Donate Help Contact The AHA Sign In Home
American Heart Association
Hypertension
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents | Next Article »
Hypertension. 2004;44:655-661
Published online before print October 4, 2004, doi: 10.1161/01.HYP.0000144400.49062.6b
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
44/5/655    most recent
01.HYP.0000144400.49062.6bv1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chen, K.
Right arrow Articles by Mehta, J. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chen, K.
Right arrow Articles by Mehta, J. L.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH
Related Collections
Right arrow Other heart failure
Right arrow Remodeling
Right arrow ACE/Angiotension receptors
Right arrow Other Treatment
Right arrow Chronic ischemic heart disease
Right arrow Oxidant stress

(Hypertension. 2004;44:655.)
© 2004 American Heart Association, Inc.

Scientific Contributions

Angiotensin II Regulation of Collagen Type I Expression in Cardiac Fibroblasts

Modulation by PPAR-{gamma} Ligand Pioglitazone

Kui Chen; Jiawei Chen; Dayuan Li; Xingjian Zhang; Jawahar L. Mehta

From the Departments of Internal Medicine and Physiology and Biophysics, University of Arkansas for Medical Sciences, and the Central Arkansas Veterans Healthcare System, Little Rock.

Correspondence to J.L. Mehta, MD, PhD, Division of Cardiovascular Medicine, University of Arkansas for Medical Sciences, 4301 W. Markham St, Slot 532, Little Rock, AR 72205. E-mail MehtaJL{at}uams.edu


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Angiotensin II (Ang II)–mediated stimulation of fibroblast growth and collagen type I synthesis is believed to be an important component of the cardiac remodeling process in hypertension and chronic ischemia. Ang II–mediated oxidative stress could be important in enhanced fibroblast growth and collagen formation. Accordingly, we postulated that the PPAR-{gamma} ligand, pioglitazone, which is known to modulate oxidative stress, would alter Ang II–induced formation of collagen type I in cardiac fibroblasts. Cardiac fibroblasts were treated with different concentrations (10–8 to 10–6 M) of Ang II for different times (6 hours, 12 hours, and 24 hours). Ang II increased the expression of collagen type I in a concentration- and time-dependent fashion (P<0.01 versus control). Ang II also decreased the expression and activity of matrix metalloproteinase (MMP)-1 (MMP-1, P<0.05 versus control). These effects of Ang II were attenuated by pretreatment of cells with pioglitazone (10 µmol/L). Ang II stimulated the intracellular generation of reactive oxygen species (ROS), and this effect was also attenuated by pioglitazone. Ang II treatment activated the redox-sensitive transcription factor NF-{kappa}B, and pioglitazone pretreatment blocked this effect of Ang II. Ang II also activated another transcription factor, AP-1, but this effect of Ang II was not modulated by pioglitazone. In other experiments, we observed that trolox, the water soluble analog of vitamin E, attenuated the effects of Ang II on the expression of collagen type I and MMP-1, in a manner similar to pioglitazone. Thus, pioglitazone attenuates Ang II-mediated collagen type I synthesis in cardiac fibroblasts. The effects of pioglitazone are mediated by the modulation of ROS release and redox-sensitive transcription factor NF-{kappa}B.


Key Words: angiotensin II • cardiac function • collagen


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Heart failure is the end result of cardiovascular disease states, such as hypertension and myocardial ischemia.1 Heart failure is characterized by abundant accumulation of matrix proteins in the extracellular space. Among the extracellular matrix (ECM) proteins, collagens constitute up to 85%.2,3 Collagens are proteins that consist of a triple helix of polypeptide chains and globular domains, and comprise a family of proteins of at least 19 genetically distinct types, among which type I and type III constitute two-thirds.2,3 Collagen type I is usually present in the form of thick fiber with a high tensile strength. Therefore, collagen type I content is considered a major determinant of myocardial stiffness.3,4 Fibroblasts are the major source of collagen in the myocardium.3,5 Proliferation of cardiac fibroblasts and deposition of collagen are directly associated with both systolic and diastolic (especially the latter) heart failure.6 Collagen type I accumulation in the heart depends not only on its production, but also on its degradation, which is performed by proteinases, such as matrix metalloproteinase-1 (MMP-1).3

