以基因改造小鼠研究PPARr 於血壓調節之角色

(1)

成功大學補助優秀新進教師學術研究計畫成果報告

Role of PPARr in the blood pressure regulation using genetic mouse models

計畫類別：; 個別型計畫 □ 整合型計畫計畫編號：D97-3500

執行期間： 97 年 3 月 1 日至 98 年 2 月 28 日

計畫主持人：蔡曜聲共同主持人：

計畫參與人員：戴豪志，葉芳好

執行單位：國立成功大學臨床醫學研究所

中華民國 98 年 4 月 25 日

(2)

一、中文摘要

Peroxisome proliferator-activated receptor γ (PPARγ) 是屬於核內的接受器，對於脂肪細胞的生成及維持體內葡萄糖的恆定佔有關鍵角色，同時它也是一群胰島素敏感性藥物的結

合標的。然而PPARγ 不僅表達於脂肪細胞，人類 PPARγ 基因突變也會導致高血壓。最近研

究成果顯示在老鼠及人類的動物模式中，PPARγ 活化劑的作用可以降低血壓。我們先前已

經證實以老鼠為動物模式將PPARγ 基因進行 dominant-negative 突變，導致老鼠的血壓升高

但卻未改變胰島素敏感度，所以我們推測PPARγ 對於血壓的控制上扮演重要角色，且對於

血壓控制與胰島素敏感性經由不同的控制機轉。然而在in vivo 上 PPARγ 如何影響血壓及血

管的反應性仍然未清楚了解。為了研究PPARγ 在高血壓及心血管疾病病程所扮演的角色，

發展以基因轉殖小鼠的技術，建立 PPARγ高量表達及低量表達之小鼠模式，藉由改變

PPARγ 的非轉譯區域影響基因產物層次，來探討 PPARγ 表達量的影響對於這些疾病病程所扮演的角色。

具體目標一、研究在 in vivo 中 PPARγ表現量差異對於鹽類平衡及血壓調控的影響 我們運

用3’端非轉譯區域 (3’-UTR)可調節mRNA 穩定性的特性，已經在 in vivo 中正向或負向的

調控PPARγ 的表達量。藉由改變老鼠 Pparg 基因的 3’-UTR，建立 PPARγ高量表達(穩定

的PPARγ訊息)及低量表達(不穩定的 PPARγ訊息)之小鼠模式。藉由此類不同 PPARγ表

達量的小鼠，使我們可以詳細的探討PPARγ 表達量的差異如何影響血壓的調控。我們並藉

由餵予高鹽及低鹽的飲食來探討PPARγ 表達量的差異如何影響腎臟鹽類和水分再吸收和對

鹽類的敏感性。

具體目標二、研究在 PPARγ極低量表達的小鼠中高脈壓(pulse pressure)的機制我們初步

的實驗結果顯示老鼠在PPARγ表現量非常低的情況僅會增加收縮壓，而舒張壓沒有任何的

改變。由於脈壓的定義是收縮壓減掉舒張壓，因此所得到的脈壓顯著增加。脈壓過高是心血管疾病很重要的危險因子。包含動脈硬化及發炎、甲狀腺機能亢進、嚴重的貧血和動脈

瓣膜失調，這些情況被認為與脈壓增加有關聯。為了研究PPARγ極低表現量的老鼠高脈壓

的機制，我們將分析老鼠動脈的硬度、心臟的功能、甲狀腺荷爾蒙的功能、血管的發炎狀況以及血球細胞計數。

我們的計畫將與成功大學生理所任卓穎教授合作關於血管反應性的研究，和北卡大學

病理所Dr. Nobuyo Maeda 合作研究關於血壓上生理的變化。藉由這些基因轉殖鼠系統化的

分析PPARγ表現量的差異，可以讓我們了解在血壓的控制上 PPARγ複雜的調節機制。這

些研究的方向最終將提供高血壓和心血管疾病的小鼠致病模式，並且解答PPARγ在高脈壓

疾病的致病機制以及心血管疾病之中的疾病生理學角色。

關鍵詞: 核內接受器、血壓調節、脈壓、動脈硬化

Keywords: PPARγ, blood pressure regulation, pulse pressure, arterial stiffness

(3)

二、英文摘要

Peroxisome proliferator-activated receptor γ (PPARγ), a nuclear receptor that regulates adipocyte development and glucose homeostasis, is the molecular target of a class of insulin-sensitizing drugs. Expression of PPARγ, however, is not limited to adipocytes, and loss-of-function mutations in the PPARγ gene are implicated in human hypertension.

