Gene therapy of hypertension requires long-term expression of a therapeutic gene to achieve stable reduction of blood pressure. Human tissue kallikrein (HK) cleaves kininogen to produce a potent vasoactive peptide kinin, which plays an important role in the regulation of the cardiovascular and renal functions. In the present study, we have delivered human kallikrein cDNA with an rAAV vector to explore the potential therapeutic effects of kallikrein on hypertension and related secondary complications. A single tail vein injection of the rAAV-HK vector into the adult spontaneously hypertensive rats resulted in a significant reduction (12.0±2.55 mmHg, P<0.05, n=6, ANOVA) of the systolic blood pressure from 2 weeks after vector injection, when compared with the control rAAV-lacZ vector-injected rats. Weekly blood pressure monitoring showed stable hypertension-reduction effect throughout the course of the 20-week experiments. In addition, total urine microalbumin contents decreased as a result of rAAV-HK treatment. Histological analysis of various tissues showed remarkable amelioration of cardiovascular hypertrophy, renal injury and collagen depositions in the rAAV-treated group. Finally, persistent expression of the transgene product HK was confirmed by the enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction. We conclude that rAAV-mediated HK delivery rendered a long-term and stable reduction of hypertension and protected against renal injury, cardiac remodeling in the spontaneously hypertensive rat model. Further studies are warranted for the development of a gene therapy strategy for human hypertension.
Hypertension is a major disease, which affects 15–25% or more individuals in the world by increasing both morbidity and mortality of cardio-cerebral vascular disorders such as peripheral vascular disease, coronary heart disease, congestive heart failure and strokes.1, 2, 3 Clinical trials conducted in the last two decades have demonstrated that a reduction in blood pressure of 10/5 mmHg will result in the decrease in stroke mortality by 40% and coronary heart disease by 20%, regardless of the classes of antihypertensive drugs prescribed.1, 2, 3 However, the ratio of goal blood pressure control is very low, especially in the developing countries (about 4%), because current drugs, although effective, have poor compliance, are expensive and short-lasting (hours or one day). Gene therapy may have the potential to solve these problems and provide an effective and stable control of blood pressure for hypertensive patients.
The recombinant adeno-associated virus (rAAV) vector offers many unique advantages over other vectors, including its stable transduction and long-term expression of the transgene, the ability to infect both the dividing and nondividing cells, the lack of pathogenesis or inflammatory response, which are very important requirements for the treatment of chronic diseases such as hypertension. The recent establishment of package and replication systems independent of adenovirus helper and improvement of packaging capacity4, 5, 6 make rAAV the most attracting vector for long-term gene therapy.
Tissue kallikreins belong to a subgroup of serine proteinases which cleave low-molecular-weight kininogen substrate to produce the vasodilator kinin peptide.7 Binding of kinins to the bradykinin (BK) B2 receptor activates second messengers in target tissues and triggers a wide spectrum of biological effects such as vasodilation, vasoconstriction, increase in vascular permeability and inhibition or stimulation of cell growth.8, 9, 10, 11 The vasodilatory action of the kallikrein–kinin system counterbalances the vasoconstrictive action of the renin–angiotensin system (RAS). The observation that urinary excretion of tissue kallikrein is significantly reduced in hypertensive patients was reported in 1934.12 Extensive epidemiological studies have documented an inverse relationship between urinary or renal kallikrein levels and blood pressure elevation in essential hypertensive patients.13, 14, 15 Reduced urinary kallikrein excretion has also been described in a number of genetically hypertensive rat models.14, 16, 17, 18 These findings suggest that low kallikrein levels may contribute to hypertension. The possible physiological roles of tissue kallikrein in cardiovascular and renal functions have been widely studied in humans and various animal models. The findings indicate that the expression of tissue kallikrein–kinin system components are crucially important for cardiovascular and renal function such as the kidney, heart, brain and aorta.7, 19, 20, 21
Gene therapy for hypertension is a viable strategy.1, 2, 3 The application of kallikrein gene therapy to reduce blood pressure has been tested in several hypertensive rat models, and the results showed that the injection of various kallikrein gene constructs with adenovirus or plasmids caused a significant reduction in systemic blood pressure in SHRs, one-clip Goldblatt hypertensive rats and Dahl-salt-sensitive hypertensive rats.22, 23, 24, 25, 26, 27 All those studies, however, were performed using transient expression vectors and provided short-term control of blood pressure (a few weeks), a duration not meeting the requirement for clinical therapy of hypertension. Therefore, in the present study, we used rAAV as gene therapy vector to carry human tissue kallikrein cDNA (HK) to explore whether rAAV-mediated HK delivery can offer long-term and stable blood pressure control, as well as prevent the complications of hypertension for potential clinical applications. Our results showed that the delivery of HK via rAAV not only stably expressed HK and caused long-term blood pressure reduction, but also offered protection of animals from secondary injuries of heart, kidneys and vessels.
