PROPHYLACTIC PROOPIOMELANOCORTIN EXPRESSION ALLEVIATES CAPSAICIN-INDUCED NEUROGENIC INFLAMMATION IN RAT TRACHEA

Copyright @ 2009 by the Shock Society. Unauthorized reproduction of this article is prohibited.

PROPHYLACTIC PROOPIOMELANOCORTIN EXPRESSION ALLEVIATESCAPSAICIN-INDUCED NEUROGENIC INFLAMMATION IN RAT TRACHEA

Guei-Sheung Liu,*† Hung-Tu Huang,‡ Che-Jen Lin,* Jhih-Yin Shi,‡ Li-Feng Liu,§

Rue-Tseng Tseng,|| Wen-Tsan Weng,* Hing-Chung Lam,¶ Zhi-Hong Wen,**Tian-Lu Cheng,†† Kuei-Sen Hsu,† and Ming-Hong Tai*‡‡‡

*Department of Medical Education & Research, Kaohsiung Veterans General Hospital, Kaohsiung;†Departments of Pharmacology, College of Medicine, National Cheng Kung University, Tainan; ‡Instituteof Biological Sciences, National Sun Yat-Sen University; §Department of Biological Science and Technology,I-Shou University; ¬Department of Allergy, Immunology, and Rheumatology, Kaohsiung Veterans GeneralHospital; ¶Department of Metabolism, Kaohsiung Veterans General Hospital; **Department of MarineBiotechnology and Resources, National Sun Yat-Sen University; ††Department of Biomedical Scienceand Environmental Science, Kaohsiung Medical University; and ‡‡Institute of Biomedical Sciences,

National Sun Yat-Sen University, Kaohsiung, Taiwan

Received 21 Jan 2009; first review completed 3 Feb 2009; accepted in final form 16 Mar 2009

ABSTRACT—Neurogenic inflammation frequently causes acute plasma leakage in airways and life-threatening pulmonaryedema. However, limited strategies are available to alleviate neurogenic inflammation. Proopiomelanocortin (POMC) is theprecursor of anti-inflammatory melanocortins, which have been proposed of therapeutic potential for various inflammatorydiseases. The present study aimed to evaluate whether peripheral POMC expression ameliorated capsaicin-induced acuteneurogenic inflammation in rat trachea. Prophylactic POMC expression was achieved by intravenous injection ofadenovirus encoding POMC (Ad-POMC), which led to POMC expression in livers and elevated plasmaadrenocorticotropin levels for approximately 60 days. After gene delivery for 7 days, neurogenic inflammation wasinduced in rats by capsaicin injection. The extent of capsaicin-evoked plasma leakage in trachea was alleviated in Ad-POMCYtreated rats compared with animals of control groups (P G 0.01). Moreover, the number of endothelial gaps intracheal venules was also significantly decreased in Ad-POMCYtreated animals (P G 0.01). Prophylactic POMCexpression, however, did not alter the basal substance P (SP) expression or the capsaicin-induced SP elevation intrachea and circulation. Instead, cell cultures studies revealed that POMC overexpression or application of POMC-derivedmelanocortins potently inhibited the SP-induced migration of endothelial cells (P G 0.01), thereby possibly contributing tothe attenuation of endothelial gap formation and plasma leakage. The present study indicates that the anti-inflammatoryPOMC gene vector or melanocortins may constitute a therapeutic alternative for neurogenic inflammation.

KEYWORDS—Proopiomelanocortin, neurogenic inflammation, substance P, plasma leakage, endothelial cells

INTRODUCTION

Neurogenic pulmonary edema is a life-threatening compli-

cation. However, pathogenic mechanism and management

strategies for pulmonary edema due to neurogenic inflamma-

tion are still insufficient. Neurogenic inflammation results

from the release of bioactive substances from peripheral

terminals of primary afferent neurons via axon reflexes or

dorsal root reflexes (1). The inflammation is characterized by

vasodilation, edema formation, and hypersensitivity (2), and

has been implicated in a variety of diseases, including

bronchial inflammatory diseases (3). Cumulative evidence

indicates that peripheral release of neuropeptides such as

substance P (SP) is responsible for neurogenic inflammation.

