In Vivo klotho Gene Delivery Protects against Endothelial Dysfunction in Multiple Risk Factor Syndrome

Biochemical and Biophysical Research Communications 276, 767–772 (2000) doi:10.1006/bbrc.2000.3470, available online at http://www.idealibrary.com on

In Vivo klotho Gene Delivery Protects against Endothelial Dysfunction in Multiple Risk Factor Syndrome Yuichiro Saito,* Tetsuya Nakamura,* Yoshio Ohyama,* Toru Suzuki,† Akihiro Iida,‡ Takako Shiraki-Iida,‡ Makoto Kuro-o,§ Yo-ichi Nabeshima, ¶ Masahiko Kurabayashi,* and Ryozo Nagai† ,1 *Second Department of Internal Medicine, Gunma University School of Medicine, 3-39-22 Showa, Maebashi, Gunma 371-8511, Japan; †Department of Cardiovascular Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8865, Japan; ‡Tokyo Research Laboratories, Kyowa Hakko Kogyo Company, Limited, 3-6-6 Asahi Machida-ku, Tokyo 194-8533, Japan; §University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, Texas 75235-9072; and ¶Department of Pathology and Tumor Biology, Kyoto University Graduate School of Medicine, Yoshida Konoe, Sakyo-ku, Kyoto 606-8501, Japan

Received August 16, 2000

The klotho gene, originally identified by insertional mutagenesis in mice, suppresses multiple aging phenotypes (e.g., arteriosclerosis, pulmonary emphysema, osteoporosis, infertility, and short life span). We have previously shown that mice heterozygous for a defect in the klotho gene upon parabiosis with wild-type mice show improved endothelial function, suggesting that the klotho gene product protects against endothelial dysfunction. In the present study, using the Otsuka Long-Evans Tokushima Fatty (OLETF) rat which demonstrates multiple atherogenic risk factors (e.g., hypertension, obesity, severe hyperglycemia, and hypertriglyceridemia) and is thus considered an experimental animal model of atherosclerotic disease, we show that adenovirus-mediated klotho gene delivery can (1) ameliorate vascular endothelial dysfunction, (2) increase nitric oxide production, (3) reduce elevated blood pressure, and (4) prevent medial hypertrophy and perivascular fibrosis. Based on these findings, klotho gene delivery improves endothelial dysfunction through a pathway involving nitric oxide, and is involved in modulating vascular function (e.g., hypertension and vascular remodeling). Our findings establish the basis for the therapeutic potential of klotho gene delivery in atherosclerotic disease. © 2000 Academic Press Key Words: klotho; nitric oxide; endothelium; hypertension; diabetes mellitus; hyperlipidemia; obesity; vascular remodeling; atherosclerosis; gene therapy.

Abbreviations used: OLETF, Otsuka Long-Evans Tokushima Fatty; LETO, Long-Evans Tokushima Otsuka; NO, nitric oxide; L-NAME, N ␻-nitro-L-arginie methyl ester; lacZ, Escherichia coli ␤-galactosidase gene; NOS, nitric oxide synthase; eNOS, endothelial NOS. 1 To whom correspondence should be addressed. Fax: ⫹81-3-38152087. E-mail: [email protected].