Pharmaceutical activations of PPARγ are reported to decrease blood pressure in the animal models, as well as human patients. We have previously demonstrated that the dominant-negative mutation in PPARγ results in increased blood pressure without any changes in insulin sensitivity in mice, suggesting PPARγ plays a critical role in blood pressure regulation that is not dependent on altered insulin sensitivity. How PPARγ affects blood pressure and vascular reactivity is not clear in vivo. To study the role of PPARγ in the pathogenesis of hypertension and vascular diseases, we propose to develop genetic mouse models with overexpressed and underexpressed PPARγ by manipulating the non-coding region of the PPARγ gene in such a way that will affect the steady state gene product levels.

Specific Aim 1 is to study quantitative effects of PPARγ on sodium balance and blood pressure regulation in vivo. We have taken the advantage of mRNA stabilizing/destabilizing features of 3’-untranslated region (3’-UTR) to modulate the PPARγ expression in either a positive or a negative way in vivo. 3'-UTR of the mouse Pparg gene has been modified through gene targeting to make mice with stabilized (overexpressed) or destabilized (underexpressed) PPARγ message. These quantitative PPARγ variants will enable us to test in details how PPARγ variations affect blood pressure regulation. The salt and water reabsorption in the kidney, as well as the salt sensitivity in blood pressure, will be examined by feeding these quantitative PPARγ variants high-salt and low-salt diets.

Specific Aim 2 is to dissect the mechanism of increased pulse pressure in the PPARγ hypomorphic mice. Our preliminary data showed that mice with extremely low PPARγ exhibit increased systolic blood pressure without any change in diastolic blood pressure. Thus, the resulting pulse pressure, defined as the systolic pressure minus diastolic pressure, is significantly increased. High pulse pressure is an important risk factor for cardiovascular diseases. Certain conditions have been suggested for increased pulse pressure, including stiffness and inflammation of the aorta, hyperthyroidism, severe anemia, and aortic valve disorders. To dissect the mechanism of increased pulse pressure in the PPARγ hypomorphic mice, we will examine the arterial stiffness, heart function, thyroid hormone function, vascular inflammation, and complete blood cell count.

Our project involves close collaborations with Dr. Chau-Ying Jen (任卓穎; Department of Physiology, NCKU) for vascular reactivity and Dr. Nobuyo Maeda (Department of Pathology, UNC-Chapel Hill) for blood pressure physiology. The systemic analysis of quantitative variants in the defined genetic background of mice will allow us to unveil the complexity of PPARγ regulatory pathways in blood pressure regulation. These approaches would ultimately generate mouse models for hypertension and vascular diseases, and lend insight into the underlying mechanisms of increased pulse pressure and the role of PPARγ in the pathophysiology of cardiovascular diseases.

(4)

三、前言與研究目的

Pulse pressure, defined as the difference between the systolic and diastolic pressures, is the change in blood pressure seen during the contraction of the heart. Increased pulse pressure has been thought to be a consequence of aging, and confers a substantial cardiovascular risk (1, 2).

Pulse pressure is a better predictor of cardiovascular risks than systolic or diastolic pressure in people aged over 50 (3).

Pulse pressure also can be calculated by [Stroke volume/compliance]. According to this formula, pulse pressure is determined not only by stroke volume and cardiac output, but also by vascular compliance. In the arterial system, the aorta has the highest compliance, due in part to a relatively greater proportion of elastin fibers versus smooth muscle and collagen. This serves the important function of dampening the pulsatile output of the left ventricle, thereby reducing the pulse pressure. Thus, if the aorta were a rigid tube, the pulse pressure would be very high.