Exogenously administered SP enhances the permeability of

synovial blood vessels (4) and increases the severity of joint

injury in experimental arthritis in rats (5). Substance P also

stimulates early angiogenesis in the knee joint during acute

neurogenic inflammation, which is a key step in the transition

from acute to persistent inflammation (6). Because SP induces

plasma extravasation in the cutaneous tissues and visceral

organs, application of selective antagonists to blockade

neurokinin receptors has been shown to effectively reduce

the plasma extravasation in vivo (7, 8).

Capsaicin is an excellent agent to induce neurogenic

inflammation in trachea. Capsaicin stimulates airway C-fibers

and is one of the most potent stimuli known to induce the

release of SP and other neurotransmitters from sensory nerves,

causing plasma leakage via the activation of tachykinin NK1

receptors on endothelial cells (9). After capsaicin challenge,

SP elicits local vasodilatation and alters vascular permeabil-

ity, thus enhancing the delivery and accumulation of leuko-

cytes to tissues (10). In addition, SP stimulates the chemotaxis

of various types of cells, including lymphocytes, monocytes,

neutrophils, and fibroblasts (11, 12), and has been implicated

in inflammatory reactions in diverse tissues, including the

lung, gut, and joints (10).

POMC is a multifunctional gene located on human chromo-

some 2p23.3 and encodes a 31-kDa prohormone that is processed

into various neuropeptides, including adrenocorticotrophin

(ACTH), melanotrophins (!Y, "Y and +Ymelanocyte stimulating

645

SHOCK, Vol. 32, No. 6, pp. 645Y650, 2009

Address reprint requests to Ming-Hong Tai, Ph.D. or Kuei-Sen Hsu, Ph.D.,

Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung 804,

Taiwan. E-mail: [email protected] or [email protected].

This work was supported in part by the National Science Council, Taiwan (NSC

94-2752-B-075B-001-PAE and NSC 94-2622-B-075B-001-CC3), Kaohsiung

Veterans General Hospital, Taiwan (VGHKS97-CT3-25), and National Sun Yat-

Sen University-Kaohsiung Medical University Joint Research Center and Asia-

Pacific Ocean Research Center.

DOI: 10.1097/SHK.0b013e3181a5aa10

Copyright � 2009 by the Shock Society


hormone [MSH]), lipotropins, and "-endorphin ("-EP) (13, 14).

Proopiomelanocortin peptides possess pleiotrophic functions,

including anti-inflammation, pigmentation, adrenocortical func-

tion, energy homeostasis, and immune modulation (14). Proopio-

melanocortin processing was thought to occur primarily in the

central nervous system. Recently, POMC neuropeptides such as

!-MSH and "-EP are identified in various non-neuronal cells,

including memory T cells, macrophages, and melanocytes (15),

indicating that processing of POMC into functional peptides also

occurs in the peripheral system. In this study, we evaluated the

preventive effect of prophylactic expression of anti-inflammatory

POMC gene on the capsaicin-induced acute neurogenic inflam-

mation in rat trachea.

MATERIALS AND METHODS

Adenovirus vectors and cell culturesRecombinant adenovirus vectors encoding proopiomelanocortin (Ad-

POMC), green fluorescent protein (Ad-GFP), and luciferase (Ad-luciferase)were prepared as previously described (16). For production and propagation ofadenovirus vectors, E1a-transformed human embryonic kidney 293 cells werepurchased from Microbix Biosystems, Inc. (Toronto, Canada). HumanEA.hy926 endothelial cells (kindly provided by Dr. C.J.S. Edgell at Universityof North Carolina, NC) were maintained at low passage and cultured inDulbecco modified Eagle medium (Gibco BRL, Rockville, Md) containing 10%fetal calf serum (PAA, Austria), 100 2M sodium hypoxanthine, 0.4 2Maminopterin, 16 2M thymidine (Gibco BRL), 2 mM glutamine, 100 U mLj1