Hypotensive effects of rAAV-HK delivery in SHR
After the injection of the rAAV vectors into SHR, the systolic blood pressure was measured weekly. A significant reduction in blood pressure was observed 2 weeks post rAAV injection (by 12.0±2.55 mmHg, P<0.05, n=6, ANOVA) in rAAV·HK-treated rats compared with rAAV·LacZ- and saline-treated rats. The hypotensive effects continued till the end of the experiments (Figure 1). These results indicated that rAAV·HK delivery induced a stable and long-term reduction in blood pressure in SHRs.
Expression of human tissue kallikrein mRNA in SHR
Human tissue kallikrein mRNA in SHR after rAAV delivery was analyzed by RT–PCR. Total RNA was prepared from livers, hearts, kidneys, lungs and skeletal muscles at week 19 after intravenous injection of rAAV-HK or rAAV-lacZ. Human kallikrein mRNA was detected in the livers, hearts, kidneys, lungs and skeletal muscles, and the most abundant in liver and subsequently in lungs, kidney and heart, and a little in skeletal muscle, in rAAV·HK-treated rats, but not in rAAV·LacZ-treated rats (Figure 2). The results show that long-term expression of human tissue kallikrein was achieved in tissues relevant to cardiovascular and renal functions after rAAV·HK transfer in SHR.
Time course of immunoreactive human tissue kallikrein in urine and serum in SHR
Immunoreactive human tissue kallikrein levels in the serum and urine of rAAV·HK- and rAAV·LacZ-treated rats were measured by an ELISA specific for human tissue kallikrein. No immunoreactive human tissue kallikrein was detected in urine of control rats. After intravenous injection of rAAV-HK, the urine level of immunoreactive kallikrein reached 3.84±0.241 ng/ml in 3 weeks and the level was maintained at about 5.12±0.125 ng/ml till the end of the experiment (Figure 3a). As well, serum kallikrein measurements show lower HK levels than urine, but they are parallel to urine levels in all the durations of the experiment and significantly higher than control animals (Figure 3b). The results show that rAAV-mediated gene transfer can drive long-term and stable expression of the HK gene in vivo.
Detection of human kallikrein in tissues of SHR after gene delivery
Expression levels of tissue kallikrein in various tissues of SHR were analyzed by ELISA after animals were killed and tissue protein was extracted. Results show that significant level of expressed tissue kallikrein was detected in various tissues in rAAV-HK-treated rats and expression level in liver is the highest and all other tissues are slightly lower. In control animals, very low kallikrein level was detected due to cross-reaction of antibodies (Figure 4).
Effect of rAAV·HK virus delivery on urinary microalbumin in SHR
The increased albumin in urine is a marker of kidney injuries as well as an independent predictor of cardio-cerebral vascular diseases. In this study, microalbumin level in urine was measured by an ELISA specific for human urinary microalbumin. The urine microalbumin level in rAAV-lacZ- and saline-treated SHR was maintained at about 0.238±0.0298 and 0.241±0.131 mg/l, respectively. But, in rAAV-HK-treated rats, the microalbumin level was reduced from 0.248±0.012 to 0.165±0.0123 mg/l. There was a significant difference between the two groups, beginning from week 12 until the end of the experiment (Figure 5). The results indicated that HK gene delivery can significantly attenuated the renal dysfunction due to hypertension in SHR.