Because the aorta is compliant, as blood is ejected into the aorta, the walls of the aorta expand to accommodate the increase in blood volume. The more compliant the aorta, the smaller the pressure change during ventricular ejection (ie, smaller pulse pressure). Therefore, aortic compliance, as well as stroke volume, is a major determinant of the pulse pressure.

Significance Human genetic studies have identified several PPARγ variants that span a wide spectrum of PPARγ activity (4-6). Both gain-of-function and loss-of-function mutations in the PPARγ gene are implicated in human hypertension and vascular diseases (7). However, studies addressing the relationship between PPARγ activity and disease processes did not give unequivocal answers. Therefore, the systematic analysis of quantitative variants in defined genetic background of mice will yield information on the impact of the PPARγ activity in disease processes. To this end, we have generated mice with overexpressed and underexpressed endogenous PPARγ by manipulating the non-coding region of the Pparg gene in such a way that affects the steady state gene product levels. The resulting mice with different levels of PPARγ gene expression may represent variations in humans caused by common polymorphic variations in genomic sequences. These approaches would test the hypothesis that the level of a gene (Pparg) can be modulated in a predicted fashion in mice. Ultimately this proposal would generate mouse models for hypertension and vascular diseases, and allow us to investigate the pleiotropic nature of PPARγ and its impact on blood pressure and vascular diseases.

(5)

C/- C/C C/+ +/+ B/+ B/B 0.0

0.5 1.0 1.5 2.0 2.5

*

**

Adipose tissue PPARγ expression 20% 52% 77%100%128%183%***

WAT Kidney Liver Heart 0.0

0.2 0.4 0.6 0.8 1.0

1.2 +/+

C/+

C/C

** ** *

***

**

***

Relative mRNA

WAT Kidney Liver Heart 0.0

0.5 1.0 1.5 2.0

2.5 +/+

B/+

B/B

*

** *

Relative mRNA

四、結果與討論

Modifications of the mouse Pparg 3’-UTR. To generate mice with various steady state levels of PPARγ mRNA, we replaced PPARγ 3’-UTR with β-globin 3’-UTR for stabilization of PPARγ message; and inserted c-fos ARE into the PPARγ 3’-UTR for destabilization of PPARγ message.

Breeding of the respective Pparg^B/+ (for β-globin replacement) and Pparg^C/+ (for the c-fos ARE insertion) heterozygous mice produced homozygous mice in a Mendelian ratio and the Pparg^B/B and Pparg^C/C homozygotes grow normally.

To test the effectiveness of 3’-UTR modifications to alter gene expression in vivo, the steady state levels of PPARγ mRNA were measured by quantitative RT-PCR. In adipose tissue, the primary site of PPARγ function, of homozygous Pparg^C/C mice, expression of PPARγ reduced to 52% of wildtype (Figure 1). Expressions of PPARγ in Pparg^C/C mice were decreased to 48%, 30%, and 52% of wildtype in heart, liver, and kidney, respectively (Figure 1). On the other hand, expression of PPARγ in homozygous Pparg^B/B mice increased to 183% in adipose tissue (Figure 1), but only 149%, 143%, and 128% in kidney, liver, and heart, respectively (Figure 1). These results indicate that the modification of 3’-UTR does change the steady state PPARγ mRNA in vivo, but is not as efficient as our expectation based on our previous in vitro results.