penicillin, and 100 2g mLj1 streptomycin (Gibco BRL) in 5% CO2 at 37-C.To investigate the optimal route, rats were administrated with of

adenovirus vectors (1 � 109 plaque-forming units [pfu] per rat in 100 2Lsaline) by intramuscular or intravenous injection via tail vein for 7 days, thenthe blood samples were collected for measurement of plasma ACTH level. Todetermine the dose effect, rats were administrated with escalating doses ofadenovirus vectors (5 � 107, 5 � 108, 5 � 109 pfu per rat in 100 2L saline) byintravenous injection for 7 days before collecting blood samples formonitoring ACTH level.

Bioluminescence imagingAfter anesthesia with a cocktail of ketamine:xylazine (4:1) in phosphate-

buffered saline (PBS), rats were injected intraperitoneally with 200 2L of D-luciferin (Promega, Madison, Wis; 20 mg mLj1) and placed in the imagingchamber (IVIS Imaging System 200 Series; Caliper Life Sciences; Hopkinton,Mass) on a platform warmed to 37-C. A gray-scale body surface image wasobtained in the chamber under dim illumination, followed by 5-minacquisition and overlay of the pseudocolor images. The spatial distributionand quantity of photon counts from cells expressing luciferase were presentedwith colored pixels produced by the computer.

Radioimmunology assay and enzyme immunoassayThe plasma ACTH levels in rats at different time intervals after injection

of adenovirus vectors were determined using a radioimmunology assay (RIA)kit (Nichols Institute Diagnostics, San Juan Capistrano, Calif) as previouslydescribed (16). The plasma SP levels were measured using an enzymeimmunoassay kit (Cayman; Ann Arbor, Mich) following the instructions ofthe manufacturer. To measure plasma SP concentration, the blood samples(0.1 mL) were collected from the anesthetized rats after injection ofadenovirus vectors for 7 days at 30 min before and 5 min after capsaicininjection, respectively.

Capsaicin-induced plasma leakageSprague-Dawley rats (male; 350 T 50 g) were purchased from The National

Animal Center (Taipei, Taiwan) and housed in the animal center of theKaohsiung Veterans General Hospital (Kaohsiung, Taiwan) with 12-h/12-hlight-dark cycle and temperature maintained in 22-C. The study was approvedand conducted according to the Guidelines by the Committee of AnimalExperiments, Kaohsiung Veterans General Hospital (Kaohsiung, Taiwan). Toinduce plasma leakage and neurogenic inflammation in tracheae, rats wereanesthetized with pentobarbital (50 mg mLj1 kgj1, i.p.) and receivedintravenous injections of capsaicin (Sigma; 90 2g mLj1 kgj1, over 2 min)and India ink (Chroma-Gesellschaft; 1 mL kgj1, over 5 s) as previously

described (17). Rats were perfused through the aorta 5 min after capsaicininjection with 0.05 M PBS containing 2% paraformaldehyde and 0.1%glutaraldehyde, pH 7.4, for 2 min. The whole mounts of trachea weredehydrated with ethanol, cleared with toluene, mounted with Permount, andexamined under the microscope. After recording the extents of vascularleakage by photograph, the area density of mucosal surface occupied by theleaky blood vessels was determined by measuring the India inkYlabeledmicrovessels as previously described (18, 19).

Silver staining of endothelial gapsThe sliver staining was used to mark the endothelial gaps as sliver rings to

visualize the change of gap formation as previously described (20). Briefly,after capsaicin injection, the rats were perfused through the aorta with salinefor 3 min then fixatives containing 0.5% glutaraldehyde, 1% paraformalde-hyde in 0.075 M cacodylate buffer (pH 7.4) for 5 min. Subsequently, theanimals were perfused with saline over 2 min, 5% glucose over 10 s, and0.2% AgNO3 in water over 7 s. The whole mounts of trachea were dehydratedwith ethanol for 24 to 48 h, cleared with toluene, and mounted with Permount.The microcirculation and endothelial gaps with silver dots were viewed ata projected magnification of 1,400 on the charge coupled device system(Pro 150ES, Pixera). For each rat, the total number of endothelial gapswere measured from the vascular area of 10 postcapillary venules (diameter,10Y30 2m) and five collecting venules (diameter, 30Y50 2m) by Simple PCIsoftware (Compix Inc., Imaging Systems, Cranberry, Pa). Data wereexpressed as the number of endothelial gaps per square micrometer ofendothelium.