Activation of the renin-angiotensin system with synthesis and release of angiotensin II (Ang II) is a key feature of hypertension and myocardial ischemia, and both conditions lead to cardiac remodeling comprising myocyte hypertrophy and fibroblast proliferation.7,8 Most of the known effects of Ang II in the heart are believed to be mediated by the activation of its type 1 (AT1) receptors.8 AT1 receptors are widely present on cardiac fibroblasts, and Ang II via AT1 activation mediates fibroblast proliferation and ECM production.9,10 Indeed, long-term intravenous infusion of Ang II is associated with the development of hypertension and cardiac fibrosis.10,11 In recent studies, we showed that Ang II via AT1 activation stimulates oxidative stress in human coronary artery endothelial cells.12 Whether inhibition of oxidative stress can modulate the profibrotic effects of Ang II is not known.

Peroxisome proliferator-activated receptors (PPAR) are a family of at least 3 nuclear receptors ({alpha}, ß/{delta}, and {gamma}).13,14 After activation by the ligand, PPARs heterodimerize with retinoic X receptor and recognize PPAR response element which exists in the promoter of target genes.15 The ligand-activated PPAR functions as a transcription factor and regulates target gene expression. It has been shown that PPAR plays a critical role in lipid catabolism, peroxisome proliferation,13 and adipogenesis.16 Several investigators have shown that PPAR-{gamma} ligands reduce myocardial infarct size after coronary artery ligation.17,18 Shiomi et al19 provided evidence that pioglitazone attenuates cardiac remodeling after chronic ischemia. In recent studies, we showed that the PPAR-{gamma} ligand pioglitazone blocks the effect of several mediators of tissue injury, such as oxidized low density lipoprotein (ox-LDL), Ang II, and tumor necrosis factor-{alpha} (TNF-{alpha}), on endothelial cells.12

In the present study, we examined the hypothesis that pioglitazone blocks the effect of Ang II on collagen type I and MMP-1 expression in cardiac fibroblasts as a basis of its effect on cardiac remodeling.


*    Materials and Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Cell Culture
Rat cardiac fibroblasts were prepared and cultured as described earlier.20 In brief, rat hearts were removed from anesthetized Sprague-Dawley male rats (200 to 250 g, Jackson Laboratories, Bar Harbor, ME). The ventricles were isolated, minced, and digested in 0.25% collagenase solution (37°C, 1 hour). After digestion, the cells were pelleted and suspended in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 1% penicillin and streptomycin and 10% fetal bovine serum (FBS). The suspension was transferred to 56.7 cm2 dishes (Nuncbrand). After a 60-minute incubation period, cells that were weakly attached or unattached were removed, and the attached cells were grown to confluence. After 2 to 3 days, the confluent cells were detached by trypsin and seeded on new dishes. The purity of these cultured cardiac fibroblasts was greater than 95% on the basis of positive staining for vimentin and negative staining for smooth muscle actin and von Willebrand factor (vWF, Sigma). Third to fifth passage cardiac fibroblasts were used for all experiments. Cardiac fibroblasts were treated with different concentrations of Ang II (10–8, 10–7, and 10–6 M) for 6 to 24 hours to determine the expression of collagen type I and MMP-1.

In parallel experiments, cells were pretreated with pioglitazone (10 µmol/L) for 30 minutes followed by treatment with Ang II (or left untreated) in the presence of pioglitazone. This concentration of pioglitazone in previous studies was shown to result in maximal inhibition of reactive oxygen species (ROS) release.12 In other experiments, fibroblasts were pretreated with trolox (10 µmol/L), a water soluble analog of vitamin E, before exposure to Ang II.

All experimental procedures were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee.

Measurement of Intracellular Generation of Reactive Oxygen Species
Intracellular ROS generation was measured by the method described by Ushio-Fukai et al.21 In brief, cardiac fibroblasts were cultured with DMEM containing 10% calf serum until they grew to 60% confluence; then they were cultured in DMEM containing 0.1% calf serum for 48 hours. Cells were then treated with Ang II (10–7 M) for 12 hours, and the ROS-sensitive fluorophore DCF-DA (10 µmol/L) added. Intracellular ROS generation was measured using Fluoromax-3 spectrofluorimeter with an excitation set at 485 nm and emission at 530 nm. This method involves conversion of the nonfluorescent dichlorofluorescein diacetate to the highly fluorescent compound 2,7-dichlorofluorescein (DCF) in the cells. In parallel studies, cells were pretreated with pioglitazone for 12 hours followed by treatment with Ang II or left untreated.

