BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 244, 449–454 (1998)ARTICLE NO. RC988232

Central Delivery of Human Tissue Kallikrein GeneReduces Blood Pressure in Hypertensive Rats

Cindy Wang,* Caroline Chao,* Paolo Madeddu,† Lee Chao,* and Julie Chao1

*Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston,South Carolina 29425-2211; and †Clinica Medica, University of Sassari, Sassari, Italy

Received January 26, 1998

acetate-salt hypertensive rats (5, 6). The ability of cen-The human tissue kallikrein gene, in the form of na- trally administered angiotensin-converting enzyme

ked DNA (CMV-cHK) or an adenoviral vector (Ad.CMV- (ACE) inhibitors to lower mean arterial pressure hascHK), was directly delivered by intracerebroventricu- been demonstrated in SHR (7). Kinins have been impli-lar injection into spontaneously hypertensive rats. cated in the anti-hypertensive action of ACE inhibitors,Control rats received the same amount of vector DNA suggesting that the brain kallikrein-kinin system con-(pcDNA3) or adenoviral vector (Ad.CMV-LacZ) car-

tributes to ACE inhibition. It has been shown that therying the lacZ gene. A single injection of the humanbradykinin B1 and B2 receptors are involved in thetissue kallikrein gene caused a rapid and prolongedblood pressure regulation in the central nervous sys-blood pressure-lowering effect that began 1 day posttem (CNS) by antisense inhibition strategies (8, 9). In-injection and the effect lasted for more than 7 days.hibition of kininogen or B2 receptor mRNA translationThe expression of human tissue kallikrein and itsin the CNS by antisense oligonucleotides caused a sig-mRNA was identified in the cortex, cerebellum, brainnificant blood pressure increase in SHR (8). Antisensestem, hippocampus and hypothalamus. Cellular local-inhibition of the B1 receptor mRNA translation in theization of b-galactosidase was detected by X-gal stain-CNS produced a profound blood pressure reduction foring in the thalamus, hypothalamus and third ventricle

in rats injected with Ad.CMV-LacZ. This suggests that more than 48 hours (9). These results indicate that thethe tissue kallikrein-kinin system may function in the brain kallikrein-kinin system plays a role in the centralcentral control of blood pressure homeostasis. q 1998 regulation of blood pressure and suggests that this sys-Academic Press tem may exert a protective action against further eleva-

tion of blood pressure levels in SHR.Our previous studies have shown that systemic kalli-

krein gene delivery in the form of naked DNA or adeno-Tissue kallikrein (E.C. 3.4.21.35) is a serine protein- virus dramatically delays the development of hyperten-ase that is capable of cleaving low-molecular weight sion in various animal models (10, 11). In this study, wekininogen to produce the vasoactive kinin peptide (1). present data on central delivery of the tissue kallikreinThe binding of kinins to bradykinin B2 receptors acti- gene in the brain and its effect on blood pressure ho-vates second messengers which trigger a broad spec- meostasis in SHR.trum of biological effects such as vasodilation, smoothmuscle contraction and relaxation, inflammation, pain

MATERIALS AND METHODSand cell proliferation (2). Abnormality of the tissue kal-likrein-kinin system has long been documented in the

Animals. Adult spontaneously hypertensive rats (male, 8 weekspathogenesis of hypertension (3, 4). Accumulated evi-old, 160-170 g) were purchased from Harlan Sprague-Dawley (India-dence suggests a role of the tissue kallikrein-kinin sys-napolis, ID). All experimental protocols were approved by the Institu-

tem in central blood pressure regulation. Kallikrein tional Animal Research Committee of the Medical University oflevels in cerebrospinal fluid of spontaneously hyperten- South Carolina and were carried out according to the Guide for the

Care and Use of Laboratory Animals of the National Institutes ofsive rats (SHR) have been shown to be higher thanHealth.that in Wistar-Kyoto rats and in deoxycorticosterone

Adenovirus and plasmid DNA construct. Adenoviral vector car-rying a human tissue kallikrein expression unit under the control of acytomegalovirus promoter/enhancer (Ad.CMV-cHK) was constructed1 Corresponding author. Fax: (803)-792-1627. E-mail: chaoj@

musc.edu. as previously described (12). Adenovirus harboring the LacZ gene

0006-291X/98 $25.00Copyright q 1998 by Academic PressAll rights of reproduction in any form reserved.