ImmunohistochemistryThe immunohistochemical and morphometric measurements of SP-positive

axons were performed as previously described (21). After capsaicin injectionfor 5 min, rats were perfused for 10 min through the aorta with 0.05 M PBScontaining 2% paraformaldehyde and 0.1% glutaraldehyde (pH 7.4). Gluta-raldehyde was used to ensure the fixation of the mucosal surface epithelium ofthe airways with which the SP-immunoreactive axons are closely associated.After perfusion fixation, the tracheas were removed, washed with phosphatebuffer, and immersed in picric acidYparaformaldehyde for 2 h before thetissues surrounding the intrapulmonary bronchi were cleaned. The tracheaswere cut open longitudinally along their ventral midline, pinned on theSylgard bases Corning (Wiesbaden, Germany), and immersed in fresh picricacidYparaformaldehyde solution for an additional 12 to 18 h at 4-C. Thetissues were washed with distilled water, dehydrated in a graded series ofethanol, cleared in toluene, rehydrated with a graded series of ethanol, andwashed with 0.01 M PBS (pH 7.4). The samples were incubated for 2 h inPBS containing 5% normal goat serum (Zymed Laboratories, San Francisco,Calif) and further incubated for 24 h in rabbit anti-SP (Zymed Laboratoriesdiluted 1:25 in PBS containing 1% normal goat serum), 3% Triton X-100(Sigma), and 0.01% thiemerosal (Sigma). Tissues were washed afterward withPBS for 4 h and then incubated for 12 h in goat antirabbit immunoglobulin Gantibodies (Zymed Laboratories), and were then washed in PBS and incubatedfor 24 h in rabbit peroxidase antiperoxidase complex (diluted 1:250; ZymedLaboratories) at room temperature. Finally, the whole mounts were incubatedin 0.05 M Tris-HCl containing 0.01% H2O2, 0.05% 3, 3¶-di-2, 2 amino-benzidine tetrahydrochloride (Sigma), and 1% Triton X-100 (pH 7.6) for 7 hat 4-C. The whole mounts were then dehydrated with ethanol, cleared withtoluene, and mounted with Permount.

Cell migration assayMigration assay of endothelial cells was performed as previously described

(22). Briefly, EA.hy926 endothelial cells were infected with adenovirusvectors for 24 h and seeded in triplicate in the upper compartments of theBoyden chamber (1.2 � 105 cells in 400 2L) with various concentrations ofSP (1-1000 nM). The lower compartments were filled with 200 2L of theDulbecco modified Eagle medium. A polycarbonate filter (8-2 pore size;Nucleopore, Costar, Chambridge, Mass) coated with 0.005% gelatin to allowcell adhesion separated the compartments. After incubation for 6 h in ahumidified 5% CO2 atmosphere at 37-C, cells on the upper side of the filterwere removed, and those that had migrated to the lower side were fixed inethanol, stained with 10% Giemsa solution (Merck, Germany), and counted asmean T SEM per filter under five different high-power fields. For perturbationof SP-induced migration, ACTH (1Y24), !-MSH, and "-EP were purchasedfrom BACHEM (Torrance, Calif).

Statistical analysisAll values were mean T SEM. Data were analyzed with Student t test or

one-way ANOVA, followed by Newman-Keuls method. A P value less than0.05 was considered statistically significant.