Collagen Synthesis Assay
Collagen synthesis was determined by measuring 3H-proline incorporation as described earlier.22 In brief, cardiac fibroblasts were made quiescent by culture in serum-free DMEM for 48 hours. These cells were then cultured in DMEM supplemented with 2.5% FBS, 50 µg/mL ascorbate and L-[2,3,4,5-3H] proline (1 µCi/mL, Amershan Biosciences) and treated as mentioned above. After treatment, cells were washed with PBS and ice-cold 10% trichloroacetic acid (TCA). The cells were solubilized and the cell extracts analyzed in a liquid scintillation counter. The count represented the amount of newly synthesized collagen. The data were normalized to cell number.

Protein Preparation and Analysis by Western Blot
Fibroblasts from each group were pelleted and extracted in iced cell lysis buffer (Cell Signaling Technologies). Cell lysates were centrifuged at 15 000g for 15 minutes at 4°C and the supernatants from each group (45 µg protein per lane) were separated by 10% SDS-PAGE (for MMP-1) and 8% nondenatured-PAGE (for collagen type I) and then transferred to nitrocellulose membranes. After incubation in blocking solution (5% nonfat milk, Sigma), membranes were incubated with primary antibodies overnight at 4°C. Primary antibodies used were anticollagen type I (Rockland) and anti-MMP-1 (Oncogene). Membranes were washed with 1xTBST solution and then incubated with secondary antibody (1:5000 dilution, Amersham Life Sciences) for 2 hours. The membranes were detected with the ECL system (Amersham Life Sciences) and relative intensities of protein bands analyzed by Scan-gel-it software.23

Collagenase Activity Assay
Collagenase zymography was performed as recently described.24 Essentially, the conditioned culture medium was collected from the dishes and 10 µL of the medium was subjected to electrophoresis in SDS polyacrylamide gel containing 0.1% gelatin under nonreducing conditions. The gels were soaked in 2.5% Triton-X100 for 60 minutes and then washed with water for 60 minutes to remove SDS. The gels were then incubated in a developing buffer containing 50-mmol/L Tris, pH 7.4, 5-mmol/L CaCl2, and 0.02% sodium azide for 18 hours at 37°C. After incubation, the gels were stained with Coomassie blue and photographed.

Electrophoresis Mobility Shift Assay
Nuclear extracts were prepared according to the method published previously.25 In brief, cultured cells were washed with ice-cold Tris-buffered saline twice and pelleted. Then the pellets were suspended in 400 µL of solution A (10 mmol/L HEPES, 10 mmol/L KCl, 0.1 mmol/L EDTA, 0.1 mmol/L EGTA, 1 mmol/L dTT, 1 mmol/L PMSF, 1 mmol/L leupeptin, pH 7.9) for 5 minutes. After a brief vortex and centrifugation, the pellets were suspended and incubated in 150 µL of solution B (20 mmol/L HEPES, 0.4 mol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L dTT, 1 mmol/L PMSF, 1 mmol/L leupeptin) for 30 minutes. After centrifugation, the supernatants were transferred to a clear tube and stored in –80°C. EMSA was performed as described previously. In brief, 10 µg of nuclear extract was incubated with radio-labeled probes for 30 minutes. The probes used were oligonucleotides containing nuclear factor-{kappa}B (NF-{kappa}B) binding sites (5'-AGT TGA GGG GAC TTT CCC AGG C-3', 3'-TCA ACT CCC CTG AAA GGG TCC G-5') or activator protein-1(AP-1) binding sites (5'-CGC TTG ATG AGT CAG CCG GAA-3', 3'-GCG AAC TAC TCA GTC GGC CTT-5'). The protein/DNA complexes were separated by 4% acrylamide gel. After autoradiography, the relative intensities of bands were analyzed by Scan-gel-it software.23

Data Analysis
All data represent the mean of duplicate samples from at least 3 separately performed experiments. Data are presented as mean±SD. Statistical significance was determined in multiple comparisons among independent groups of data using ANOVA followed by F-test. A probability value of ≤0.05 was considered significant.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
Ang II and the Expression of Collagen Type I and MMP-1 in Cardiac Fibroblasts
Ang II treatment increased the expression of collagen type I protein in a time- and concentration-dependant fashion in cardiac fibroblasts (Figure 1). Concurrent with the increase in collagen expression, MMP-1 expression decreased (P<0.01 versus control). This effect, ie, reduction in MMP-1, was evident when cells were treated with Ang II for as short a period of time as 6 hours (Figure 2 left). In all subsequent experiments, the concentration of Ang II was kept at 10–7 M and incubation time at 12 hours.



