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(Hypertension. 1995;25:715-719.)
© 1995 American Heart Association, Inc.

Articles

Muscle Delivery of Human Kallikrein Gene Reduces Blood Pressure in Hypertensive Rats

William Xiong; Julie Chao; Lee Chao

From the Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston.

Correspondence to Lee Chao, PhD, Department of Biochemistry and Molecular Biology, Medical University of South Carolina, 171 Ashley Ave, Charleston, SC 29425.

	Abstract

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Abstract We recently found that transgenic mice expressing human tissue kallikrein develop sustained hypotension. The result suggests that a continuous supply of human tissue kallikrein could have a prolonged effect on blood pressure reduction. In the present study, we investigated the potential of using human tissue kallikrein for gene therapy by injecting a kallikrein gene construct into the skeletal muscle of spontaneously hypertensive rats. Expression of the human tissue kallikrein messenger RNA in spontaneously hypertensive rats was identified by reverse transcription–polymerase chain reaction with Southern blot. Human tissue kallikrein was detected in the injected animals by an enzyme-linked immunosorbent assay. Injection of the human kallikrein gene into spontaneously hypertensive rats caused a significant reduction of systemic blood pressure, ranging from 15 to 26 mm Hg, compared with the control group. The differences were significant 1 week after the injection and continued for more than 2 months. Blood pressure reduction could be reversed after the administration of the bradykinin antagonist Hoe 140. The results indicate that somatic delivery of the human tissue kallikrein gene induces a sustained reduction of systemic blood pressure in spontaneously hypertensive rats. The present study raises the possibility of applying kallikrein gene therapy to the treatment of human hypertensive diseases.

Key Words: kallikrein • rats, inbred SHR • gene therapy • blood pressure

	Introduction

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Tissue kallikrein is a serine proteinase capable of generating potent vasoactive peptides, known as kinins, by selective cleavage of the kininogen substrate.¹ Kinins have a number of important physiological roles in vasodilation, increased vascular permeability, and ion transport. The tissue kallikrein-kinin system has long been implicated in blood pressure regulation.^{2 3} A significant reduction in urinary kallikrein excretion in hypertensive subjects was reported as early as 1934,⁴ and was confirmed years later.^{5 6} Studies with spontaneously hypertensive rats (SHR) have indicated that they have reduced levels of renal or urinary kallikrein.^{7 8 9} A search for mutations in the tissue kallikrein gene of SHR led to the discovery of a number of restriction fragment length polymorphisms (RFLPs) in the tissue kallikrein gene.¹⁰ Kallikrein gene RFLPs in SHR were later found to cosegregate with the hypertension phenotype.¹¹ The finding that a dominant allele, which is expressed as high total urinary kallikrein excretion, may be associated with decreased risk of essential hypertension in a number of large family pedigrees further confirms the role of kallikrein in blood pressure regulation.¹²

Transient hypotension can be induced by intravenous kallikrein injection and by high oral doses of porcine pancreatic kallikrein.^{13 14} Attempts have been made to prolong the half-life of circulating tissue kallikrein by chemical modification.¹⁵ However, no practical and reliable method of tissue kallikrein delivery has been developed for therapeutic use. We have recently established transgenic mice that carry the human tissue kallikrein gene fused to the mouse metallothionein promoter.¹⁶ These transgenic mice express high levels of human tissue kallikrein and are hypotensive compared with their littermates. The hypotensive effect can be reversed by administration of the kallikrein inhibitor aprotinin or by the bradykinin receptor blocker Hoe 140. These findings indicate that a steady supply of tissue kallikrein in experimental animals could have a prolonged blood pressure–lowering effect and offer a potential therapeutic approach to hypertension.