The klotho gene, identified by insertional mutagenesis in mice, is a suppressor of multiple aging phenotypes which include arteriosclerosis, pulmonary emphysema, osteoporosis, infertility, and short life span (1). Previously, we have shown that mice deficient for the klotho gene show endothelial dysfunction as manifested by an attenuated response of aortic relaxation in response to acetylcholine stimulation. Nitric oxide production was also significantly reduced in klotho deficient mice. Importantly, aortic relaxation in response to acetylcholine improved upon parabiosis of wild-type mice and mice heterozygously deficient for the klotho gene (2–5). These findings suggest that the klotho gene product is involved in regulating endothelial function likely through the involvement of nitric oxide. To further focus on the therapeutic implications of administration of klotho as a regulatory agent of endothelial function, we have examined the effects of klotho gene delivery by using adenovirus-mediate gene delivery on Otsuka Long-Evans Tokushima Fatty (OLETF) rats which manifest multiple atherogenic risk factors (e.g., hypertension, diabetes mellitus, hyperlipidemia, obesity) and is an experimental model of atherosclerotic disease. We show that adenovirus-mediated klotho gene delivery can (1) ameliorate vascular endothelial dysfunction, (2) increase nitric oxide production, (3) reduce elevated blood pressure, and (4) prevent medial hypertrophy and perivascular fibrosis in OLETF rats. Collectively, klotho gene delivery improves endothelial dysfunction through a pathway involving nitric oxide, and is involved in modulating vascular function (e.g., hypertension, vascular remodeling). These findings establish the basis for the therapeutic potential of gene delivery of klotho as a novel

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strategy to protect the vascular endothelium and prevent atherosclerosis.

TABLE 1

Effects of klotho Gene Delivery on OLETF Rats OLETF

MATERIALS AND METHODS Animals. All procedures were done in accordance with the institutional guidelines for animal research. Male OLETF rats and LETO rats (6 weeks old) were a kind gift from Otsuka Pharmaceuticals, Tokushima, Japan (6) and were bred at the animal laboratory facilities of Gunma University. Animals were fed standard laboratory chow and given tap water ad libitum. Blood pressure measurements and blood analysis. Systolic blood pressure was measured by the photoelectric volume oscillometric method using an automated tail cuff sphygmomanometer (Ueda, Nagano, Japan) without anesthesia (n ⫽ 8). Blood samples were drawn from the abdominal aorta under sodium pentobarbital anesthesia. Plasma samples were stored immediately at ⫺30°C. Biochemical analysis was done using an autoanalyzer (MBC, Gunma, Japan). Adenovirus construction. Adenovirus constructs expressing the membrane form of the mouse klotho gene were constructed by the COS-TPC method (7). A 3.2 kb NotI-XbaI fragment containing the full-length coding region of the membrane form of mouse klotho cDNA clone was blunted then inserted into the SwaI site of the replication-defective adenovirus cosmid pAxCawt to produce pAxCAmKlo (8, 9). Recombinant virus was generated by cotransfecting 293 cells with cosmid pAxCAmKlo and EcoT22I digested Ad5-dlX DNA-terminal protein complex (10). Virus was propagated to high titer in 293 cells and purified by two times cesium chloride density gradient centrifugation (11). AxCALacZ harboring the Escherichia coli ␤-galactosidase gene was used as a control (12). Administration of adenovirus. Purified recombinant adenovirus (5 ⫻ 10 8 plaque forming units) was delivered through a 23-gauge needle to the right hindlimb of OLETF rats (27 weeks old) once a week for 3 weeks (n ⫽ 6). Adenovirus was mixed with carbon particles to aid in subsequent localization at the injection site. Analysis of aortic relation in response to acetylcholine using aortic rings. Aortic ring (3 mm long) segments were mounted between two stainless-steel wires, and placed in organ bath containing Krebs’ bicarbonate solution bubbled with a mixture of 95% O 2 and 5% CO 2 to obtain rapid mixing of drugs (n ⫽ 6). One wire was attached to a fixed support, and the other was connected to a force-displacement transducer (model UR-50G, Minebea Co. Ltd., Nagano, Japan) (13). The preparation was allowed to equilibrate for 90 min, then preconstricted by phenylephrine (10 ⫺7 M). To obtain a dose–response curve for acetylcholine (10 ⫺8 to 10 ⫺5 M) and sodium nitroprusside (10 ⫺10– 10 ⫺7 M), agents were added cumulatively to organ bath. Data are expressed as percentage relaxation of phenylephrine-induced preconstriction. NOx (NO 2⫺ and NO 3⫺) assay. Concentration of NO 2⫺ and NO 3⫺ in urine was measured by an autoanalyzer (TCI-NOX 1000, Tokyo Kasei Kogyo, Tokyo, Japan). Deproteinized urine samples were premixed with carrier solution (0.007% EDTA and 0.03% NH 4Cl). Samples were passed through a cadmium reducer and reacted with Griess reagent (1% sulfonamide and 0.1% N-1-naphthylethylenediamine dihydrochloride in 5% HCl) to form a purple azo dye (n ⫽ 6). Absorbance was detected at 540 nm using a flowthrough visible spectrophotometer (Model S/3250, Soma-Kogaku, Tokyo, Japan) connected to a strip chart recorder. The limit of detection of NO 2⫺ was 0.2 mM (with 99% confidence) and the intra- and inter-assay coefficients of variation were 1.6 and 1.7%, respectively (14, 15). Analysis of klotho mRNA expression. RNA extraction and Northern blot analysis were done as previously described (2). Poly(A) ⫹ RNA was separated by electrophoresis on a 2% agarose-