View larger version (20K):
[in this window]
[in a new window]
  		Figure 1. Top left panel, Ang II and expression of collagen type I. Incubation with Ang II (10–8 to 10–6 M for 12 hours) increased the expression of collagen in a concentration-dependent manner in cardiac fibroblasts. Top right panel, The upregulation of collagen by Ang II (10–7 M) occurred in a time-dependent manner. Collagen protein band density was normalized by its control. Top panels are representative experiments. Bottom panels are the summary of data (mean±SD) from 5 independent experiments.



View larger version (19K):
[in this window]
[in a new window]
  		Figure 2. Ang II and expression of MMP-1. MMP-1 expression was markedly decreased by treatment of fibroblasts with low concentration of Ang II (10–7 M). The reduced expression of MMP-1 with low concentration of Ang II (10–7 M) was evident when the incubation time was only 6 hours. With the increase in incubation time, the reduction in MMP-1 expression was still evident. The right panel shows that Ang II decreases both 42 kDa (MMP-1) and 62 kDa (MMP-2) bands. Top panels are representative experiments. Bottom panels are summary of data (mean±SD) from 5 separate experiments.

Next we examined if the effect of Ang II on MMP expression was associated with change in MMP activity. We noted a significant reduction in MMP activity in cardiac fibroblasts when the cells were treated with Ang II (both P<0.05) (Figure 2, right). There were 2 distinct bands on zymography, a 62 kDa band, reflecting MMP-2, and another 42 kDa band reflecting MMP-1. The latter band was quite faint and the signal was only one-third to one-half of the MMP-2 band. Nonetheless, signals for both MMP-1 and MMP-2 fell further on treatment of cardiac fibroblasts with Ang II.

Modulation of Ang II-Induced Expression of Collagen Type I and MMP-1 by Pioglitazone
As mentioned above, Ang II treatment markedly increased the expression of collagen and decreased the expression of MMP. These effects of Ang II were almost completely blocked by the pretreatment of cardiac fibroblasts with pioglitazone (both P<0.05 versus Ang II treatment). Pioglitazone pretreatment also reversed the effect of Ang II on MMP activity in fibroblasts. Notably, pioglitazone alone had no effect on the basal expression level of collagen or MMP expression (Figure 3).



View larger version (30K):
[in this window]
[in a new window]
  		Figure 3. Pioglitazone and Ang II–induced collagen type I expression and MMP-1 expression/activity in cardiac fibroblasts. Incubation with Ang II (10–7 M) for 12 hours increased the expression of collagen and reduced the expression of MMP-1. Pretreatment of cardiac fibroblasts with pioglitazone (10 µmol/L) for 30 minutes almost completely blocked the effect of Ang II. Note that pioglitazone alone had no effect on the basal expression of collagen or MMP-1. Pioglitazone pretreatment also attenuated the effect of Ang II on the activity of both MMP-1 and -2. Top panels are representative experiments. Bottom panels are the summary of data (mean±SD) from 5 separate experiments.

Modulation of Ang II-Induced Collagen Synthesis by Pioglitazone
Compared with control, Ang II treatment (10–7 M, 12 hours) enhanced collagen synthesis in cardiac fibroblasts (P<0.01). Pretreatment of cells with pioglitazone attenuated this effect of Ang II (P<0.05 versus Ang II treatment). Notably, pioglitazone alone had no effect on basal collagen synthesis (Figure 4).



View larger version (21K):
[in this window]
[in a new window]
  		Figure 4. Pioglitazone and Ang II–induced collagen synthesis (3H-proline incorporation) in cardiac fibroblasts. Ang II treatment (10–7 M, 12 hours) markedly stimulated collagen synthesis in cardiac fibroblasts. Pretreatment of cells with pioglitazone attenuated this effect of Ang II. Again, pioglitazone alone had no effect on collagen synthesis. Protein synthesis was normalized for fibroblast cell count. The data (mean±SD) are summary of 5 separate experiments.