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under the control of a cytomegalovirus promoter (Ad.CMV-LacZ) wasobtained from the Institute for Human Gene Therapy (Wistar Insti-tute, Philadelphia, PA). Human tissue kallikrein expression plasmidCMV-cHK was constructed as previously described (13). The humantissue kallikrein cDNA was oriented under the control of the cyto-megalovirus (CMV) promoter and followed by the bovine growth hor-mone polyadenylation signal sequence. The plasmid DNA of pcDNA3

vector and CMV-cHK were purified with a plasmid purification kit(Qiagen, Chatsworth, CA) according to the manufacturer’s instruc-tion.

Intracerebroventricular (ICV) delivery of Ad.CMV-cHK adenovirusand CMV-cHK plasmid DNA. For adenovirus-mediated gene deliv-ery, SHR were randomly divided into two groups, control andAd.CMV-cHK, with 6 and 8 animals in each group, respectively. ICVinjection of adenovirus Ad.CMV-cHK (1 1 109 pfu in 10 ml of 10 mMTris-HCl, pH 7.4, 1 mM MgCl2, 10% glycerol per rat) was performedas previously described (14). Control rats received the same amountof control adenovirus Ad.CMV-LacZ. For kallikrein gene deliverycarried by naked plasmid DNA, SHR were randomly divided intotwo groups, control and CMV-cHK with 6 animals in each group.SHR in the CMV-cHK group received CMV-cHK plasmid DNA (100mg in 10 ml of phosphate-buffered saline) via ICV injection. Controlrats received the same amount of vector DNA pcDNA3.

Systolic blood pressure. Systolic blood pressure was measuredwith a photoelectric tail cuff device (Natsume Co., Tokyo, Japan)(15). For each animal, the systolic blood pressure was representedas the mean of 8 recordings.

Preparation of tissue extracts. At 1, 3, 5, and 7 days post genedelivery, rats were sacrificed and the brain was dissected to exam-ine the expression of human tissue kallikrein. Tissue extracts wereprepared as previously described (16). The total protein was deter-mined by Lowry protein assay using bovine serum albumin as thestandard (17).

Enzyme-linked immunosorbent assay (ELISA). For detection ofimmunoreactive human tissue kallikrein in tissue extracts, anELISA was performed as previously described (13).

Reverse transcription-polymerase chain reaction (RT-PCR) South-ern blot analysis of human tissue kallikrein mRNA. Total RNA wasextracted from fresh rat tissues by guanidine isothiocyanate. RT-PCR Southern blot analysis specific for human tissue kallikrein

FIG. 1. (A) Systolic blood pressure of SHR rats receiving adenovi-mRNA was performed as previously described (10).ral vectors Ad.CMV-cHK or Ad.CMV-LacZ via ICV injections. (B)

Preparation of cryosections and detection of b-galactosidase activ- Systolic blood pressure of SHR rats receiving plasmid DNA CMV-ity. Rat brain was embedded in O.C.T. compound (Tissue-Tek, cHK or vector DNA pcDNA3 (control) via ICV injections. SystolicMiles Inc., Elkhart, IN) and the sections were stained in a solution blood pressure values are expressed as mean{ SEM (nÅ6). †Põ0.01,containing 5 mM K4Fe(CN)6, 5 mM K3Fe(CN)6, 1 mM MgCl2 and 0.5 Ad.CMV-cHK vs. Ad.CMV-LacZ group. *Põ0.05, CMV-cHK vs. con-mg/ml 5-bromo-4-chloro-3-indoyl-beta-D-galactopyranoside (X-gal) trol group.at 377C for 2 hours. After staining, the sections were fixed in 2%gluteraldehyde for 5 minutes, rinsed in PBS and mounted.

