1004(50 mg/ml) was prepared in dimethyl sulfoxide and di-lutions were made in PBS.

Animals and TreatmentEight-week-old C57BL/6 mice were used in this study.The experiments were approved by the Inha Universityinstitutional animal care and use subcommittee. Dia-betes was induced by intraperitoneal injections of STZ(50 mg/kg) for 5 days consecutively as previouslydescribed.8 Eight weeks after the induction of diabetesthe animals were anesthetized with ketamine (100 mg/kg) and xylazine (5 mg/kg) intramuscularly, and thepenis was exposed by sterile technique. A 30-gauge in-sulin syringe was used to administer repeat injections ofPBS (20 ml on days e3 and 0) or Sac-1004 compound(1, 2, 5 and 10 mg, respectively, in 20 ml PBS on dayse3 and 0) in the mid portion of the corpus cavernosum.

1938 PSEUDO-SUGAR DERIVATIVE OF CHOLESTEROL RESTORES ERECTILE FUNCTION

We compressed the penis at the base with a vascularclamp immediately before injection. The clamp remainedin place for 30 minutes to restrict blood flow out of thepenis.

One week after treatment we evaluated erectilefunction during electrical stimulation of thecavernous nerve in 6 mice per group. The highesterectile response was noted in diabetic mice thatreceived Sac-1004 at the concentration of 0.25 mg/ml(5 mg in 20 ml PBS). Based on these functional resultsthe mice were divided into the 3 groups for histolog-ical examination and Western blot analysis, includingage matched control, diabetic mice that receivedrepeat intracavernous injections of PBS (20 ml on dayse3 and 0) and diabetic mice that received repeatintracavernous injections of Sac-1004 compound (5 mgin 20 ml PBS on days e3 and 0). Fasting and post-prandial blood glucose levels were determined with anAccu-Chek� blood glucose meter before the mice weresacrificed.

Physiological Erection and Inhibition StudiesICP and systemic blood pressure were measured duringelectrical stimulation of the cavernous nerve as previouslydescribed.8

For the inhibition study a separate group of diabeticmice was subcutaneously given soluble antibody to Tie2(4 mg/20 ml, R&D Systems�), a receptor tyrosine kinase forAng1. This was done to examine the role of Ang1 in Sac-1004 mediated enhancement in angiogenesis andimprovement in erectile function. Soluble Tie2 wasadministered immediately before intracavernous injectionof Sac-1004.

Histological ExaminationsFor fluorescence microscopy in 6 mice per group peniletissue was fixed in 4% paraformaldehyde for 24 hours at4C. Frozen section tissues (8 mm) were incubated withantibodies to the endothelial cell marker PECAM-1(Chemicon�, 1:50), FITC (fluorescein isothiocyanate)conjugated antibody to smooth muscle a-actin, a smoothmuscle cell marker (Sigma�, 1:200), the pericytemarker NG2 (Millipore�, 1:50), VE-cadherin (1:50),occludin (Invitrogen�, 1:50), oxidized LDL (Abcam�,1:50), phospho-eNOS (Ser1177, Cell Signaling Tech-nology�, 1:50) or eNOS (BD Transduction Labo-ratories�, 1:300) at 4C overnight. After several washeswith PBS sections were incubated with tetramethylrhodamine isthiocyanate or FITC conjugated secondaryantibodies (Zymed� Laboratories) for 2 hours at roomtemperature. Signals were visualized and digital imageswere obtained with a FV1000 confocal microscope(Olympus�).

The content of endothelial cells, smooth muscle cellsand pericytes as well as the extent of phospho-eNOS,eNOS, VE-cadherin, occludin and oxidized LDL stainingin the corpus cavernosum were quantitatively analyzedwith the Image J 1.34 image analyzer system (http://rsb.info.nih.gov/ij/index.html).

Western BlotEqual amounts of protein (100 mg per lane) were elec-trophoresed on 8% to 12% sodium polyacrylamide gels,

transferred to nitrocellulose membranes and probedwith antibodies to eNOS (BD Transduction Labora-tories, 1:200), phospho-eNOS (Ser1177, Cell SignalingTechnology, 1:200), Ang1 (NOVUS� Biologicals, 1:200),ZO-1 (Zymed Laboratories, 1:1,000), occludin (Invi-trogen, 1:1,000) or b-actin (Abcam, 1:6,000). Resultswere quantified by densitometry in 4 preparations pergroup.