Protective effects of long-term expression of HK on kidney
Hematoxylin and eosin (HE) staining shows that hypertension induced very severe damage in kidneys in control SHRs, including tubular dilatation, atrophy of partial renal tubules, loss of brush border in proximal tubules and formation of protein casts, and glomerular sclerosis and atrophy, as observed in other studies.28 In contrast, rAAV·HK delivery significantly attenuated renal damages induced by hypertension, as indicated by the normal appearance of the cortex and medulla (Figure 6).
Sirius red staining showed more intense red staining, a sign of collagen deposition, in glomerulus and medulla vascular areas of the control SHRs than that of the HK transgenic SHR (Figure 7a). To evaluate the collagen density in kidney, the Sirius red-stained sections were analyzed by using HAIPS pathologic Imagic Analysis System and was expressed as the ratio of collagen to kidney area (percentage of extracellular matrix (ECM)).25 The results showed the ECM was markedly reduced in HK-treated animals compared with the control group (4.41±1.94 vs 15.55±2.85%, n=5, P<0.01) (Figure 7b). These results suggest that HK attenuated the collagen deposition or fibrosis in SHR, thus further explaining why rAAV·HK treatment is able to protect kidney from hypertensive renal injuries.
Protection of rAAV·HK virus delivery against cardiovascular remodeling
Myocardial hypertrophy is an important part of heart injuries resulting from hypertension, as well as an independent predictor of cardiovascular events in clinical practice. It is also an important consideration in the therapy of hypertension to reverse cardiac hypertrophy.29 In this study, we determined the impact of rAAV·HK on hypertensive cardiac hypertrophy in SHR. The results show that the ratio of total heart weight to body weight of the rAAV·HK-treated SHR was significantly lower than that of the control SHR (4.26±0.362 vs 5.01±0.232 mg/g, P<0.05). rAAV·HK delivery also significantly attenuated the ratio of left ventricular weight to heart weight compared with control SHR (0.870±0.12 vs 0.912±0.008 g/g, P<0.05) (Figure 8). Sirius staining showed a lack of cardiomyocyte uniformity, structural derangement, and increased red-staining area in the left ventricle sections in control rats, indicating significant fibrosis in myocardium, while rAAV·HK treatment significantly prevented cardiomyocyte hypertrophy, derangement of structures and matrix proliferation as well as cardiac fibrosis (Figure 9a and b). The collagen density analysis showed that rAAV·HK treatment significantly reduced collagen deposition in heart (ECM: 15.58±6.25% in control group vs 4.26±3.51% in HK-treated animals, n=5, P<0.01) (Figure 9c). Sirius red staining also showed that collagen accumulation in the vasculature, a sign of vascular fibrosis, was also significantly reduced by rAAV-HK treatment and ECM, which is 24.02±2.47% in control animals and 16.31±3.22% in HK-treated animals, respectively (n=5, P<0.05; Figure 10).
In this study, we delivered human kallikrein cDNA in an rAAV-based vector into SHR. A single intravenous injection of rAAV·HK into the adult SHR induced a significant blood pressure reduction (up to 12.0±2.55 mmHg, P<0.05, n=6, ANOVA) at 2 weeks after injection and the hypotensive effect lasted throughout the duration of the experiment. In addition, kallikrein delivery significantly decreased the urine microalbumin, protected the kidneys from the injuries of glomerulus and tubules and renal arterial fibrosis induced by hypertension. Both the morphological analysis and the ratios of the heart weight to body weight and left ventricle weight to whole heart weight showed that rAAV·HK injection also significantly attenuated cardiac hypertrophy and fibrosis, as well as collagen deposition in the aorta. The rAAV·HK delivery protected the SHR from both ischemic and hemorrhagic strokes. These findings demonstrated that rAAV-mediated kallikrein therapy resulted in stable expression of human tissue kallikrein and long-term reduction in systolic blood pressure, likely responsible for the protective effects against renal injury and cardiac remodeling.