Urine collection and electrolyte analysis. Twenty-four hour urineof rats was collected using metabolic cages at 5 days post-injection. SHR, systolic blood pressure was monitored from 1 toThe rats were maintained in metabolic cages that allowed for the

10 days post gene delivery (Fig. 1A). The basal bloodseparate collection of urine and measurement of water intake.pressure before gene delivery was 172 { 2 and 175 {Statistical analysis. Repeated blood pressure measurements be-2 mmHg in control and Ad.CMV-cHK groups, respec-tween control and experimental groups were evaluated by ANOVA.

The statistical difference between two groups was estimated using tively. A single ICV injection of the recombinant adeno-Student’s t test for unpaired values. Group data is expressed as mean virus harboring the human tissue kallikrein gene{ SEM. Values were considered significantly different at Põ0.05. caused a significant blood pressure-lowering effect that

began 1 day post injection and continued for 7 days. ARESULTSmaximal difference in blood pressure occurred 5 days

Effect of Central Delivery of the Human Tissue post injection between control and Ad.CMV-cHKKallikrein Gene on the Blood Pressure of groups (195 { 4 vs. 174 { 3 mmHg, n, Põ0.01). Com-Spontaneously Hypertensive Rats pared with control rats receiving Ad.CMV-LacZ, the

body weight of the Ad.CMV-cHK group significantlyTo evaluate the effect of intracerebroventricular(ICV) injection of adenoviral vector Ad.CMV-cHK in decreased (nÅ6, Põ0.05), beginning 2 days post gene

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Markedly reduced levels of human tissue kallikreinwere detected in these extracts 7 days post gene deliv-ery (Table 1). These results suggest that direct ICVinjection of the adenoviral vector carrying the humantissue kallikrein expression unit can result in a localexpression of human tissue kallikrein in rat brain.

Expression of Human Tissue Kallikrein mRNAby RT-PCR Southern Blot Analysis

The expression of the human tissue kallikrein mRNAin SHR was detected by RT-PCR followed by Southernblot analysis 3 days after ICV injection of adenoviralvectors Ad.CMV-cHK or Ad.CMV-LacZ. Human tissuekallikrein mRNA was expressed in the cortex, cerebel-lum, brain stem, hippocampus, and hypothalamus inFIG. 2. Enzyme-linked immunosorbent assay of recombinant hu-

man tissue kallikrein in rat brain extracts and sera at 1 day post SHR receiving Ad.CMV-cHK (Fig. 3). The expressiongene delivery. The standard curve of human tissue kallikrein ranges of human tissue kallikrein mRNA was not detected infrom 0.4 to 25.0 ng/ml. Serial dilutions of rat brain extracts and sera control rats receiving adenoviral vector Ad.CMV-LacZare parallel to the human tissue kallikrein standard curve.

(Fig. 3). Similar levels of b-actin mRNA were detectedin brain tissues of both experimental and controlgroups, indicating the integrity of RNA in these sam-

delivery and continuing for 6 days. The difference in ples (Fig. 3). In addition, RT-PCR Southern blot analy-body weight between control and Ad.CMV-cHK (183 { sis was performed to examine the expression of human5 vs. 159 { 5 g, nÅ6, Põ0.05) groups reached a maxi- tissue kallikrein in various peripheral tissues includ-mum 2 days post injection. ing kidney, adrenal gland, heart, thoracic aorta, liver,