Tube Formation AssayMCECs were prepared and maintained as previouslydescribed.19 Cells at passages between 2 and 3 were usedfor experiments. Tube formation assay was performedusing 48-well plates coated with 100 ml Matrigel� perwell. MCECs were seeded on coated plates at 2 � 104

cells per well in complete Medium 199 (GIBCO�).Samples were divided into 3 groups, including MCECsexposed to a normal glucose condition (5 mmol), MCECsexposed to a high glucose condition (30 mmol) andtreated with PBS, and MCECs exposed to a high glucosecondition (30 mmol) and treated with Sac-1004 (0.1 mg/1 ml). The assay was performed in a CO2 incubator andthe plates were incubated at 37C for 24 hours. Imageswere obtained with a phase contrast microscope. Thenumber of tubes in each well of the plate was counted ata screen magnification of 40�. Only integrated tubeswere counted.

Statistical AnalysisResults are expressed as the mean � SE. Group compar-isons of parametric data were made by 1-way ANOVAfollowed by the Newman-Keuls post hoc test. We used theKruskal-Wallis test for nonparametric data. Statisticalanalysis was performed with SigmaStat� 3.5 softwarewith p <5% considered significant.

RESULTS

Metabolic Variables

Body weight was significantly lower in diabetic micethan in control mice. Fasting and postprandial bloodglucose concentrations were significantly higher indiabetic mice than in controls. No significant dif-ferences in body weight or blood glucose levels werefound between the diabetic groups regardless of thetreatment given (see table).

Sac-1004 in Diabetic Mice

Restored Erectile Function. Figure 2, A shows arepresentative intracavernous tracing afterstimulation of the cavernous nerve (5 V and 12 Hzfor 1 millisecond) for 1 minute in age matchedcontrol and diabetic mice 1 week after treatment.Ratios of maximal ICP and total ICP to MSBP weresignificantly lower in PBS treated diabetic mice thanin age matched controls. Repeat intracavernousinjections of Sac-1004 significantly improvederection parameters at the concentrations of0.1 mg/ml (2 mg in 20 ml), 0.25 mg/ml (5 mg in 20 ml)and 0.5 mg/ml (10 mg in 20 ml). The highest erectile

http://rsb.info.nih.gov/ij/index.html

http://rsb.info.nih.gov/ij/index.html

Physiological and metabolic parameters in 6 STZ induced diabetic mice per group

Mean � SE Control Mean � SE PBS

Mean � SE Sac-1004 (mg/ml)

0.05 0.1 0.25 0.5

Body wt (gm) 31.8 � 0.43 18.9 � 0.77* 23.8 � 0.63* 21.0 � 0.54* 21.6 � 0.43* 21.4 � 0.59*Glucose (mg/dl):Fasting 113.5 � 2.1 238.3 � 34.7* 293.3 � 12.2* 270.0 � 26.3* 251.3 � 12.4* 248.3 � 22.4*Postprandial 149.8 � 4.4 567.8 � 32.3* 534.8 � 12.2* 475.3 � 19.6* 505.3 � 26.9* 556.3 � 21.7*

Blood pressure (mm Hg):Systolic 90.3 � 1.9 95.0 � 3.3 99.7 � 6.4 95.4 � 4.5 99.5 � 5.3 98.6 � 4.2Mean 71.5 � 0.5 70.4 � 2.0 75.0 � 4.1 72.3 � 3.3 70.5 � 2.4 75.7 � 5.4Diastolic 61.9 � 1.4 57.3 � 2.1 63.5 � 4.6 56.1 � 2.6 55.7 � 2.8 62.1 � 5.9

*p <0.01 vs control.


function recoverywas observedat the concentrationof0.25 mg/ml (5 mg in 20 ml), which reached up to 96% ofcontrol values (fig. 2, B and C ). No detectabledifferences were found in MSBP among the 6experimental groups (see table). However erectilefunction recovery was not obvious 2 weeks after Sac-1004 administration (fig. 2, D to F ).