SHR is a genetic hypertensive model, and a number of studies have shown that kallikrein gene delivery in plasmid or adenovirus vector can cause a prolonged reduction in systemic blood pressure for few weeks in SHR. But, these studies could neither gain long-term and stable expression of human tissue kallikrein nor last reduction in blood pressure due to the transient expression of the transgene and the immunogenic or toxic nature of the adenoviral vector. In the present study, we used rAAV, a vector capable of long-term gene delivery in vivo. Thus, a bolus intravenous injection of rAAV·HK resulted in a long-term and stable reduction of blood pressure in SHR. A long-term expression of human tissue kallikrein was detected by ELISA and the reverse transcription-polymerase chain reaction (RT-PCR) showed that HK mRNA expresses in the liver, heart, lung, kidney and skeletal muscle in SHR. These results suggested that rAAV-mediated stable and long-term expression of human tissue kallikrein gene leads to a stable reduction of blood pressure in SHR.
Our study also showed that rAAV·HK delivery significantly attenuated both the ratios of total heart weight to body weight and left ventricular weight to heart weight compared with SHR injected with LacZ gene. SHR that received the LacZ gene had large areas of intense interstitial fibrosis. HK gene delivery attenuated fibrosis as observed by reduced focal extracellular matrix (ECM) staining. As well, morphologic analysis showed that the damages in the rAAV·LacZ-treated control SHR included tubular dilatation, loss of brush border in proximal tubule and glomerular sclerosis, and also large areas of intense focal fibrosis in the cortex and medulla vascular area observed by red staining. In contrast, HK gene delivery greatly attenuated all these renal damages and reduced the size and number of protein casts present in the tubules. These results showed that the long-term and stable expression of HK gene significantly protects the kidney and heart against the deterioration of structures and functions of the kidney and heart in SHR. These protective effects may be mediated via NO-cGMP and/or PGE-cAMP signaling pathways and reducing apoptosis.22, 23, 24, 25, 26, 27, 30, 31
In summary, the results of the present study show that rAAV-mediated HK cDNA delivery has great potential for the treatment of hypertension. Throughout the experiments, it is also interesting to see that HK expression did not cause the blood pressure to drop below healthy levels. No detectable adverse effects were found in rAAV-injected animals. These results lend strong support to the potential application of rAAV·HK as a safe and effective therapeutic approach in the treatment of hypertension, or other cardiovascular and renal diseases.32, 33
Materials and methods
Production of rAAV serotype-2 vectors
The rAAV vector plasmid pXXUF1 and an rAAV plasmid containing the reporter gene pdx11-lacZ were previously described.4 An 860-bp HK fragment (NotI to NotI) containing the open reading frame was subcloned into pXXUF1 downstream of CMV to produce plasmid pUF1·HK. Packaging plasmid pXX2 (for serotype 2) and adenovirus helper plasmid pXX6 were also previously described.4
For the packaging and production of the rAAV serotype 2, human 293 cells were grown in Dulbecco's modified Eagle medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco) and antibiotics in a 5% CO2 atmosphere at 37°C. Immediately before transfection, each 15-cm-diameter plate of the cells (70–80% confluent) was fed with 15 ml of fresh DMEM containing 10% fetal bovine serum. A total of 85 μg of plasmid DNA (ratio of pUF1·HK or pdx11-lacZ:pXX2:pXX6 was 1:1:1 in molar) was dissolved in 0.25 M CaCl2 and then quickly mixed with 2 × BES-buffered saline and added to the cells. At 8–12 h after transfection, the medium was replaced with fresh DMEM containing 10% FBS. The cells were harvested at 48–72 h post transfection. After low-speed centrifugation on a tabletop centrifuge, the cell pellets were resuspended in 100 mM NaCl, 10 mM Tris–HCl (pH 8.0) and subjected to three cycles of freeze-thaw, and cell debris was removed by centrifugation. For large-scale rAAV preparation, 40 × 15-cm-diameter plates were used and a single-step gravity-flow column purification method was carried out by a previously published method.34
In all, 18 male spontaneously hypertensive rats (SHR, 3-month old), weighing 250−280 g, were randomly divided into three groups (six for each group), and used in the experiments. The rats were housed at room temperature with a 12-h light/dark cycle and allowed normal rat chow and tap water. All animal experimental protocols were approved by the Institutional Animal Research Committees of the Medical University of South Carolina and Tongji Medical College, and were carried out according to the guidelines of the National Institutes of Health.