646 SHOCK VOL. 32, NO. 6 LIU ET AL.


RESULTS

Intravenous injection of Ad-POMC led to systemicproduction of POMC neuropeptides

Recombinant Ad-POMC was generated for systemic produc-

tion of anti-inflammatory POMC peptides. To determine the

optimal route, rats were administrated with adenovirus vectors

by intramuscular or intravenous injection then monitored for

plasma ACTH level. It was found that, although injection of Ad-

POMC through either route led to prominent rise in plasma

ACTH level, intravenous injection resulted in a significantly

higher ACTH level in circulation than that in intramuscular route

(P G 0.01; Fig. 1A). Subsequently, we evaluated the dose-

dependency of intravenous POMC gene delivery and revealed

a dose of 5 � 108 pfu was required to elicit significant incre-

ment of plasma ACTH level (P G 0.05; Fig. 1B). Therefore,

intravenous injection of adenovirus vectors at 5 � 109 pfu was

adopted in the following studies.

To evaluate the profile of adenovirus-mediated transgene

expression, we measured the luciferase expression by bio-

luminescence analysis in Sprague-Dawley rats after injection

of Ad-luciferase and found that the bioluminescence was

localized mainly in the livers of rats (Fig. 1B). Subsequently,

to evaluate the duration of liver-based POMC expression, the

plasma was collected at different time intervals after adeno-

virus injection to measure the circulating ACTH level by RIA.

It was found that the circulating ACTH level in Ad-POMCYtreated rats raised within 24 h after injection and reached the

maximal level between days 7 to 14, yet remained higher than

that of control groups for at least 56 days (Fig. 1C). Thus,

intravenous injection of Ad-POMC resulted in liver-based

POMC expression and elevated ACTH levels in circulation.

Prophylactic POMC expression alleviated the plasmaleakage in capsaicin-induced neurogenic inflammation

To evaluate the effects of prophylactic POMC expression

on the capsaicin-induced neurogenic inflammation, rats were

injected with adenovirus vectors for 7 days then administrated

with capsaicin to induce plasma leakage. The plasma leakage

in trachea was monitored using India ink labeling. It was

found that capsaicin induced extensive plasma leakage as

noted by leaky blood vessels in control and Ad-GFPYtreated

rats (Fig. 2A). In contrast, Ad-POMCYtreated rats showed

significantly reduced extent of plasma leakage in the trachea.

By morphometric analysis, Ad-POMCYtreated rats had sig-

nificantly decreased dye accumulation in trachea by 48% of

control groups (Fig. 2B; P G 0.01). Therefore, past POMC

expression attenuated the capsaicin-induced plasma leakage in

trachea.

POMC gene transfer inhibited the capsaicin-inducedendothelial gap formation in trachea

To further validate the protection of POMC gene transfer

on the capsaicin-induced neurogenic inflammation, the extent

of capsaicin-stimulated endothelial gap formation was inves-

tigated by silver staining. The endothelial gaps marked with

sliver deposits were prominent among endothelial cells of

venules in control or Ad-GFPYtreated rats (Fig. 3A), whereas

the Ad-POMCYtreated rats displayed reduced endothelial gap

formation in tracheal venules. Quantification analysis showed

FIG. 1. The adenovirus-mediated transgene expression in rats after intravenous administration. A, Effect of injection route on systemic ACTHproduction by adenovirus-mediated POMC gene transfer. The plasma ACTH levels were measured by RIA after intramuscular (i.m.) or intravenous (i.v.)injection of adenovirus vectors (1 � 109 pfu) for 7 days and expressed as mean T SEM (n = 5). LP G 0.01 vs. Ad-POMC (i.m.). B, Dose-dependent effect ofintravenous POMC gene delivery on circulating ACTH levels. Rats were treated by injection of adenovirus vectors (5 � 107, 5 � 108, 5 � 109 pfu, i.v.) andmonitored for ACTH level at day 7 (n = 5). C, Bioluminescence images of rats treated with PBS (left) or Ad-luciferase (right; 5 � 109 pfu) for 7 days. Thebioluminescence signals after lucigenin injection were localized mainly in the liver as indicated by the arrow. D, Time-dependent effect of plasma ACTH levels inrats after injection of adenovirus vectors (5 � 109 pfu, i.v.) at day 0. Data were mean T SEM (n = 6). *P G 0.05, **P G 0.01 vs. Ad-GFP group.