Figure1

PPARγ expression and BP. Blood pressure was assessed in conscious, 3~4 month old mice by tail-cuff blood pressure measurement. The decrease of PPARγ expression in Pparg^C/C and Pparg^C/+ mice and its increase in Pparg^B/B mice did not significantly altered their BP compared to their respective wildtype littermates as shown in Figure 2. Both male and female Pparg^B/B mice had approximately 3 mmHg lower systolic blood pressures but the difference was not no

statistically significant (P=0.12 by ANOVA with sex and genotype as two factors). Heart rate was also not different between genotypes in both males and females. BP measurements have large standard deviations within each experiment as well as different sets of experiment. For example, in 161 wild type mice, of which BP was measured in 23 sets of experiments, the genetic

background was the single most important factor influencing their BP (P<0.001). Since the blood pressure was measured in mice with the C allele that are F1 and F2 between 129/SvEv and C57BL/6 while that in mice with the B allele was measured in those with more than 95% of their genome from C57BL/6, the assessment of overall effects of PPARγ expression on BP by

combining the effects of the C and B alleles is not simple. However, although none of the individual experiments provided significant difference, the mean BP of the Pparg^B/B mice was lower than that in the wildtype mice in 6 of 7 sets of measurements. The mean BP of the Pparg^C/C mice was higher than that in the wildtype littermates in all 4 sets of measurements. Figure 4 illustrates the differences between the mean BP of mice of each genotype and their wild type

(6)

WAT BAT KidneyLiver Heart 0.0

0.2 0.4 0.6 0.8 1.0 1.2 1.4

*** ** ***

*

+/+

C/-

Relative mRNA

littermates in individual experiments. Thus there is an inverse relationship between the PPARγ expression levels and BP, and we estimate that a 50% change in the level of PPARγ mRNA alters BP by about 3 mmHg (P<0. 01).

Figure 2

BP adjusted by wt as 107 in each experiment C/- 38 110.4±1.4

+/- 25 110.2±1.9 C/C 15 108.1±2.7 C/+ 55 107.8±1.0 +/+ 78 107.0±0.7 +/+ 76 107.0±0.9 B/+ 15 105.3±2.1 B/B 46 103.2±1.2 R2=0.04, P<0.0001

N=412

two-fold difference in expression causes 4mmHg difference in BP

Mice with extremely low (hypomorphic) PPARγ levels To further generate mice with extremely low PPARγ levels, we bred Pparg^+/- mice (from Dr. Ronald Evans (8)) with our Pparg^C/+ mice. Mating of Pparg^+/- and Pparg^C/+ mice produced Pparg^+/+, Pparg^C/+, and Pparg^+/- pups at the expected Mendelian ratio (56:65:64), but the number of Pparg^C/- pups was significantly reduced (37; Chi-square test, P=0.028). Expressions of PPARγ in Pparg^C/- mice were decreased to 20%, 38%, 57%, 16%, and 23% of wildtype in white adipose tissue, brown adipose tissue, heart, liver, and kidney, respectively (Figure 3). Thus, these PPARγ variants span a wide spectrum of PPARγ levels from 25% to 180% (Figure 1).

Figure 3

Increased pulse pressure in the PPARγ hypomorphic (Pparg^C/-) mice. To further record continuous BP in Pparg^C/- mice, we implanted a telemetric device in both Pparg^C/- and wildtype mice (n=5 each). Consistent with the tail-cuff BP measurements, telemetric BP monitoring showered higher systolic BP in the Pparg^C/- mice (Figure 4). However, the diastolic BP in Pparg^C/- mice is not different from that of wildtype mice. Thus, the resulting pulse pressure, which is defined as the systolic pressure minus the diastolic pressure, in Pparg^C/- mice is significantly increased throughout the measurement, compared to that of wildtype mice. Thus, the inverse correlation between the PPARγ level and BP indicates that the level of PPARγ is

0 50 100 150 200

95 100 105 110 115 120

C/- +/- C/C C/+ +/+ B/+ B/B

BP (mmHg)

Pparg mRNA (adipose tissue)

*

%

(7)

Systolic pressure

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 115

120 125 130 135 140

145 wtC/-

NS HS LS NS

Day

Systolic pressure (mmHg)

Diastolic pressure

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 85

90 95 100 105

110 C/-wt

NS HS LS NS

Day

Diastolic pressure (mmHg)