Animals were anesthetized by intraperitoneal injection of pentobarbital at a dose of 50 mg/kg body weight. A single tail vein injection of rAAV·HK, rAAV·lacZ (about 1 × 1011 transducing units in 1 ml of saline solution) or equal volume of saline solution into the SHRs was performed. After the injection, the rats were kept warm by an infrared lamp until they regained their consciousness. All the animals were killed 19 weeks after rAAV delivery under pentobarbital anesthesia (50 mg/kg body wt) and the rat hearts, lungs, kidneys, brains, aorta and livers were collected in liquid nitrogen and then stored at −80°C until further experimentation.
Systolic blood pressure
After rAAV injection, the systolic blood pressure of the rats was measured weekly with a manometer–tachometer (Rat Tail NIBP System, ADI Instruments, Australia) using the tail-cuff method as described previously.22, 23 This device requires minimal warming of rats (usually 15 min) before blood pressure determination and a brief period of restraint in a plastic cage. For each animal, the systolic blood pressure was represented by the mean of five recordings.
Collection of urine and serum
The 24-h urine samples were collected in metabolic cages with 500 μl toluene for preventing the decay of urine every 2 weeks. Approximately 1 ml blood was drawn from the tail vein and sera were collected after coagulation and centrifugation. All the samples were stored at −80°C until assayed. Then the samples were subjected to measurements of kallikrein levels in urine and sera, and microalbumin in urine.
Tissue extract preparation
In all, 50–100 mg of liver, heart, lung, kidney and spleen tissues were homogenized in TRIZOL buffer and tissue protein was extracted according to the manufacturer's instructions. Protein concentration was measured spectrophotometrically by the method of Bradford with bovine serum albumin as standard and stored at −20°C until assay for kallikrein.
Weight measurements of the whole heart and left ventricle
At 20 weeks after human tissue kallikrein gene delivery, the heart was removed and the whole heart was weighed and left ventricle weighed after the right ventricular free wall and both atria were carefully dissected from the left ventricle. The inter-ventricular septum was included in the left ventricular weight. The ratios of both whole heart/body weight and left ventricle/whole heart were calculated.
RT–PCR analysis of human tissue kallikrein mRNA
Total RNA was extracted from fresh rat tissues by TRIZOL reagent (GIBCO). RT–PCR analysis specific for human tissue kallikrein mRNA (HK primer, 5′ primer, 5′-IndexTermCCACCATGGGGTTCCTGGTT-3′; 3′ primer, 5′-IndexTermCGCGGATCCACATTTGATTT-3′); For β-actin primer, 5′ primer, 5′-IndexTermGGAGAAGATGACCCAGATC-3′, 3′ primer, 5′-IndexTermGATCTTCATGAGGTAGT CAG-3′). The RT-PCR method was performed as described in the RT-PCR kit from TAKARA biotechnology Co. Ltd. The first-strand cDNA synthesis was carried out in a 20 μl reaction volume with 5 μg of total RNA and the following components: 1 μl 50 pmol/μl of random nine primers, 1–5 μg total RNA (1 μl), 8.5 μl RNA free ddH2O, 2 μl 10 × RNA PCR buffer (250 mM Tris-Cl, pH 8.3, 375 mM KCl, 15 mM MgCl2), 2 μl 10 mM dNTP Mix (10 mM each dATP, dGTP, dCTP and dTTP at neutral pH), 0.5 μl RNase Inhibitor and 1 μl AMV Reverse Transcriptase. The mixture was incubated at 30°C for 10 min, at 42°C for 30 min and at 99°C for 5 min. Then, PCR reaction was performed in a final reaction volume of 100 μl using 2 μl of the reverse transcription reaction product in a thermal cycler with a denaturing phase of 1 min at 94°C, annealing phase of 40 s at 65°C and extension phase of 1 min at 72°C for 20 cycles.