Systolic BP (mmHg) Body weight (g) Glucose (mg/dl) Triglyceride (mg/dl) Total cholesterol (mg/dl)

LETO

156 ⫾ 3 146 ⫾ 3* 681 ⫾ 14 502 ⫾ 11* 364 ⫾ 34 133 ⫾ 5* 233 ⫾ 38 39 ⫾ 6* 133 ⫾ 7 90 ⫾ 5*

Ad-kl

Ad-lacZ

139 ⫾ 6* 582 ⫾ 21* 232 ⫾ 17* 150 ⫾ 25 109 ⫾ 4

152 ⫾ 7 527 ⫾ 11* 235 ⫾ 19* 99 ⫾ 30* 107 ⫾ 8

Note. OLETF; Otsuka Long-Evans Tokushima Fatty rats, LETO; Long-Evans Tokushima Otsuka, Ad-kl; OLETF rats treated with klotho gene, Ad-lacZ; OLETF rats treated with lacZ gene. Value are mean ⫾ SE. *P ⬍ 0.05 vs OLETF rats.

formaldehyde denaturing gel and transferred to a nylon membrane (n ⫽ 6). The membrane was hybridized with DNA probes labeled with [ 32P]dCTP by random oligonucleotide priming. The membrane was washed and exposed to X-ray film. Glyceraldehyde-3-phosphate dehydrogenase was used as a control probe. Histochemical analysis. After rats were sacrificed, thoracic aorta and hearts were rapidly extracted (n ⫽ 6). Hearts were sectioned from apex to base along a horizontal plane, then fixed with formalin. Paraffin-embedded sections were cut into 7-␮m-thick sections, then stained with hematoxylin– eosin and Masson’s trichrome. Perivascular fibrosis was assessed using planimetry on an automatic image analyzer (HC-2500, Fuji Film, Chiba, Japan). Statistical analysis. All values are presented as mean ⫾ SEM. Data were evaluated by the unpaired t test or analysis of variance (ANOVA) for repeated measures. P ⬍ 0.05 was considered statistically significant.

RESULTS Atherosclerotic and Metabolic Disorders in OLETF Rats The Otsuka Long-Evans Tokushima Fatty (OLETF) rat was selected for the present study as an experimental animal model of atherosclerotic disease to test the effects of klotho administration given that this experimental rat manifests multiple atherogenic risk factors such as hypertension, diabetes mellitus, hyperlipidemia and obesity. OLETF rats (30 weeks old) showed mild hypertension, obesity, severe hyperglycemia, and hypertriglyceridemia as compared with control LETO rats as shown in Table 1. Body weight of the OLETF rat was significantly increased as compared to control LETO rats (681 ⫾ 14 mmHg vs 502 ⫾ 11 mmHg, P ⬍ 0.05) as was systolic blood pressure (156 ⫾ 3 mmHg vs 146 ⫾ 3 mmHg, P ⬍ 0.05). Plasma glucose was also significantly increased (2.7-fold increase) in OLETF rats as compared with control LETO rats, as was tryglyceride (6.0-fold increase) and total cholesterol levels (1.5-fold increase). These baseline measurements confirmed presence of hypertension, obesity, severe hyperglycemia, and hypertriglyceridemia in addition to hypercholesterolemia under our experimental conditions.