Ang II-Induced ROS Generation in Cardiac Fibroblasts and Modulation by Pioglitazone
There was minimal ROS generation in cardiac fibroblasts in the control state. However, on treatment with Ang II, there was a marked increase in intracellular production of ROS, which was inhibited by pretreatment of cardiac fibroblasts with pioglitazone. Pioglitazone alone had no effect on basal ROS generation. Results of a representative experiment, as well as summary of data from multiple experiments are shown in Figure 5.



View larger version (66K):
[in this window]
[in a new window]
  		Figure 5. Pioglitazone and Ang II–induced ROS generation in cardiac fibroblasts. Ang II treatment (10–7 M, 12 hours) markedly stimulated intracellular production of ROS compared with control cells. Pretreatment of cardiac fibroblasts with pioglitazone (10 µmol/L) for 30 minutes significantly inhibited Ang II–mediated generation of ROS. Pioglitazone alone had no effect on the basal level of ROS generation. Results of a representative experiment as well as summary of fluorescence intensity are shown in this figure.

To determine whether a known antioxidant would alter the effects of Ang II, fibroblasts were treated with trolox before exposure to Ang II. As shown in Figure 6, trolox pretreatment significantly blocked the effect of Ang II on collagen type I and MMP-1 expression. These effects of trolox and pioglitazone were qualitatively similar.



View larger version (14K):
[in this window]
[in a new window]
  		Figure 6. Pretreatment of fibroblasts with trolox (10 µmol/L) for 30 minutes attenuated the effects of Ang II on collagen type I and MMP-1 expression. Top panel is a representative experiment, and the bottom panel is summary of data from 5 separate experiments.

Pioglitazone and Ang II-Induced Activation of NF-{kappa}B and AP-1 in Cardiac Fibroblasts
Ang II treatment significantly enhanced the activation of transcription factor NF-{kappa}B (P<0.05 versus control). This effect of Ang II was blocked by the pretreatment of cardiac fibroblasts with pioglitazone (P<0.05 versus no pretreatment).

Ang II treatment also significantly induced the activation of another transcription factor AP-1 (P<0.05 versus control), but this effect was not modulated by the pretreatment of cells with pioglitazone. Pioglitazone alone had no effect on the basal activation of either NF-{kappa}B or AP-1 (Figure 7).



View larger version (26K):
[in this window]
[in a new window]
  		Figure 7. Pioglitazone and Ang II–induced activation of NF-{kappa}B and AP-1 in cardiac fibroblasts. Compared with control, Ang II treatment (10–7 M, 12 hours) induced the activation of transcription factors NF-{kappa}B and AP-1. The effect of Ang II on NF-{kappa}B activation was blocked by the pretreatment of cardiac fibroblasts with pioglitazone. The effect of Ang II on the activation of AP-1 was not blocked by pioglitazone. Pioglitazone alone had no effect on the activation of either NF-{kappa}B or AP-1 in cardiac fibroblasts. Top panels are representative of 3 separate experiments. Bottom panels are summary of data (mean±SD) from 3 separate experiments.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
In this study, we show that Ang II increases the expression and synthesis of collagen type I in cardiac fibroblasts. Concurrently, Ang II decreases MMP-1 expression and activity. Importantly, we show that the PPAR-{gamma} ligand pioglitazone almost completely blocks these effects of Ang II. We also show a marked increase in ROS release when fibroblasts are exposed to Ang II, and pioglitazone reduces Ang II-induced ROS generation. Lastly, we show that Ang II activates the transcription factors NF-{kappa}B and AP-1, but pioglitazone pretreatment only inhibits the activation of NF-{kappa}B.

Effect of Ang II on Collagen Synthesis and Degradation
Studies in rat and human cardiac fibroblasts have shown that Ang II stimulates cell proliferation and collagen synthesis.26–29 The increase in synthesis and expression of collagen in response to Ang II in cardiac fibroblasts was confirmed in the present study. Because collagen content of ECM depends not only on its production, but also on its degradation, which relies on enzymes such as MMP-1,3 we conducted studies to determine the expression of MMP-1. We found that Ang II decreased the expression of MMP-1, even when cardiac fibroblasts were incubated with Ang II for a short period of time (6 hours). The 10–7 M concentration of Ang II, which is likely to be present in the ischemic heart, significantly inhibited MMP-1 expression. Importantly, collagenase activity was reduced by the treatment of fibroblasts with Ang II in concert with a decrease in its protein expression. It is logical to assume that the reduction in collagenase activity would result in less collagen degradation. This may well be the explanation for the profibrotic effect of Ang II in the myocardium in hypertension, as well as during chronic ischemia, when the renin-angiotensin-system is activated.