FIG. 2. Effect of POMC gene transfer on the capsaicin-inducedplasma leakage in trachea. A, The representative photographs of capsai-cin-induced plasma leakage in Ad-GFPY and Ad-POMCYtreated rats. Afterinjection of adenovirus vectors (5 � 109 pfu per rat) for 7 days, rats wereadministrated with capsaicin for 5 min. The leaky blood vessels (arrows) inthe tracheas were labeled with India ink after capsaicin injection. Scale bar,200 2m. B, Quantification analysis of the capsaicin-induced plasma leakagein different groups of rats. Data were mean T SEM (n = 6) percentages ofvessels labeled by India ink. *P G 0.05, **P G 0.01 (n = 6) vs. Ad-GFP andcontrol groups.

SHOCK DECEMBER 2009 POMC FOR NEUROGENIC INFLAMMATION 647


that the endothelial gaps in Ad-POMCYtreated rats were

decreased by 43% of control groups (Fig. 3B; P G 0.01). Thus,

POMC gene transfer attenuated the capsaicin-induced plasma

leakage through perturbation of endothelial gap formation in

trachea.

Prophylactic POMC gene transfer had no effect on thecapsaicin-induced SP expression in trachea and SPrelease in circulation

We attempted to elucidate the protective mechanism

underlying POMC-mediated suppression of neurogenic

inflammation. Because SP is the primary mediator of neuro-

genic inflammation, it was investigated whether POMC

expression perturbed the SP expression or release after

capsaicin injection. Thus, we examined the SP expression

level in trachea by whole mount immunohistochemistry.

Without capsaicin injection, the SP-immunoreactive axons

were scarce in trachea from all groups of rats (Fig. 4A).

Capsaicin injection elicited the presence of numerous SP-

immunoreactive axons at the base of mucosal surface

epithelium in trachea. However, the number or intensities of

SP-positive axons in Ad-POMCYtreated rats was not different

from that in control animals. Similarly, measurement of

plasma SP level revealed that capsaicin administration evoked

a rapid increase in plasma SP level within 5 min after

injection (P G 0.01; Fig. 4B). However, there was no

difference in circulating SP concentration between Ad-POMC

and control groups after capsaicin injection. Therefore, it

seemed unlikely that POMC inhibited the neurogenic inflam-

mation through modulating the endogenous or capsaicin-

induced SP expression.

Past POMC expression perturbed the SP-induced migrationof cultured endothelial cells

Because migration of endothelial cells plays an important

role in endothelial gap formation, we subsequently evaluated

whether POMC expression affected the chemotaxis function

of SP in cultured endothelial cells. To determine the effect of

SP on endothelial motility, we examined the migration of

cultured endothelial cells toward various doses of SP in the

Boyden chamber. It was found that exogenous SP effectively

promoted the migration of endothelial cells at as low as 1 nM

and in a dose-dependent manner with a maximal increment

of 60% of control (P G 0.01; Fig. 5A). Subsequently, we

evaluated the effect of POMC gene transfer on the SP-induced

migration in endothelial cells. Despite the lack of influence on

cell growth (data not shown), POMC gene delivery signifi-

cantly attenuated the SP-stimulated migration that Ad-

POMCYinfected cells showed substantially retarded migration

(102.2 T 6.8 cells at multiplicity of infection [MOI] of

500) compared with cells of control groups (245.3 T 15.5

and 247.2 T 12.2 cells for control and Ad-GFP group at MOI

of 500, respectively; P G 0.01; Fig. 5B). To delineate the

POMC peptide(s) responsible for such inhibition, several

POMC peptides, including ACTH, !-MSH, and "-endorphin,

were applied to investigate their influences on the SP-induced

FIG. 3. Effect of POMC gene transfer on capsaicin-induced endo-thelial gaps formation in rats. A, The representative profile ofcapsaicin-induced endothelial gap formation in the trachea of Ad-GFPYand Ad-POMCYtreated rats. After injection of adenovirus vectors (5 � 109

pfu per rat) for 7 days, rats were administrated with capsaicin for 5 min,and the endothelial gaps (as indicated by asterisk) were visualized bysilver staining. Scale bar, 10 2m. B, Quantification analysis of capsaicin-induced endothelial gap formation in different groups of rats. Thedensities of gaps in venues of microcirculation were expressed asnumber per square micrometer. Data were mean T SEM (n = 6). *P G0.05, **P G 0.01 (n = 6) vs. Ad-GFP and control groups.