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FIG. 1. Expression of klotho mRNA in OLETF rats. (a) Expression of klotho mRNA in kidney is significantly decreased in OLETF rats as compared to LETO rats. (b) Local expression of the klotho mRNA in skeletal muscle after repeated transfection of the klotho gene (Ad-kl) or the lacZ gene (Ad-lacZ).

Expression Levels of klotho mRNA in OLETF Rats klotho mRNA is abundantly expressed in kidney, but downregulated under sustained cardiovascular or metabolic stress, such as hypertension, diabetes mellitus, and hyperlipidemia (2). To determine mRNA expression levels in OLETF rats, Northern blot analysis was done which showed significant reduction of klotho mRNA expression in the kidneys of OLETF rats (Fig. 1a) consistent with our previous data that klotho mRNA expression is attenuated under metabolic stress. As parabiosis experiments showed improvement in endothelial function of klotho deficient mice, and as endogenous levels of klotho mRNA are reduced in animals under metabolic stress (e.g., atherosclerosis), we reasoned therefore that supplementation of the klotho gene product in OLETF rats which showed reduced endogenous klotho levels may improve their atherosclerotic phenotype.

P ⬍ 0.05). Acetylcholine-induced vasorelaxation was not affected in OLETF rats administered the adenovirus lacZ construct confirming that the specificity of this effect is attributable to the klotho construct and not the administration of adenovirus alone. After treatment with L-NAME, aortic relaxation in response to acetylcholine was completely abolished in each group, and no difference was seen in maximum relaxation induced by the endothelium-independent vasodilator, sodium nitroprusside (data not shown). Effects of Adenovirus-Mediated klotho Gene Delivery on Nitric Oxide As previous experiments showed levels of nitric oxide metabolites (NO 2⫺ and NO 3⫺) in urine to be reduced in mice heterozygously deficient for the klotho gene, we next examined whether adenovirus-mediate klotho delivery would improve NO levels. Nitric oxide metabolites (NO 2⫺ and NO 3⫺) in urine were significantly increased in OLETF rats treated with the klotho adenovirus as compared to untreated OLETF rats or those treated with the lacZ gene (Fig. 2b, P ⬍ 0.05). Although this increase in nitric oxide metabolite levels cannot be determined to be attributed solely to the vasculature because we examined systemic levels, these findings support the involvement of nitric oxide in the effects of klotho. Effects of Adenovirus-Mediated klotho Gene Delivery on Physiological Indexes Furthermore, to examine the effects of klotho gene delivery on the atherosclerotic phenotype of OLETF rats, systolic blood pressure, body weight, glucose, triglyceride and total cholesterol levels were examined.

Adenovirus-Mediated klotho Gene Delivery To address whether administration of klotho would improve the atherosclerotic phenotype of OLETF rats, a replication-defective adenovirus construct harboring the full-length murine klotho membrane-form cDNA was constructed to allow for adenovirus-mediated klotho gene delivery. The adenovirus construct was administered once a week for three weeks by intramuscular injection. klotho mRNA expression was induced at the injection site as confirmed by Northern blot analysis (Fig. 1b). Effects of Adenovirus-Mediated klotho Gene Delivery on Endothelial Function Endothelial dysfunction in OLETF rats was improved in rats administered klotho adenovirus on the basis that aortic relaxation in response to acetylcholine (10 ⫺5 M) was significantly increased from 62 ⫾ 3% to 82 ⫾ 5% as compared with control LETO rats (Fig. 2a,

FIG. 2. (a) Endothelium-dependent relaxation of aorta in response to acetylcholine. Transfection with the klotho gene (Ad-kl) resulted in restoration of acetylcholine-induced relaxation in OLETF rats, but not in OLETF rats transfected with the lacZ gene (Ad-lacZ). *P ⬍ 0.05 vs OLETF rats. (b) NO metabolites in urine are significantly increased in OLETF rats after transfection with the klotho gene. *P ⬍ 0.05 vs OLETF rats.