A comment needs to be made regarding the identification of MMP-1 and MMP-2 on zymography here. The signal for MMP-1 was considerably weaker than that for MMP-2, but there was a consistent reduction in both signals when fibroblasts were treated with Ang II, and pioglitazone pretreatment reversed the effects of Ang II. We were guided in the detection and recognition of MMP-1 and MMP-2 bands by the work of Yu et al28 and Watanabe et al.29

Brilla et al30 in 1994 first showed that Ang II decreases collagenase activity in cardiac fibroblasts. This effect of Ang II could be blocked by the AT1 receptor antagonist losartan. More recently, Peng et al31 showed that Ang II decreases TNF-{alpha}-stimulated MMP-2 activity and increases the expression of MMP inhibitor (TIMP-1) in neonatal rat cardiac fibroblasts. These effects of Ang II on cardiac fibroblasts are different from the effects of Ang II in endothelial cells32 and cardiac myocytes,33,34 wherein Ang II enhances MMP expression/activity. Thus, Ang II may affect MMP expression and activity differently in different cell types. Nonetheless, its effects on fibroblasts tend to favor collagen synthesis.

Ang II–Induced Oxidative Stress and the Modulatory Effect of Pioglitazone
NADPH oxidase is a major source of ROS in vascular and cardiac tissues, and Ang II stimulates NADPH oxidase.35 It has been postulated that the increase in ROS is an important mechanism by which Ang II contributes to the pathogenesis of vascular disease,36,37 and perhaps cardiac remodeling. Sano et al38 showed that Ang II stimulates ROS generation in cardiac fibroblasts, and we confirmed this phenomenon in the present study. In another recent study,12 we observed that pioglitazone blocks ROS generation in response to ox-LDL, TNF-{alpha}, and Ang II in human coronary artery endothelial cells. Several investigators have shown that pioglitazone attenuates the expression and activity of NAD(P)H oxidase subunits (p22phox, p47phox, gp91phox).39,40 We postulate that Ang II induces ROS generation in cardiac fibroblasts by activating NADPH oxidase and possibly other pathways, but this remains to be defined. Nonetheless, we observed that pioglitazone attenuated Ang II–enhanced ROS generation. In parallel experiments, pioglitazone modulated the effect of Ang II on collagen as well as MMP-1 expression in cardiac fibroblasts. We suggest that the inhibition of ROS generation is, in large part, the basis of the observed effects of pioglitazone. The evidence for this suggestion comes from experiments wherein fibroblasts were pretreated with trolox. This water soluble analog of vitamin E exerted qualitatively similar effects as pioglitazone on collagen type I and MMP-1 expression in response to Ang II.

Many cytokines can transduce intracellular signals that stimulate the proteolytic breakdown of I{kappa}B{alpha}, a cytoplasmic inhibitor of transcription factor NF-{kappa}B.41,42 In turn, NF-{kappa}B is rapidly translocated to the nucleus, where it regulates the expression of many related genes. NF-{kappa}B is a redox-sensitive transcription factor, which is usually activated by oxidative stress.43 We have previously shown that NF-{kappa}B is involved in the activation of endothelial cells in response to Ang II through increased intracellular oxidative stress.12 Now, we show that Ang II activates NF-{kappa}B in cardiac fibroblasts. It was the activation of NF-{kappa}B, which was blocked by the pretreatment of cells with pioglitazone. This observation, together with the data on the measurement of ROS generation, suggests that oxidative stress is one of the important mediators of the effects of Ang II on cardiac fibroblasts, and pioglitazone attenuates the effects of Ang II by blocking ROS release and related transcription factor activation.