FIG. 4. Effect of POMC gene transfer on SP expression in trachea andcirculation after capsaicin injection. A, Representative profiles of SPexpression in the trachea by whole mount immunohistochemical analysis.After injection of adenovirus vectors (5 � 109 pfu per rat) for 7 days, ratswere either left untreated (control) or administrated with capsaicin for 5 min.Subsequently, the tracheas were removed for immunohistological analysis ofSP-positive axons. The SP-positive axons were barely found in rats withoutcapsaicin challenge (control). After capsaicin injection, the SP-positive axons(as indicated by arrows) were readily detected in rat tracheas. However,there was no significant difference in the number of SP-positive axonsbetween Ad-GFPY and Ad-POMCYtreated rats after capsaicin injection. B,Enzyme immunoassay analysis of plasma SP level in rats at 30 min before(control) and 5 min after capsaicin treatment. Data were mean T SEM (n = 6per group). **P G 0.01 vs. control. NS indicates not significant.



endothelial migration. It was noted that adding melanocortins

such as ACTH or !-MSH potently perturbed the SP-induced

endothelial migration (P G 0.01; Fig. 5C). In contrast,

application of "-endorphin had no such effect. Therefore,

POMC overexpression or application of POMC-derived

melanocortins inhibited the SP-induced endothelial migration,

which would possibly perturb the subsequent endothelial gap

formation and plasma leakage in the trachea.

DISCUSSION

Neurogenic inflammation is a pathophysiological process

characterized by increased vascular plasma leakage. The

present study validated the novel protection conferred by

anti-inflammatory POMC expression, which potently allevi-

ates the capsaicin-induced neurogenic inflammation in rat

trachea. In fact, intravenous capsaicin injection leads to severe

neurogenic inflammation that approximately 40% to 60%

experimental animals died immediately after capsaicin admin-

istration probably due to pulmonary complication. In contrast,

none of the Ad-POMCYtreated rats experienced such sudden

mortality after capsaicin injection. This finding may serve as

additional proof supporting the POMC-mediated protection

from aggravated trachea inflammation.

Because SP is an important mediator of neurogenic

inflammation (23, 24), we initially hypothesized that POMC

therapy might confer protection through down-regulation of

endogenous or capsaicin-mediated SP expression. However,

the number of SP-positive axons in Ad-POMCYtreated rats

was approximate to that in control animals before or after

capsaicin injection. Likewise, measurement of plasma SP

levels showed the capsaicin-stimulated rise in circulating SP

level. However, no difference was found among all groups of

rats. Therefore, prophylactic POMC expression had no

influence on basal SP expression and capsaicin-induced SP

release in the trachea and circulation. Subsequently, we

examined whether POMC influenced the functions of SP on

the vessel-lining endothelial cells. It is unveiled that SP

stimulates the motility of endothelial cells in a dose-depend-

ent manner, consistent with recent reports depicting the

functions of SP in mediating the permeability and migration

of endothelial cells (25Y27). These results are also in agree-

ment with our previous study that POMC expression modu-

lates the properties of endothelial cells and suppresses the

angiogenic processes such as migration in cultured endothelial

cells (28).