Pulse pressure

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 26

28 30 32 34 36 38

40 wtC/-

NS HS LS NS

* * **

*

* * **

** * *

******

**

*

**

*

* ** *

*

Day

Pulse pressure (mmHg)

Diastolic pressure

NS HS LS NS

80 85 90 95 100 105

110 wt

C/-

*

Diastolic pressure (mmHg)

Pulse pressure

NS HS LS NS

20 25 30 35

40 wt

C/-

***

*** ***

Pulse pressure (mmHg)

important in maintaining BP. In addition, once the level of PPARγ drops too low, it results in a special form of hypertension, increased pulse pressure.

Blood pressure is ultimately determined by the filtration of blood in kidney glomeruli and reabsorption of the filtrate in renal tubules. In kidney, PPARγ is mainly expressed in the inner medulla (9), where sodium and water reabsorption takes place. If the blood pressures in these PPARγ quantitative variants are not salt-sensitive, this suggests that the role of PPARγ in the renal medulla on hypertension is not significant. This can be further confirmed by examination of systemic and kidney renin-angiotensin-aldosterone (RAS) system, since no detectable change in the systemic and kidney RAS usually indicates the absence of salt sensitivity.

Effects of altered dietary salt on blood pressure To determine the effects of the altered PPARγ levels on the adaptation to changes in dietary salt intake, we measured mean blood pressures in mice that were sequentially fed diets of different sodium chloride content. Pparg^C/- and wiltype mice (n=5 each) were first fed a control diet containing 0.7% NaCl (LabDiet 5P76). Mice were then fed a high-salt diet (8% NaCl, TD 92012, Harlan Teklad, Madison, Wisconsin, USA) or a low-salt diet (0.05% NaCl, TD 94267, Harlan Teklad, Madison, Wisconsin, USA) for several days, and BP were real-time measured by telemetry recording system.

Figure 4

Systolic pressure

NS HS LS NS

110 115 120 125 130 135

140 ^wt

C/-

*** *

*** **

Systolic pressure (mmHg)

(8)

In Figure 4, the telemetry recording results demonstrated that the pulse pressure in Pparg^C/- mice was increased similarly regardless the content of the dietary salt, implicating that the change in the blood pressure is likely mediated through the vascular resistance in the peripheral.

Dissect the mechanism of increased pulse pressure In the year 2, we focused on dissecting the mechanism of increased pulse pressure in PPARγ hypomorphic mice. This will help understand how PPARγ is involved in the regulation of vascular reactivity and blood pressure.

Certain conditions have been suggested for increased pulse pressure, including stiffness and inflammation of the aorta, hyperthyroidism, severe anemia, and aortic valve disorders. To dissect the mechanism of increased pulse pressure in the PPARγ hypomorphic mice, we performed a series of comprehensive studies.

Arterial stiffness One of the most important factors in the development of increased pulse pressure is believed to be a loss of elasticity of the aorta and peripheral arteries. Histo-pathologic examination of the aorta of an elderly person in Western society typically reveals thickening of the aorta and media due to the accumulation of collagen fibers (10). Inflammation and lipid deposition also contribute to the arterial stiffness.

Measurement of Pulse Wave Velocity (PWV) The determination of arterial pulse wave velocity (PWV) is an index of arterial stiffness (Hartley et al. 1997). Stiffer vessels propagate pressure and velocity waves faster than more compliant vessels. By recording velocity signals from two sites separated by a known distance (Δd) and measuring the difference in pulse arrival times (Δt), we can determine the pulse transit time and calculate PWV by this equation (PWV = Δd/Δt).

We found that C/- mice have significantly higher PWV than wildtype, suggesting PPARγ deficiency increased arterial stiffness. The increase of PWV is not associated with changes in the heart rate (Figure 1).

(9)

Figure 1

Morphological analysis of aorta Serial 5 μm paraffin sections from five different levels of aorta (100 μm apart) isolated from both Pparg^C/- and wildtype mice were cut and stained with H&E.