When the human tissue kallikrein gene in the form pancreas, spleen, and salivary gland of SHR after cen-of naked plasmid DNA (CMV-cHK) was injected into tral delivery of the human kallikrein gene. The expres-the brain of SHR via ICV cannula, a similar blood pres- sion of human tissue kallikrein mRNA was not de-sure lowering-effect was observed (Fig. 1B). The basal tected in peripheral tissues examined (data not shown).blood pressure before gene delivery was 177 { 4 vs. These results show that the sites of human tissue kalli-178 { 4 mmHg in control and CMV-cHK groups, re- krein synthesis are localized in the CNS of SHR afterspectively. A maximal difference in blood pressure oc- ICV injection of adenovirus Ad.CMV-cHK.curred 3 days post injection between control and CMV-cHK groups (200 { 6 mmHg vs. 182 { 2 mmHg, nÅ6, Cellular Localization of b-Galactosidase ActivityP0.05). The blood pressure lowering effect lasted for 7

Fig. 4 shows the b-galactosidase staining of rat braindays (control, 205 { 6 mmHg; CMV-cHK, 190 { 4frozen sections in the hypothalamus and thalamus 1mmHg; n Å 6, Põ0.05).day after ICV injection of Ad.CMV-LacZ. In addition,intense blue staining was observed in the third ventri-Levels of Immunoreactive Human Tissue Kallikreincle indicating the expression of b-galactosidase (data

Recombinant human tissue kallikrein levels in ex- not shown). The staining was clustered in isolated re-tracts of the cortex, cerebellum, brain stem, hippocam- gions but not uniformly distributed throughout thepus, and hypothalamus of rats receiving adenovirus brain section, indicating that the uptake of adenovirusAd.CMV-cHK were measured with an ELISA specific by the brain may be limited to regions that are ac-for human tissue kallikrein. Immunoreactive human cessable to direct ICV injection.tissue kallikrein detected in these extracts displayedparallelism to the human tissue kallikrein standard, Effect of Central Delivery of Human Tissue Kallikreinindicating their immunological identity (Fig. 2). No im- on Physiological Parameters in Urinemunoreactive human tissue kallikrein was detected inbrain extracts of control rats receiving Ad.CMV-LacZ, Urine excretion, water intake, and food consumption

of SHR receiving Ad.CMV-LacZ and Ad.CMV-cHKdemonstrating the specificity of the assay (data notshown). In the cortex, cerebellum, brain stem, and hy- were measured at 5 days after central gene delivery.

There was no significant difference between Ad.CMV-pothalamus extracts, human kallikrein levels werehighest 3 days post gene delivery (Table 1). A similar cHK and control groups in urine excretion (4.3 { 0.5

ml/100 g body weight vs. 4.6 { 0.6 ml/100 g bodylevel of human kallikrein was found in hippocampusextracts 1, 3, and 5 days post gene delivery (Table 1). weight, nÅ5 or 6), water intake (3.8 { 0.6 ml/100 g

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TABLE 1

Levels of Immunoreactive Human Tissue Kallikrein in SHR Brain Extracts after Direct Injection of Replication-DeficientAdenoviral Vector Ad.CMV-cHK via an Intracerebroventricular Cannula

Human tissue kallikrein (ng/mg protein)

Tissue extracts 1 day 3 days 5 days 7 days

Cortex 1.44 { 0.10 1.92 { 0.12 1.05 { 0.08 0.12 { 0.00Brain stem 0.77 { 0.10 0.89 { 0.09 0.71 { 0.09 0.09 { 0.00Cerebellum 0.91 { 0.08 0.98 { 0.04 0.73 { 0.03 n.d.Hippocampus 1.62 { 0.32 1.48 { 0.29 1.60 { 0.33 0.12 { 0.00Hypothalamus 0.82 { 0.12 2.02 { 0.36 1.57 { 0.22 0.45 { 0.02

The content of immunoreactive tissue kallikrein in tissue extracts was measured by a specific ELISA at 1, 3, 5, and 7 days post kallikreingene delivery. Total protein concentrations were measured by Lowry’s method. Specific activity of human tissue kallikrein in rat brainextracts is expressed as ng tissue kallikrein/mg protein. Immunoreactive tissue kallikrein was not detected in control samples which receivedAd.CMV-LacZ. Values represent mean { SEM (nÅ 3 or 4). n.d., not detected.