The science of gene therapy is still in its early stages of development. A simple approach for gene delivery was reported by Wolff et al,^{17 18} who used direct intramuscular injection of naked plasmid DNA. We investigated the possibility of tissue kallikrein gene delivery by intramuscular injection in vivo. A DNA construct containing the coding region of the human tissue kallikrein gene fused to the mouse metallothionein promoter¹⁶ was injected intramuscularly into SHR. The impact of kallikrein gene expression on systemic blood pressure was characterized. The findings raise the possibility that intramuscular delivery of a tissue kallikrein gene could be used in gene therapy for treating hypertensive diseases.

	Methods

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DNA Constructs
The DNA construct used for this study contains the entire 5.6-kb human tissue kallikrein gene coding sequence and 300 bp of its 3' flanking region, under the control of a mouse metallothionein metal-responsive element.¹⁶ To generate this construct, the mouse metallothionein metal-responsive element (MRE) in mMT-1¹⁹ was excised with Kpn I (-650) and Bgl II (+65). This fragment was then fused to the human tissue kallikrein gene construct (PHK) to form the final plasmid construct MRE-PHK. Detailed procedures were described previously.¹⁶

The plasmid DNA was isolated by the alkaline lysis method. The supercoiled plasmid DNA was purified by cesium chloride–ethidium bromide gradient centrifugation²⁰ and diluted to 1 mg/mL in 0.9% NaCl for muscle injection.

Animals
Three-month-old male SHR (n=27) from Harlan Sprague Dawley, Inc, were used in the experiments. Rats were housed at 20°C with a 12-hour light/dark cycle and allowed normal rat chow and tap water ad libitum. All animal experimental protocols were approved by the Institutional Animal Research Committee of the Medical University of South Carolina and were carried out according to the guidelines of the National Institutes of Health.²¹

Somatic Gene Delivery
Intramuscular injections were performed according to a protocol described by Wolff et al,¹⁸ with modifications. Animals were anesthetized intraperitoneally with pentobarbital at a dose of 50 mg/kg body wt. DNA was dissolved in normal saline at a concentration of 1 µg/µL. A total of 1 mg DNA was injected into the left and right quadriceps of each SHR at multiple sites with a 27-gauge needle. The depth of injection was 2 to 5 mm. The same amount of vector DNA (pUC18) was injected into each control rat. After the procedure, rats were kept warm by the infrared lamp until they regained consciousness.

Messenger RNA Detection
The quadriceps muscle previously injected with DNA was removed. RNA was extracted from the tissues by guanidine–cesium chloride gradient centrifugation²² and was then reverse- transcribed. The reverse transcription reaction mixture (20 µL) contained 0.5 µg total RNA, 10 pmol of the 3' primer (CTTCACATAAGACAGCAC), 2 µL of 2.5 mmol/L deoxynucleoside triphosphates, 4 µL of 5x reverse transcription buffer (250 mmol/L Tris-HCl, pH 8.3, 375 mmol/L KCl, 15 mmol/L MgCl₂), and 10 U avian myeloblastosis virus reverse transcriptase (BRL GIBCO Laboratories). The reaction mixture was incubated at 37°C for 1 hour to allow synthesis of the first strand of complementary DNA. The products were then amplified according to the Perkin-Elmer Ampliwax protocol (Roche Molecular Systems, Inc). Fifty picomoles of the 5' primer (AACACAGCCCAGTTTGT) and 3' primer (as above), 10 µL 10x polymerase chain reaction buffer, 5 µL of 2.5 mmol/L deoxynucleoside triphosphates, and 2.5 U Taq DNA polymerase (BRL GIBCO Laboratories) were added to the reverse transcription mixture, which underwent 30 cycles (94°C, 1 minute; 55°C, 2 minutes; and 72°C, 3 minutes) in a thermal cycler. The 5' and 3' primers were designed in such a way that they correspond to a portion of the sequence at exons 3 and 5 of the human tissue kallikrein gene, respectively. The polymerase chain reaction product generated from the human tissue kallikrein messenger RNA (mRNA) using these primers contains 503 bp. One fifth of the samples were electrophoresed in a 0.8% agarose gel and analyzed by Southern blot, as described previously.¹⁶ The blot was hybridized with the end-labeled oligonucleotide GACCTCAAAATCCTGCC, the sequence of which was derived from the fourth exon of the human tissue kallikrein gene. The filter was washed in 2x SSC (1x SSC equals 0.15 mol/L sodium chloride and 0.015 mol/L sodium citrate, pH 7.0) at 45°C and exposed to Kodak X-Omat film at -80°C.