In addition to NF-{kappa}B, we showed the activation of AP-1 in response to Ang II. In at least one previous study,34 activation of AP-1 by Ang II in cardiac tissues was described, although it is not clear if the preparation contained fibroblasts. Hattori et al44 showed that Ang II activates AP-1 in vascular smooth muscle cells, and this effect is not affected by pioglitazone. In our study in pure cardiac fibroblasts, we observed that Ang II stimulated the activation of AP-1, and pioglitazone pretreatment did not modulate this effect of Ang II. Schenk et al45 found that transient expression or exogenous application of thioredoxin (an important cellular protein oxidoreductase with antioxidant activity) resulted in a dose-dependent inhibition of NF-{kappa}B activity. AP-1-dependent transactivation, in contrast, was strongly enhanced by thioredoxin. As such, a possible explanation for the lack of effect on AP-1 is that pioglitazone inhibits the ROS production induced by Ang II not only by enhancing antioxidant capacity of cells, but also by its own antioxidant effect.

Perspectives
This study shows that Ang II stimulates collagen synthesis and activity, and attenuates the expression of collagen-degrading enzyme MMP-1 in rat cardiac fibroblasts. We propose that these effects of Ang II are mediated, in large part, by Ang II-stimulated ROS generation. Because the hemodynamic stress of hypertension can be excluded in the present in vitro studies, it can be safely assumed that it is the presence of Ang II (and associated ROS generation) that is the basis of enhanced collagen synthesis by cardiac fibroblasts. Importantly, modulation of the effects of Ang II in cardiac fibroblasts by pioglitazone may have important therapeutic implications in disease states characterized by enhanced collagen synthesis in the heart.


*    Acknowledgments
 
These studies were supported in part by funds from Takeda Pharmaceuticals.