body weight vs. 4.4 { 0.8 ml/100 g body weight, nÅ5 we showed that systemic delivery of the human tissuekallikrein gene via intravenous injection caused a pro-or 6), or food consumption (8.63 { 0.25 g/100 g body

weight vs. 8.92 {0.36 g/100 g body weight, nÅ5 or 6). longed blood pressure-lowering effect, prevention ofcardiac hypertrophy and increased renal function inNo apparent changes in urinary excretion of Na/ (232

{ 17 vs 242 { 23 mmol/100 g/day), K/ (424 { 29 vs various hypertensive rat models (18, 19). Because thebrain-blood barrier impedes the passage of adenoviral437 { 38 mmol/100 g/day), and protein levels (7.16 {

0.49 vs 7.03{ 0.33 mg/100 g/day) were observed in SHR vectors from the blood to the brain, the expression ofthe human tissue kallikrein gene introduced via intra-receiving central kallikrein gene delivery as compared

with control rats receiving Ad.CMV-LacZ. venous injection was limited to peripheral tissues suchas liver, kidney, heart and aorta. In this study, we deliv-ered the human tissue kallikrein gene into the brain ofDISCUSSIONSHR by direct injection of an adenoviral vector express-

The tissue kallikrein-kinin system has been shown ing human tissue kallikrein. A single ICV injection ofto be involved in vasodilation, blood pressure reduction, adenovirus containing the human tissue kallikreindiuresis, and natriuresis (3,4). In our previous studies, gene into the brain caused a rapid and profound effect

on the blood pressure of SHR and the effect lasted formore than 7 days. A similar effect on blood pressurewas observed in SHR injected with the plasmid DNAvector containing the human tissue kallikrein expres-sion unit. Together, these findings provide a direct linkbetween the expression of tissue kallikrein in the brainand central blood pressure regulation. The delivery ap-proach using nonviral vectors, such as naked plasmidDNA, is orders of magnitude less efficient than viralvector. However, nonviral vectors raise none of the con-cerns of viral vectors such as limited carrier capacityand immunological responses. Although gene transferefficiency is much higher for adenoviral vectors, immu-nological responses preclude the readministration of vi-ruses and the long-term expression of exogenous genes.

A number of studies have demonstrated that cen-tral gene delivery in viral vectors can be achieved inthe rat after administration of virus to the brain byfocal intracerebral inoculation (20, 21). In these

FIG. 3. Expression of human tissue kallikrein by RT-PCR South- studies, the primary target of focal inoculation ofern blot analysis. Total RNA samples extracted from rats receiving virus was limited to 1 to 3 mm surrounding the injec-Ad.CMV-cHK adenovirus injection showed a band on the blot after

tion site. After focal intracerebral inoculation, ade-autoradiography. Control RNA samples extracted from rats receivingAd.CMV-LacZ adenovirus yielded negative results. novirus was found in some neuronal cells but was

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FIG. 4. In vivo Ad.CMV-LacZ-mediated gene transfer into SHR brain. Microscopic picture of the b-galactosidase activity. Blue nuclearstaining indicates b-galactosidase activity. Magnification: 1 40.

predominantly found in cells of glial morphology The expression of the kallikrein gene in varioustissues following intravenous injection of adenovirus(21). The mechanisms of infection and uptake of ade-

novirus in the CNS are not clear. In this study, the carrying the human tissue kallikrein gene, Ad.CMV-cHK, produces a high level of recombinant kallikreinexpression of recombinant human tissue kallikrein

introduced by ICV injection was detected in the cor- in the plasma and urine. Systemic delivery of thekallikrein gene also caused significant increases intex, brain stem, cerebellum, hippocampus, and hypo-