Tissue Extract Preparation
Muscle tissue surrounding the injection sites (approximately 1.5 g) was excised and isolated. The tissue was immediately homogenized with a polytron in PBS, pH 7.2. The homogenate was centrifuged at 400g for 10 minutes. The supernatant was incubated with 0.5% sodium deoxycholate and then centrifuged at 31 000g for 30 minutes. The content of human tissue kallikrein in the extract was measured by an enzyme-linked immunosorbent assay (ELISA). Total protein concentration was determined by the method of Lowry et al.²³

Human Tissue Kallikrein ELISA
According to the method of Guesdon et al,²⁴ 2 mg/mL purified rabbit anti-human tissue kallikrein IgG was dialyzed against 0.1 mol/L sodium bicarbonate buffer, pH 9.5, at 4°C for 24 hours and added to 10 mL freshly prepared 0.1 mol/L biotinyl-N-hydroxysuccinimide ester (dissolved in dimethyl formamide). The reaction was carried out at room temperature for 1 hour and the mixture was dialyzed against PBS. An equal volume of double-distilled glycerol was added, and the biotin-labeled anti-human tissue kallikrein IgG was stored at -80°C. Microtiter plates (96-well) were coated with nonlabeled anti-human tissue kallikrein IgG (2 µg/mL, 100 µL per well) overnight at 4°C. The plates were then blocked with 200 µL PBS (10 mmol/L sodium phosphate, pH 7.4, 150 mmol/L NaCl) containing 1% bovine serum albumin at 37°C for 1 hour. The plates were washed three times with PBS containing 0.1% Tween-20 (washing solution). Purified human tissue kallikrein standard (0.04 to 2.5 ng) and samples were added to individual wells in a total volume of 100 µL PBS containing 0.05% Tween-20 and 0.5% gelatin (dilution buffer). The plates were incubated at 37°C for 90 minutes. After incubation, the plates were washed three times with the washing solution. One hundred microliters of 1 µg/mL biotin-labeled anti-human tissue kallikrein IgG diluted in the dilution buffer was added to each well. The reaction was carried out at 37°C for 1 hour. After incubation, excess labeled IgG was washed off three times with the washing solution. One hundred microliters of 1 µg/mL peroxidase-avidin diluted in the dilution buffer was added to each well, and the plates were incubated at 37°C for 1 hour. After incubation, the plates were washed five times with the washing solution and once with PBS. The color reaction was performed by adding 100 µL freshly prepared substrate solution [0.03% 2,2'-azino-bis(3-ethyl benzthiazoline-6-sulfonic acid) and 0.03% H₂O₂ in 0.1 mol/L citrate buffer, pH 4.3] to each well and incubating at room temperature for 30 minutes. The plates were read at 414 nm on an Titertek Multiskan ELISA reader (Flow Laboratories).

Detection of Serum Antibodies in Rats
Serum antibodies against human tissue kallikrein DNA or protein were monitored by ELISA. Microtiter plates were coated with either purified human tissue kallikrein at 5 µg/mL at 4°C or with purified human tissue kallikrein DNA at 5 µg/mL at 37°C overnight. Serial dilutions of rat serum were added to the plates. After 45 minutes of incubation at room temperature, plates were washed and peroxidase-conjugated goat anti-rat IgG was added. Substrate solution was applied and the plates were analyzed with an ELISA reader at 414 nm.