The medial thickness of aorta and internal diameter of lumen were scored using Nikon NIS-Elements D 3.0 software. Elastin/collagen content is a major determinant for aorta stiffness.

To evaluate the elastin content, we performed Weigert’s stain. For the collagen content, we performed Masson's Trichrome stain to differentiate collagen from other fibers, particularly smooth muscle and elastin.

Our preliminary data found that no significant abnormality was observed in C/- mice. These results are summarized in Figure 2 and Figure 3.

(10)

Figure 2

Figure 3

Gene expression analysis To identify the potential molecular mechanisms underlying the

(11)

increased pulse pressure in the Pparg^C/- mice, I measured the steady-state mRNA levels of genes in the aorta of mutant mice, compared to those of wildtype mice. We focused on 1) genes that have PPRE(s) in its promoter region; and/or 2) genes that are important in regulation of arterial compliance and vascular reactivity. The principal pathways to be investigated in aorta include:

vascular elasticity/stiffness, inflammation, lipid metabolism (including fatty acid oxidation and lipid uptake/storage), and the results are summarized in Figure 4.

Figure 4

五、參考文獻

1. Staessen, J.A., Fagard, R., Thijs, L., Celis, H., Arabidze, G.G., Birkenhager, W.H., Bulpitt, C.J., de Leeuw, P.W., Dollery, C.T., Fletcher, A.E., et al. 1997. Randomised double-blind comparison of placebo and active treatment for older patients with isolated systolic hypertension. The Systolic Hypertension in Europe (Syst-Eur) Trial Investigators. Lancet 350:757-764.

2. Franklin, S.S., Khan, S.A., Wong, N.D., Larson, M.G., and Levy, D. 1999. Is pulse

pressure useful in predicting risk for coronary heart Disease? The Framingham heart study.

Circulation 100:354-360.

3. O'Rourke, M., and Frohlich, E.D. 1999. Pulse pressure: Is this a clinically useful risk factor? Hypertension 34:372-374.

4. Deeb, S.S., Fajas, L., Nemoto, M., Pihlajamaki, J., Mykkanen, L., Kuusisto, J., Laakso, M., Fujimoto, W., and Auwerx, J. 1998. A Pro12Ala substitution in PPARgamma2 associated with decreased receptor activity, lower body mass index and improved insulin sensitivity. Nat Genet 20:284-287.

5. Ristow, M., Muller-Wieland, D., Pfeiffer, A., Krone, W., and Kahn, C.R. 1998. Obesity associated with a mutation in a genetic regulator of adipocyte differentiation. N Engl J Med 339:953-959.

6. Barroso, I., Gurnell, M., Crowley, V.E., Agostini, M., Schwabe, J.W., Soos, M.A., Maslen, G.L., Williams, T.D., Lewis, H., Schafer, A.J., et al. 1999. Dominant negative mutations in

(12)

human PPARgamma associated with severe insulin resistance, diabetes mellitus and hypertension. Nature 402:880-883.

7. Willson, T.M., Lambert, M.H., and Kliewer, S.A. 2001. Peroxisome proliferator-activated receptor gamma and metabolic disease. Annu Rev Biochem 70:341-367.

8. Barak, Y., Nelson, M.C., Ong, E.S., Jones, Y.Z., Ruiz-Lozano, P., Chien, K.R., Koder, A., and Evans, R.M. 1999. PPAR gamma is required for placental, cardiac, and adipose tissue development. Mol Cell 4:585-595.

9. Yang, T., Michele, D.E., Park, J., Smart, A.M., Lin, Z., Brosius, F.C., 3rd, Schnermann, J.B., and Briggs, J.P. 1999. Expression of peroxisomal proliferator-activated receptors and retinoid X receptors in the kidney. Am J Physiol 277:F966-973.

10. Asmar, R., Safavian, A., Tual, J.L., and Safar, M.E. 1995. Arterial and cardiac changes in hypertension in the elderly. Blood Press Suppl 3:31-37.