It remains unclear how the prophylactic POMC expression

exerts the inhibitory function on the capsaicin-induced plasma

leakage. The function of POMC during stress response and

energy homeostasis in the central nervous system is well

established. Nevertheless, the peripheral role of POMC has

begun to be elucidated and explored for the treatment of

various inflammation-related diseases. The POMC-derived

melanocortins, particularly ACTH and !-MSH, are the

plausible candidate molecules to mediate the protective

function of POMC due to their potent efficacy to inhibit

inflammation (29). Indeed, inhibition of SP-induced endothe-

lial migration by ACTH and !-MSH (Fig. 5C) seems to sup-

port such notion. Adrenocorticotrophin stimulates the adrenal

gland to release cortisol, which exerts anti-inflammatory

functions directly or via the synthesis of glucocorticoid. The

potent anti-inflammatory nature of !-MSH has been proposed

for the treatment of various inflammation-related diseases

(29, 30). In addition to POMC neuropeptides, the involvement

of ACTH-induced glucocorticoid production cannot be

ignored. Cumulative evidence indicates that topical or intra-

lesional glucocorticoids confers protection to alleviate neuro-

genic inflammation (31). Besides, glucocorticoids inhibit

plasma extravasation and reduce formation of endothelial

gaps in the rat tracheal mucosa by SP and other tachykinins.

Therefore, it seems plausible that POMC gene delivery

FIG. 5. Effect of POMC gene transfer or POMC-derived peptides on theSP-induced migration of endothelial cells. A, Dose-dependent effect of SPon migration of endothelial cells. After incubation in Boyden chamber for 6 h,the migrated endothelial cells were counted and expressed as mean T SEMpercentages of control in triplicates. B, Effect of POMC gene transfer on theSP-induced migration of endothelial cells. After infection with adenovirusvectors at MOI of 500 or 100 for 24 h, the migration of infected endothelial cellsin the presence of SP (100 nM) was measured in Boyden chamber, and themigrated cells were counted and expressed as mean T SEM percentages ofcontrol in triplicates. *P G 0.05, **P G 0.01. C, Effect of POMC peptides,including ACTH, !-MSH, and "-endorphin, on the SP-induced endothelialmigration. By simultaneous application of various POMC peptides (10 nM), themigration of SP-treated endothelial cells in Boyden chamber was counted andexpressed as mean T SEM percentages of control in triplicates. **P G 0.01 vs.PBS control, LP G 0.05 between SP control and SP plus POMC peptide.

SHOCK DECEMBER 2009 POMC FOR NEUROGENIC INFLAMMATION 649


alleviates the neurogenic inflammation in trachea through

systemic production of melanocortins and cortisol. Future

studies are warranted to dissect the POMC neuropeptides(s)

and pathway(s) responsible for the control of neurogenic

inflammation.

The present study provides the proof-of-principle data to

support the potential of anti-inflammatory POMC therapy for

prevention of neurogenic inflammation. Although gene ther-

apy may not be practical at present, adequate increment of

POMC expression can be attained through programmed

exercise in experimental animals (23, 24) and in human.

Besides, cumulative evidence indicates regular exercise

reduce the risk and devastating effect of stroke (32, 33),

another form of neurogenic inflammation. In addition, a

recent report indicates that post-administration of synthetic

analogs of melanocortins confers long-lasting effect neuro-

genic protection and shows promising efficacy for treatment

of stroke (34). Future studies should be performed to evaluate

the therapeutic efficacy of melanocortins for management of

acute lung edema induced by neurogenic inflammation.

In summary, peripheral POMC gene delivery inhibits the

capsaicin-induced acute inflammation in trachea. Although

the mechanism underlying the alleviated inflammatory

responses by POMC gene transfer remains to be delineated,

future studies are warranted to investigate the synthetic

analogs of !-MSH such as MTII for control of the pulmonary

neurogenic inflammation. Alternatively, the basal POMC

expression could be enhanced by physical training or exercise

to confer protection against the neurogenic inflammation

diseases such as asthma and lung edema. Therefore, we are

cautiously optimistic that the information gained from this

study can be used clinically for neurogenic inflammation.

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PROPHYLACTIC PROOPIOMELANOCORTIN EXPRESSION ALLEVIATES CAPSAICIN-INDUCED NEUROGENIC INFLAMMATION IN RAT TRACHEA