Blood Pressure Measurements
Tail-cuff measurements of systolic pressure were performed with a programmed electrosphygmomanometer (PE-300; Narco Bio-Systems, Division of International Biomedical, Inc) according to the manufacturer's instructions. Unanesthetized rats were introduced into a plastic holder mounted on a thermostatically controlled warm plate maintained at 37°C to 38°C during measurement.

Basal blood pressures were measured twice before the animals received intramuscular injections. Animals were randomly divided into experimental and control groups. There was no significant difference in blood pressure levels between these two groups. Measurements were taken at different intervals after the intramuscular injection. In the control group, all procedures were the same except that pUC18 was used as the injection solution.

We performed direct blood pressure measurements by carotid artery cannulation by anesthetizing animals with pentobarbital (50 mg/kg body wt IP). A polyethylene cannula (PE-50; Clay Adams, Division of Becton Dickinson and Co) filled with heparinized saline was inserted into the proximal end of the carotid artery until it reached the aortic arch. The catheter was secured and its distal end was connected to a physiological pressure transducer (Statham Laboratories Inc) that was coupled to a model 7 polygraph (Grass Instrument Co).

Hoe 140 Administration
Left carotid artery cannulation was performed for blood pressure measurements as described above. The right jugular vein was cannulated for intravenous delivery. The level of blood pressure was allowed to stabilize after the procedure. Hoe 140 (Hoechst Roussel Pharmaceuticals Inc) was dissolved in normal saline and administered intravenously at a dose of 100 nmol/kg body wt in a total volume of 0.25 mL. Four readings were taken before and 20 minutes after Hoe 140 administration.

Statistical Analysis
Results are expressed as mean±SEM. Single-variable comparisons were made with a paired or unpaired Student's t test, and all other data were analyzed by ANOVA, as appropriate. A value of P<.05 was considered to indicate a significant difference.

	Results

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The expression of human tissue kallikrein mRNA and protein in SHR was monitored after the injection of the DNA constructs. Fig 1 shows the results of reverse transcription–polymerase chain reaction with Southern blot analysis of total muscle RNA at the site of injection 10 days after the gene delivery. Human tissue kallikrein mRNA was detected as a single band (lane 1) with the same size as the human tissue kallikrein mRNA positive control (lane 4). The expression of human tissue kallikrein was measured by a specific ELISA. Human tissue kallikrein in the muscle was 5.54±0.58 ng/mg (mean±SEM, n=3). Fig 2 shows the immunological identity of the human enzyme found in the rat muscle extract compared with the human tissue kallikrein standard. The parallelism of the linear displacement curves for the muscle kallikrein and the human tissue kallikrein standard indicates that rat muscle extract injected with MRE-PHK DNA contains human tissue kallikrein. In control rats, no human tissue kallikrein was detected in the muscle extract.

View larger version (63K): [in this window] [in a new window]   Figure 1. Photograph shows human tissue kallikrein messenger RNA by reverse transcription–polymerase chain reaction (RT-PCR) with Southern blot analysis. RT-PCR was performed using 0.5 µg total RNA and a pair of oligonucleotide primers specific to human tissue kallikrein. A third oligonucleotide was used for Southern blotting of the RT-PCR products. Lane 1, muscle RNA from spontaneously hypertensive rat with injection of a human tissue kallikrein gene construct fused to a mouse metallothionein metal-responsive element (MRE-PHK); lane 2, same RNA sample as in lane 1 but without reverse transcriptase in the reaction mixture; lane 3, muscle RNA from spontaneously hypertensive rat without MRE-PHK injection; lane 4, human salivary gland RNA (total RNA, 40 ng) as positive control.

View larger version (13K): [in this window] [in a new window]   Figure 2. Line graph shows human tissue kallikrein content in muscle from spontaneously hypertensive rat, assessed by enzyme-linked immunosorbent assay, after gene delivery. , Human tissue kallikrein standard curve, ranging from 0.4 to 25.0 ng/mL; , serial dilutions of rat muscle extract.