Figure 2. Sac-1004 restored erectile function. A, representative ICP r

stimulus interval. B and C, ratio of mean maximal ICP and total IC

Asterisk indicates p <0.01 vs control. Single pound sign indicates

p <0.05 vs control and diabetic plus PBS. D, representative ICP re

maximal ICP and total ICP to MSBP in 6 mice per group. Single aster

Restored Cavernous Endothelial and Smooth Muscle

Contents. Immunohistochemical staining ofcavernous tissue with antibody to PECAM-1 orsmooth muscle-a actin was performed in agematched control and diabetic mice 1 week aftertreatment. We found significantly lower cavernousendothelial and smooth muscle contents in PBS

esponses 1 week after treatment. Solid bar indicates 1-minute

P (AUC) to MSBP in 6 mice per group. DM, diabetes mellitus.

p <0.01 vs diabetic plus PBS. Double pound signs indicate

sponses of 2 weeks after treatment. E and F, ratios of mean

isk indicates p <0.01 vs control.

Figure 3. Sac-1004 restored cavernous endothelial and smooth muscle contents. A, immunohistochemical staining of cavernous tissue

with antibodies to PECAM-1 (red areas) and smooth muscle a-actin (green areas) 1 or 2 weeks after treatment. Reduced from �200.

B and C, quantitative analysis of endothelial and smooth muscle contents in 6 mice per group. DM, diabetes mellitus. B, asterisk

indicates p <0.05 vs control. Pound sign indicates p <0.01 vs diabetic plus PBS. C, asterisk indicates p <0.01 vs control. Pound sign

indicates p <0.05 vs diabetic plus PBS. D, tube formation assay in MCECs exposed to normal glucose (NG, 5 mmol) or high

glucose (HG, 30 mmol) condition and treated with 1 ml PBS or 0.1 mg/ml Sac-1004. E, number of tubes per field at 40� screen

magnification. Asterisk indicate p <0.01 vs NG group. Pound sign indicates p <0.01 vs high glucose plus PBS.


treated diabetic mice than in control mice. Repeatintracavernous injections of Sac-1004 restoredcavernous endothelial and smooth muscle contents

in diabetic mice. However cavernous endothelialand smooth muscle contents returned to baseline 2weeks after Sac-1004 administration (fig. 3, A to C ).

Figure 4. Sac-1004 induced cavernous eNOS phosphorylation. A, immunohistochemical staining of cavernous tissue performed with

antibodies to phospho-eNOS (P-eNOS ) and eNOS 1 week after treatment. Reduced from �200. B and C, quantitative analysis of

phospho-eNOS or eNOS positive area in 6 mice per group. DM, diabetes mellitus. Pound sign indicates p <0.01 vs diabetic plus

PBS. B, asterisk indicates p <0.05 vs control. C, Asterisk indicates p <0.01 vs control. D, Western blot results were similar in 4

independent samples.


We further examined the angiogenic effect of Sac-1004 in primary cultured MCECs. In vitro Matrigelassay revealed impairments in tube formation in

MCECs exposed to high glucose conditions, whichwere completely restored by treatment with Sac-1004 (0.1 mg/ml) (fig. 3, D and E ).


Induced Cavernous eNOS Phosphorylation. Cavernousphospho-eNOS or total eNOS expression asassessed by immunohistochemical staining andWestern blot analysis was significantly lower inPBS treated diabetic mice than in age matchedcontrols. Repeat intracavernous injections of Sac-1004 significantly increased cavernous phospho-eNOS and total eNOS expression in diabetic mice(fig. 4).

Restored Cavernous EC Junction Proteins. We per-formed immunohistochemical staining and West-ern blot to evaluate cavernous expression ofadherens junction protein (VE-cadherin) andtight junction proteins (ZO-1 and occludin) in agematched control and diabetic mice 1 week aftertreatment. Cavernous expression of EC junctionproteins was significantly lower in PBS treateddiabetic mice than in age matched controls.Repeat intracavernous injections of Sac-1004significantly restored EC junction proteins inthe corpus cavernosum tissue of diabetic mice(fig. 5).

Figure 5. Sac-1004 restored cavernous EC junction proteins. A, immu

occludin and VE-cadherin 1 week after treatment. Reduced from �2

positive area in 6 mice per group. DM, diabetes mellitus. Pound sig

p <0.05 vs control. C, asterisk indicates p <0.01 vs control. D, Wes

b-actin in 4 mice per group. E, asterisk indicates p <0.05 vs control

asterisk indicates p <0.01 vs control. Pound sign indicates p <0.01 vs

Restored Cavernous Pericyte Content. Pericytes areknown to have a crucial role in vascular develop-ment and the regulation of vascular perme-ability.20,21 We performed immunohistochemicalstaining of cavernous tissue with antibody to NG2in age matched control and diabetic mice 1 weekafter treatment. We observed a significantly lowerNG2 positive cavernous pericyte content in PBStreated diabetic mice than in controls. Repeatintracavernous injections of Sac-1004 completelyrestored the cavernous pericyte content in diabeticmice (fig. 6).