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FIG. 3. (a) Vascular remodeling in the thoracic aorta and the coronary artery of OLETF rats, stained with hematoxyline– eosin (upper) and Masson’s trichrome (lower). Original magnification, ⫻200. The klotho gene delivery reduced medial hypertrophy and perivascular fibrosis in OLETF rats. (b) Assessment of fibrosis using image analyzer showed a decrease in fibrosis after the klotho gene therapy. *P ⬍ 0.05 vs OLETF rats.

Systolic blood pressure of OLETF rats administered klotho were reduced to 139 ⫾ 7 mmHg (Table 1, P ⬍ 0.05) which were similar to levels in control LETO rats whereas administration of the lacZ adenovirus to OLETF rats showed no effects on blood pressure suggesting the specific effect of klotho adenovirus on blood pressure reduction. We were, however, unable to determine a specific effect of klotho adenovirus administration on body weight or blood levels of glucose, triglyceride and cholesterol. Body weight and glucose levels were significantly reduced in OLETF rats administered klotho, but lacZ treated OLETF rats also showed reductions. We attribute this reduction in body weight and glucose to stress associated with needle injection; however, further investigations will be needed to clarify the cause. Further, triglyceride and cholesterol levels showed marginal but not significant reductions in klotho treated OLETF rats. Although the reason for this is unknown, it is possible that klotho preferentially affects the vascular endothelium as com-

pared to the metabolic system. This issue will need to be addressed in further studies as well. Effect of the klotho Gene Delivery on Vascular Remodeling To examine the histological effects of klotho administration on the vasculature, histological analysis was done. OLETF rats showed medial hypertrophy of the thoracic aorta as well as perivascular fibrosis of the coronary arteries. Medial hypertrophy in the aorta and perivascular fibrosis in the coronary artery (19), are findings commonly seen in patients with hypertension or diabetes mellitus (20). OLETF rats administered klotho adenovirus showed suppression of medial thickness of the thoracic aorta and perivascular fibrosis in the coronary artery (Fig. 3a). In rats administered klotho adenovirus, perivascular fibrosis area was significantly reduced from 10995 ⫾ 1303 ␮m to 6448 ⫾ 986 ␮m (Fig. 3b, P ⬍ 0.05).

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DISCUSSION In the present study, using OLETF rats as an experimental animal model of atherosclerosis, we have shown that adenovirus-mediated klotho gene delivery ameliorates vascular endothelial dysfunction and increases NO production, and further improves the atherosclerotic phenotype by decreasing blood pressure and through vascular remodeling. Our findings show klotho gene expression is associated with improvement of endothelial function at the cellular level, and with improvement of atherosclerosis at the organ level. Importantly, our results show the direct effects of klotho administration, and establishes the basis for the therapeutic potential of klotho gene delivery for atherosclerotic vascular disease. klotho Regulates Endothelial Function Disruption of klotho gene expression causes multiple aging phenotypes including arteriosclerosis, pulmonary emphysema, osteoporosis, infertility, and skin atrophy (1). As mentioned above, we have previously shown that endothelium-dependent vasodilatation of the aorta and arterioles and urinary excretion of NO are significantly attenuated in klotho deficient mice (5), and that parabiosis between wild-type mice and mice heterozygously deficient for the klotho gene show improvement of endothelial function. We have also shown that klotho mRNA is abundantly expressed in kidney but downregulated under sustained cardiovascular or metabolic stress such as hypertension, diabetes mellitus, or hyperlipidemia which are atherogenic risk factors (2). The limitations of the past studies were that they did not address a direct role of the klotho gene product in the described phenomenon. Parabiosis experiments, while suggestive, were insufficient to confirm a causeand-effect role between klotho gene expression and phenotype modulation. Furthermore, the past studies showed vascular phenotypes which included attenuated aortic relaxation in response to acetylcholine stimulation and NO metabolites at the cellular and molecular levels in mice deficient for the klotho gene, and a decrease in klotho gene expression in animals under sustained circulatory and metabolic stress (e.g., atherosclerosis); while these findings were suggestive of a correlation relationship between klotho gene expression levels and vascular function did not answer whether quantitative and/or qualitative supplementation of klotho would improve atherosclerotic phenotype. Although klotho supplementation has been shown to improve the aging phenotype of klotho deficient mice which as would be expected (7), it remained to be addressed whether klotho supplementation in a heterogeneous experimental animal of vascular dysfunction would improve vascular function which is a