The effect of the injected human tissue kallikrein gene construct on blood pressure was determined by tail-cuff measurements. The systolic pressure of the SHR was monitored before and after gene delivery. Fig 3 shows blood pressure levels of SHR that received the MRE-PHK construct and of the control group that received the pUC18 vector alone. There was no difference in blood pressure levels between the MRE-PHK group and the control group before the intramuscular injection. The blood pressure levels of the MRE-PHK group were significantly lower than those of the control group 1 week after injection, and the difference between the MRE-PHK group and the control group remained significant for more than 2 months. The reductions ranged from 15 to 26 mm Hg. The tail-cuff method was then compared with the direct intra-arterial cannulation. The results obtained from both methods were consistent with each other (data not shown).

View larger version (14K): [in this window] [in a new window]   Figure 3. Line graph shows reductions of systolic pressure after injection of human tissue kallikrein gene fused to a mouse metallothionein metal-responsive element (MRE-PHK) into the quadriceps muscle of spontaneously hypertensive rats. Vector DNA was used in a control group. Blood pressure measurement was carried out by the tail-cuff method. Blood pressure levels of spontaneously hypertensive rats injected with MRE-PHK DNA () and vector DNA alone () are shown. Values are the average from each group, expressed as mean±SEM (n=4).

The effect of Hoe 140 is shown in Fig 4. Intravenous bolus delivery of 100 nmol/kg body wt of Hoe 140 caused an average of 10.9±1.4 mm Hg (mean±SEM, n=5) elevation of blood pressure compared with its level before Hoe 140 administration (P<.05, n=5). In the control group, there was no significant change in blood pressure (P>.1, n=4). These data suggest that the expression of human tissue kallikrein in SHR is responsible for lowering systemic blood pressure.

View larger version (11K): [in this window] [in a new window]   Figure 4. Bar graph shows the reversal of the effect of the human tissue kallikrein gene by Hoe 140. DNA for human tissue kallikrein fused to a mouse metallothionein metal-responsive element (MRE-PHK) or vector DNA was injected into the muscle of spontaneously hypertensive rats. Four weeks after gene delivery, the bradykinin receptor blocker Hoe 140 was given intravenously at 100 nmol/kg body wt in 0.25 mL normal saline. Systolic blood pressure (BP) was monitored by direct carotid artery cannulation. The bars represent the change in BP after Hoe 140 administration. Values are expressed as mean±SEM (n=5 in the MRE-PHK group, n=4 in the control group).

To determine whether there was any immunoresponse to the MRE-PHK gene delivery, antibodies against human tissue kallikrein DNA and protein were measured by ELISA. There were no antibodies detected against either human tissue kallikrein protein or its DNA in rat sera after human tissue kallikrein gene injection.

	Discussion

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The intramuscular plasmid DNA injection has been shown to be an effective method of somatic gene delivery, and its characteristics have been studied previously.^{17 18 25 26 27 28} The possible mechanisms of DNA uptake in skeletal muscle have been discussed.²⁹ The present study demonstrates that the delivery of an exogenous tissue kallikrein gene by single intramuscular injection has a blood pressure–lowering effect in SHR. Our result is consistent with results of previous studies showing that kallikrein can lower blood pressure in transgenic animals¹⁶ and in humans.¹³ It also substantiates the possibility that low tissue kallikrein level in SHR is a contributing factor in determining the hypertensive phenotype.³⁰ The SHR model was chosen for this experiment because it is regarded as one of the best experimental animal models for the study of human essential hypertension.^{31 32 33 34} The hemodynamics and onset of hypertension in SHR are similar to those in human essential hypertension. Chronic effects of high blood pressure in SHR on organ deterioration and tissue degeneration have high levels of resemblance to those of human essential hypertension. Because of the use of this model, the findings of the current study are likely to be applicable to the prevention and treatment of human hypertension.