Decreased Cavernous Oxidized LDL Extravasation.

Immunohistochemical double staining of cavernoustissue with antibodies to PECAM-1 and oxidized LDLwas performed in age matched control and diabeticmice 1 week after treatment. Extravasation ofoxidized LDL was significantly higher in the corpuscavernosum tissue of PBS treated diabetic mice thanin control mice. Repeat intracavernous injection ofSac-1004 significantly reduced cavernous oxidizedLDL leakage in diabetic mice (fig. 7).

nohistochemical staining of cavernous tissue with antibodies to

00. B and C, quantitative analysis of occludin and VE-cadherin

n indicates p <0.01 vs diabetic plus PBS. B, asterisk indicates

tern blot. E to G, relative density of each protein compared to

. Pound sign indicates p <0.05 vs diabetic plus PBS. F and G,

diabetic plus PBS.

Figure 6. Sac-1004 restored cavernous pericyte content. A, immunohistochemical double staining of cavernous tissue with antibodies

to NG2 (green areas) and PECAM-1 (red areas) 1 week after treatment. Reduced from �200. B, quantitative analysis of NG2 positive

area in 6 mice per group. Asterisk indicates p <0.05 vs control. Pound sign indicates p <0.05 vs diabetes plus PBS. DM, diabetes

mellitus.


Sac-1004 Induced Cavernous Angiogenesis and

Recovery of Erectile Function was Ang1-Tie2

Pathway Dependent

The Ang1-Tie2 signaling pathway has an importantrole in generating a nonleaky, stable and functionalvasculature.22 Cavernous Ang1 protein expressionwas significantly lower in PBS treated diabetic micethan in control mice. The expression of Ang1 proteinreturned to control values after treatment with Sac-1004 (fig. 8, A and B).

Cavernous nerve mediated erection studies indi-cated that recovery of erectile function was attenu-ated after treatment with Sac-1004 in diabetic micetreated with soluble Tie2 (fig. 8, C to E ). MoreoverSac-1004 induced restoration of cavernous endo-thelial cell and pericyte content. The subsequentdecrease in extravasation of oxidized LDL wasdiminished by treatment with soluble Tie2 in dia-betic mice (fig. 8, F and G).

DISCUSSIONThe major findings of this study are that 2 suc-cessive injections of Sac-1004 in the corpus cav-ernosum of diabetic mice significantly increased

cavernous endothelial and smooth muscle contents,and induced eNOS phosphorylation (Ser1177) anddecreased extravasation of oxidized LDL byrestoring EC junction proteins and pericyte con-tent. These changes restored erectile function indiabetic mice. Sac-1004 mediated cavernous angio-genesis and erectile function recovery depended onthe Ang1-Tie2 pathway. Sac-1004 also promotedtube formation in primary cultured MCECsin vitro. Figure 9 summarizes a proposed mecha-nism of action by which Sac-1004 restores erectilefunction.

In agreement with the findings of our previousstudies in STZ induced mice15,23,24 we found asignificant decrease in cavernous endothelial areain PBS treated diabetic mice compared with that inage matched controls. Intracavernous injection ofSac-1004 completely recovered cavernous endothe-lial content in diabetic mice. The presence ofnormal corporeal smooth muscle content is a pre-requisite for a normal erectile response and areduction in smooth muscle content leads to ED inthe diabetic condition.8,9 In the current studyintracavernous injection of Sac-1004 also restoredcavernous smooth muscle content in diabetic mice,

Figure 7. Sac-1004 decreased cavernous oxidized LDL extravasation. A, immunoflourescent double staining of cavernous tissue with

antibodies to oxidized LDL (green areas) and PECAM-1 (red areas) 1 week after treatment. Reduced from �200. B, quantitative analysis

of oxidized LDL positive area in 6 mice per group. Asterisk indicates p <0.01 vs control. Pound sign indicates p < 0.01 vs diabetic plus

PBS and control. DM, diabetes mellitus.


although it did not reach the level in age matchedcontrols.