thalamus by RT-PCR Southern blot analysis and by electrolyte output and urine excretion (18, 19). Inthis study, urine excretion, water intake, and foodELISA. The duration of expression of the adenovi-

rus-mediated kallikrein gene that we observed in the consumption were similar between SHR receivingcentral kallikrein gene delivery and control virusbrain is similar as that described with adenoviral

vectors in rat brain (20, 21). In addition, adenovirus- Ad.CMV-LacZ. These results indicate that the acti-vation of the central kallikrein-kinin system was re-mediated gene delivery was demonstrated in the

thalamus, hypothalamus, and third ventricle by b- sponsible for the reduction of blood pressure in SHRfollowing central kallikrein gene delivery and thegalactosidase activity staining after ICV injection of

Ad.CMV-LacZ. There was no evidence of necrosis or peripheral kallikrein-kinin system was not involvedin this process.inflammation in b-galactosidase-stained regions or

in adjacent cells of the brain (data not shown). ICV injection of bradykinin caused a biphasic re-sponse where an initial depressor effect is followedBiochemical and histochemical studies have iden-

tified components of the kallikrein-kinin system in by a presser response (24). The injection site is animportant factor for the direction of the cardiovascu-the brain. Our previous study showed the expression

of endogenous tissue kallikrein in various regions of lar response to bradykinin. Microinjection of brady-kinin into the nucleus hypothalamus anterior causesthe brain (22). Kallikrein was localized in the epithe-

lial cells lining the third ventricle as well as in cell hypotension and bradycardia (25), whereas microin-jection into the hypothalamus dorsalis and rostralbodies of arcuate, supraoptic, paraventricular, and

ventromedial nuclei. Immunoreactive bradykinin B2 ventrolateral medulla causes hypertension andtachycardia (25, 26). It is likely that regions of thereceptor has been demonstrated on neurons of the

cortex, hypothalamus, caudate nucleus, and brain brain where kinins could exert predominant vaso-pressor effects might not be reached by ICV injectionstem (23). The increased activity of the brain kalli-

krein-kinin system following central delivery of hu- of adenovirus Ad.CMV-cHK.In conclusion, the central gene delivery of humanman tissue kallikrein may exert a protective action

against blood pressure elevation in SHR. tissue kallikrein by adenoviral or plasmid DNA vec-

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10. Wang, C., Chao, L., and Chao, J. (1995) J. Clin. Invest. 95, 1710–tors causes a long-lasting blood pressure reduction1716.in SHR. These results indicate that activation of the

11. Jin, L., Zhang, J., Chao, L., and Chao, J. (1997) Human Genecentral kallikrein-kinin system by adenovirus-medi-Ther. 8, 1753–1761.

ated gene delivery may exert a systemic vasodilator12. Wang, C., Chao, C., Chao, L., and Chao, J. (1997) Immunophar-

action in SHR. macology 36, 221–227.13. Chao, J., Jin, L., Chen, L. M., Chen, V. C., and Chao, L. (1996)

Human Gene Ther. 7, 901–911.ACKNOWLEDGMENTS14. Madeddu, P., Glorioso, N., Soro, A., Tonolo, G., Manunta, P.,

Troffa, C., Demontis, M. P., Varoni, M. V., and Anania, V. (1990)We thank Dr. Kris Fisher (Institute of Human Gene Therapy) forHypertension 15, 407–412.his kindness in providing the adenoviral shuttle plasmid pAdLink1

15. Xiong, W., Chao, J., and Chao, L. (1995) Hypertension 25, 715–and the Wistar Institute, Institute of Human Gene Therapy for pro-719.viding Ad.CMV-cHK and Ad.CMV-LacZ. This work was supported

by National Institutes of Health Grants HL29397 and HL56686. 16. Xiong, W., Chen, L. M., and Chao, J. (1990) J. Biol. Chem. 265,2822–2827.

17. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J.REFERENCES(1951) J. Biol. Chem. 193, 265–275.

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