Human tissue kallikrein cleaves kininogen substrates to generate vasoactive kinins, which account for many of tissue kallikrein's physiological functions.^{1 3 35 36} In our laboratory, we observed that human tissue kallikrein could effectively cleave rat kininogens to produce bradykinin in vitro, and intravenous administration of human tissue kallikrein into rat could cause transient reduction of blood pressure (W.X. et al, unpublished data, 1988). Kinin receptor antagonist Hoe 140 provides us with a powerful tool in studying the physiological consequences of tissue kallikrein in vivo.^{37 38} Because of its high specificity and strong potency, Hoe 140 was used in our experiment to investigate whether the decrease of blood pressure in SHR injected with MRE-PHK is caused by human tissue kallikrein gene expression. Hoe 140 administration caused an increase in blood pressure in SHR with MRE-PHK injection but not in control SHR. These results suggest that the expression of human tissue kallikrein may exert its effect on blood pressure reduction through kinin generation. However, other pathways such as the renin-angiotensin system and mineralocorticoid metabolism may also be affected by exogenous human tissue kallikrein. Such interactions may change the balance of blood pressure homeostasis and hence lead to the phenotypic change of the experimental animals.³⁹ Determination of the underlying mechanism and exact sites of action for this physiological change awaits further studies. Hoe 140 administration did not restore levels of blood pressure to those of control rats. Several possibilities exist. The pharmacokinetic characteristics of Hoe 140 may impose a limit to its ability to reach sufficient levels in certain tissues or organs to exert its full effect. On the other hand, some of the human kallikrein's hypotensive effects may be the results of a long-term effect due to chronic changes of renal hemodynamics, such as water and electrolyte metabolism. The effect may not be corrected within such a short period of time even if Hoe 140 is sufficient to block the kinin action produced by human kallikrein. Finally, if kallikrein functions through interaction with other hormonal systems, Hoe 140 would have little influence on its effects. The partial reversal of blood pressure after Hoe 140 administration may be attributed to one of these factors or a combination thereof. Further studies are needed before a definitive conclusion can be reached. The low levels of human kallikrein in rat plasma may represent low expression level, low secretion rate into circulation, or a fast plasma clearance rate. Our previous report indicated that human tissue kallikrein can be metabolized quickly in the rat circulatory system with an 8-minute half-life.⁴⁰

The current method of delivering tissue kallikrein is clearly less than optimal. The large amount of DNA used for injection was designed to ensure that a measurable physiological effect could be produced. Studies are under way to optimize the method of delivery. There are several promoters that may have higher expression levels than that of the metallothionein promoter. We have recently generated several DNA constructs under different promoter control and are in the process of comparing their expression efficiency in cell culture systems. The information gained from these in vitro studies will be used for future studies to improve foreign gene expression in the muscle and other tissues. In addition to promoter selection, it may also be important to explore different routes of delivery. We have recently investigated, with considerable success, the feasibility of intravenous delivery of DNA constructs.⁴¹

There has been an intense interest in the therapeutic value of tissue kallikrein. Oral administration of porcine pancreatic kallikrein has been used for treating human essential hypertension, with some promising results.^{13 14} However, the absorption of protein molecules via the gastrointestinal route is poor. Transient reduction in blood pressure can also be induced by intravenous kallikrein delivery. This blood pressure–lowering effect cannot be sustained, because of the short half-life of tissue kallikrein in the circulation.^{40 42} Efforts to prolong the enzyme's half-life by protein modification¹⁵ have resulted in lengthening the effect by only several hours. Here, we have demonstrated that direct delivery of the tissue kallikrein gene by a single muscle injection can cause a sustained blood pressure reduction. This prolonged expression of gene constructs by muscle injection is in agreement with previous reports.¹⁷ Our study demonstrates that the muscle delivery system is an attractive model for human gene therapy in hypertensive diseases.

	Acknowledgments

 
This work was supported by grant HL-29397 from the National Institutes of Health, Bethesda, Md.

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