EC junction proteins have an important role inregulating vascular permeability and derangementssince these proteins are involved in a variety ofdiabetes related microangiopathies, such as diabeticretinopathy25 and diabetic ED.15,16 Similar to thesefindings cavernous expression of EC junction pro-teins was significantly decreased in the diabeticcondition. Intracavernous injection of Sac-1004restored cavernous EC junction proteins in dia-betic mice.

A previous study showed that Sac-1004 blocksvascular leakage by enhancing endothelial integ-rity via the cAMP/Rac/cortactin pathway.17

Therefore we speculate that the cAMP/RAC/cor-tactin pathway may also be involved in Sac-1004mediated restoration of endothelial junctionalintegrity in the diabetic penis. Furthermore peri-cytes, which were discovered as a population ofcontractile cells surrounding endothelial cells, arealso known to be involved in the regulation ofvascular or cavernous endothelial perme-ability.20,21 Pericyte loss or dropout and subse-quent capillary leakage are the main features of

diabetic retinopathy.26 Intracavernous injection ofSac-1004 significantly restored the NG2 positivecavernous pericyte content in diabetic mice so thatit was comparable to levels in age matchedcontrols.

In the current study the extravasation ofoxidized LDL in the corpus cavernosum wassignificantly increased in diabetic mice. Thevascular endothelium serves as a barrier to LDL inthe physiological condition.27 Diabetes mellitus isknown to promote LDL oxidation, which inducesinflammatory responses, oxidative stress andendothelial cell apoptosis.28 It was also reported inpatients with diabetic retinopathy that theseverity of retinopathy is proportional to theextent of oxidized LDL leakage.29 Sac-1004 signif-icantly decreased oxidized LDL leakage in thecorpus cavernosum tissue of diabetic mice. Webelieve that the restoration of cavernous EC junc-tion proteins and pericytes is responsible for theSac-1004 mediated decrease in oxidized LDLextravasation.

NO is a major mediator of penile erection.30 Inthe current study eNOS phosphorylation (Ser1177)was significantly decreased in PBS treated diabetic

Figure 8. Sac-1004 induced cavernous angiogenesis and

recovery of erectile function was Ang1-Tie2 pathway

dependent. A, Western blot. B, relative density of Ang1

protein vs b-actin in 4 mice per group. Asterisk indicates

p <0.05 vs control. Pound sign indicates p <0.05 vs diabetic

plus PBS. DM, diabetes mellitus. C, representative ICP

responses 1 week after treatment. Solid bar indicates 1-minute

stimulus interval. D and E, ratios of mean maximal ICP and

total ICP (AUC) to MSBP in 4 mice per group. Asterisk

indicates p <0.01 vs control. Pound sign indicates p <0.01 vs

PBS þ Fc treated diabetic. Dagger indicates p <0.05 vs Sac-

1004 treated diabetic. F, immunohistochemical staining of

cavernous tissue with antibodies to PECAM-1 (red areas), NG2

(green areas) and oxidized LDL (green areas). Reduced from

�200. G, quantitative analysis of endothelial cell and pericyte

content, and oxidized LDL positive area in 4 mice per group.

Asterisk indicates p <0.01 vs control. Pound sign indicates

p <0.01 vs PBS þ Fc treated diabetic. Dagger indicates

p <0.05 vs Sac-1004 treated diabetic.

Figure 9. Proposed mechanism of action of how Sac-1004

restores erectile function in diabetic mice.


mice compared with the control group. Increasedreactive oxygen species generation is known toinactivate eNOS in the penis of STZ induced dia-betic mice.6 Sac-1004 restored endogenous eNOSphosphorylation in diabetic mice. Regeneration ofcavernous endothelial cell content and protection ofendothelial cell damage by reducing oxidized LDLexpression may account for the restoration of eNOSactivity.

A lack of long-term efficacy of Sac-1004 toimprove erectile function is a limitation of thisstudy. Further studies are necessary to deter-mine whether periodic injections or chronicadministration of Sac-1004 as an oral formulationwould induce long-lasting recovery of erectilefunction.

CONCLUSIONSIntracavernous administration of Sac-1004 suc-cessfully restored erectile function in diabetic micevia healthy cavernous angiogenesis as evidencedby the restoration of cavernous endothelial cellsand EC junction proteins, smooth muscle cellsand pericytes as well as decreased vascularpermeability. Reestablishment of functional andstructural cavernous vasculature using the anti-permeability factor Sac-1004 may be a highlypromising treatment modality for ED from vascularcauses.


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sac-1004, a pseudo-sugar derivative of cholesterol...

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