ELISA for human tissue kallikrein
The ELISA reagents specific for human tissue kallikrein were supplied by Dr Lee Chao (Department of Biochemistry and Molecular Biology, Medical University of South Carolina). The procedures for ELISA analysis of HK in animal urine samples were performed according to the method described previously.35, 36 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 h 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 h, 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 h. The plates were washed three times with PBS containing 0.1% Tween-20 (washing solution). Purified human tissue kallikrein standard (0.04–2.5 ng) and rat urine 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 min. After incubation, the plates were washed three times with the washing solution. A volume of 100 μl 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 h. After incubation, excessive labeled IgG was washed off three times with the washing solution. In all, 100 μl 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 h. 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 min. The plates were read at 405 nm on an Elx 800 ELISA reader (Bio-tek instruments, Inc.).
Urinary microalbumin measurement
Urinary microalbumin was measured by an ELISA method. The ELISA kit was purchased from Shanghai Debo biotechnology Inc. The albumin (Alb)-coated and blocked microtiter plates (96 wells), the labeled albumin antibody, the albumin Standards and the OPD substrates were stored at 4°C. Alb standard (0.1–6.4 μg/ml) and rat urine samples were added to individual wells in a total volume of 50 μl dilution buffer, followed by adding 50 μl diluted labeled Alb antibody (dilution was according to the instruction for the kit), and mixed gently. The plates were incubated at 37°C for 90 min, followed by washing four times with the washing solution. The color reaction was performed by adding 100 μl freshly prepared OPD substrate solution at 37°C for 20 min. The plates were read at 490 nm on an Elx 800 ELISA reader (Bio-tek instruments, Inc.).
Analysis of morphology and collagen deposition
A part of the kidney heart and aorta for each animal were preserved in 4% PBS-buffered formaldehyde solution and embedded in paraffin. Sections, 4 μm-thick, were cut and stained with hematoxylin-eosin (H&E), and Sirius red (collagen stains red with Sirius red staining), using the method described by Dr Chao.37 To evaluate the collagen density in tissues, sections of heart, kidney and aorta were analyzed by using HAIPS pathologic Imagic Analysis System after Sirius red staining, and were expressed as the ratio of collagen to non-collagen area (percentage of extracellular matrix (ECM)).25 All sections were evaluated by investigators under blind conditions without previous knowledge of section groups.
The statistical significance of the difference in systolic blood pressure between SHRs receiving rAAV·LacZ and SHRs receiving rAAV·HK was determined by analysis of variance between groups (ANOVA). In addition, we used an unpaired Student's t-test to assess the differences in urine microalbumin levels, HK levels in urine, the ratios of both whole heart/body weight and left ventricle/total heart and the result of ECM quantification between rAAV·LacZ and rAAV·HK groups after gene delivery. The results were expressed as means±s.e.
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This work was supported by the National ‘863’ Plan project of China (No. 2001AA217121) and International Collaboration Grant of Wuhan City (No. 997005135G).
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Cite this article
Wang, T., Li, H., Zhao, C. et al. Recombinant adeno-associated virus-mediated kallikrein gene therapy reduces hypertension and attenuates its cardiovascular injuries. Gene Ther 11, 1342–1350 (2004). https://doi.org/10.1038/sj.gt.3302294
- human tissue kallikrein
- cardiovascular remodeling
- spontaneously hypertensive rat
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