prerequisite to address the generality of the effects of klotho on the vasculature. The present study, therefore, significantly adds to our understanding of the effects of the klotho gene expression on the vasculature. Importantly, we used adenovirus-mediated klotho gene transfer to address a direct cause-and-effect role between klotho gene expression and modulation of vascular phenotype. We further employed an established experimental animal model of atherosclerosis (e.g., OLETF rats) to show that supplementation of klotho leads to improvement in vascular function. The finding that klotho gene transfer improves endothelial function and increases NO metabolites strongly suggests a direct and possibly cooperative regulatory pathway between NO and klotho in the vasculature. Equally important is the finding that klotho gene expression improved blood pressure and induced vascular remodeling. These findings strongly suggest an underlying role of the klotho gene product in the regulation of endothelial function and of atherosclerosis. Vasodilatation induced by endothelium-derived NO has been reported to be impaired in hypertension (16), diabetes mellitus (17) and hyperlipidemia (18). These observations lead to the hypothesis that a decrease in the klotho gene expression may be atherogenic due to direct or indirect involvement with reduced NO production in these common diseases. Our findings collectively show a coupling between klotho gene expression and NO as a general phenomenon in vascular diseased states which establishes and confirms a regulatory interactive pathway between NO and klotho in the vasculature. Although the cellular and molecular mechanisms of the relationship between klotho and regulation of NO need to be clarified in further studies, involvement of the klotho gene in decreased NO formation by down-regulated eNOS or accelerated degeneration of NO by NO scavenger such as superoxide anion (23) are plausible regulatory pathways. Another interesting finding of the present study is that klotho gene transfer significantly prevented perivascular fibrosis in the coronary arteries of OLETF rats as chronic inhibition of NOS with L-NAME has been reported to be associated with an increase in wall thickening and coronary perivascular fibrosis in the rat heart. These histological findings also further support an association between NO and klotho. In line with our data relating NO and klotho, although speculation, an increase in systemic NO production may be involved in reduction in medial hypertrophy of aorta and coronary perivascular fibrosis. Our study indicates that supplementation of the klotho gene restores endothelial-dependent aortic dilatation and accelerates systemic NO production, resulting in a decrease in blood pressure and vascular remodeling in OLETF rats. klotho gene therapy is a potentially viable and novel therapeutic strategy for

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preventive treatment of atherosclerotic cardiovascular diseases. 9.

ACKNOWLEDGMENTS We thank S. Saiki, Y. Nonaka and M. Yamazaki for technical and secretarial assistance. This study was supported by the Program for Promotion of Fundamental Studies in Health Sciences of the Organization for Drug ADR Relief, R&D Promotion and Product Review of Japan (R.N.); Kimura Memorial Heart Foundation Grant for Research on Autonomic Nervous System and Hypertension; Japan Heart Foundation Grant for Study Group of Molecular Cardiology; Japan Heart Foundation Grant for Research on Hypertension and Vascular Metabolism; and Japan Heart Foundation/Pfizer Grant for Cardiovascular Disease Research and Research Foundation for Cancer and Cardiovascular Diseases (Y.S.).

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