Received June 10, 2004; first decision June 28, 2004; accepted August 17, 2004.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Wood DA. Preventing clinical heart failure: the rationale and scientific evidence. Heart. 2002; 88: 15–22.[Abstract/Free Full Text]
  2. Rekhter MD. Collagen synthesis in atherosclerosis: too much and not enough. Cardiovasc Res. 1999; 41: 376–384.[Abstract/Free Full Text]
  3. Heeneman S, Cleutjens JP, Faber BC, Creemers EE, van Suylen R-J, Lutgens E, Cleutjens KB, Daemen MJ. The dynamic extracellular matrix: intervention strategies during heart failure and atherosclerosis. J Pathol. 2003; 200: 516–525.[CrossRef][Medline] [Order article via Infotrieve]
  4. Berg TJ, Snorgaard O, Faber J, Torjesen PA, Hildebrandt P, Mehlsen J, Hanssen KF. Serum levels of advanced glycation end products are associated with left ventricular diastolic function in patients with type 1 diabetes. Diabetes Care. 1999; 22: 1186–1190.[Abstract/Free Full Text]
  5. Weber KT, Sun Y, Katwa LC, Cleutjens JP, Zhou G. Connective tissue and repair in the heart: potential regulatory mechanisms. Ann N Y Acad Sci. 1995; 752: 286–299.[Abstract]
  6. Weber KT, Brilla CG. Pathological hypertrophy and cardiac interstitium. Fibrosis and renin-angiotensin-aldosterone system. Circulation. 1991; 83: 1849–1865.[Abstract/Free Full Text]
  7. Gibbons GH. The pathophysiology of hypertension: the importance of angiotensin II in cardiovascular remodeling. Am J Hypertens. 1998; 11: 177S–181S.[CrossRef][Medline] [Order article via Infotrieve]
  8. Singh BM, Mehta JL. Interactions between the renin-angiotensin system and dyslipidemia: relevance in the therapy of hypertension and coronary heart disease. Arch Int Med. 2003; 163: 1296–1304.[Abstract/Free Full Text]
  9. Ruperez M, Lorenzo O, Blanco-Colio LM, Esteban V, Egido J, Ruiz-Ortega M. Connective tissue growth factor is a mediator of angiotensin II-induced fibrosis. Circulation. 2003; 108: 1499–1505.[Abstract/Free Full Text]
  10. Villarreal FJ, Kim NN, Ungab GD, Printz MP, Dillmann WH. Identification of functional angiotensin II receptors on rat cardiac fibroblasts. Circulation. 1993; 88: 2849–2861.[Abstract/Free Full Text]
  11. Ashizawa N, Graf K, Do YS, Nunohiro T, Giachelli CM, Meehan WP, Tuan TL, Hsueh WA. Osteopontin is produced by rat cardiac fibroblasts and mediates A(II)-induced DNA synthesis and collagen gel contraction. J Clin Invest. 1996; 98: 2218–2227.[Medline] [Order article via Infotrieve]
  12. Mehta JL, Hu B, Chen J, Li D. Pioglitazone inhibits LOX-1 expression in human coronary artery endothelial cells by reducing intracellular superoxide radical generation. Arterioscler Thromb Vasc Biol. 2003; 23: 2203–2208.[Abstract/Free Full Text]
  13. Issemann I, Green S. Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferators. Nature. 1990; 347: 645–650.[CrossRef][Medline] [Order article via Infotrieve]
  14. Dreyer C, Krey G, Keller H, Givel F, Helftenbein G, Wahli W. Control of the peroxisomal ß-oxidation pathway by a novel family of nuclear hormone receptors. Cell. 1992; 68: 879–887.[CrossRef][Medline] [Order article via Infotrieve]
  15. Schoonjans K, Staels B, Auwerx J. The peroxisome proliferators activated receptors (PPARs) and their effects on lipid metabolism and adipocyte differentiation. Biochim Biophys Acta. 1996; 1302: 93–109.[Medline] [Order article via Infotrieve]
  16. Spiegelman BM. PPAR-{gamma}: adipogenic regulator and thiazoldinedione receptor. Diabetes. 1998; 47: 507–514.[Abstract]
  17. Yue T, Chen J, Bao W, Narayanan PK, Bril A, Jiang W, Lysko PG, Gu J, Boyce R, Zimmerman DM, Hart TK, Buckingham RE, Ohlstein EH. In vivo myocardial protection from ischemia/reperfusion injury by the peroxisome proliferator–activated receptor agonist rosiglitazone. Circulation. 2001; 104: 2588–2594.[Abstract/Free Full Text]
  18. Wayman NS, Hattori Y, Mcddonald MC, Mota-Filipe H, Cuzzocrea S, Pisano B, Chatterjee PK, Thiemermann C. Ligands of the peroxisome proliferator-activated receptors (PPAR-{gamma} and PPAR-{alpha}) reduce myocardial infarct size. FASEB J. 2002; 16: 1027–1040.[Abstract/Free Full Text]
  19. Shiomi T, Tsutsui H, Hayashidani S, Suematsu N, Ikeuchi M, Wen J, Ishibashi M, Kubota T, Egashira K, Takeshita A. Pioglitazone, a peroxisome proliferator–activated receptor-{gamma} agonist, attenuates left ventricular remodeling and failure after experimental myocardial infarction. Circulation. 2002; 106: 3126–3132.[Abstract/Free Full Text]
  20. Dubey RK, Gillespie DG, Mi Z, Jackson EK. Exogenous and endogenous adenosine inhibits fetal calf serum-induced growth of rat cardiac fibroblasts: role of A2B receptors. Circulation. 1997; 96: 2656–2666.[Abstract/Free Full Text]
  21. Ushio-Fukai M, Alexander RW, Akers M, Kathy K. Griendling p38 mitogen-activated protein kinase is a critical component of the redox-sensitive signaling pathways activated by angiotensin II. J Biol Chem. 1998; 273: 15022–15029.[Abstract/Free Full Text]
  22. Dubey RK, Gillespie DG, Jackson EK. Adenosine inhibits collagen and protein synthesis in cardiac fibroblasts: role of A2b receptors. Hypertension. 1998; 31: 943–948.[Abstract/Free Full Text]
  23. Li DY, Chen HJ, Romeo F, Sawamura T, Saldeen T, Mehta JL. Statins modulate ox-LDL-mediated adhesion molecule expression in human coronary artery endothelial cells: role of LOX-1. J Pharmacol Exp Ther. 2002; 302: 601–605.[Abstract/Free Full Text]
  24. Li D, Liu L, Chen HJ, Sawamura T, Ranganathan S, Mehta JL. LOX-1 mediates ox-LDL-induced expression of matrix metalloproteinases in human coronary artery endothelial cells. Circulation. 2003; 107: 612–617.[Abstract/Free Full Text]
  25. Li D, Saldeen T, Romeo F, Mehta JL. Oxidized LDL upregulates angiotensin II type 1 receptor expression in cultured human coronary artery endothelial cells: the potential role of transcription factor NF-{kappa}B. Circulation. 2000; 102: 1970–1976.[Abstract/Free Full Text]
  26. Sadoshima J, Izuma S. Molecular characterization of angiotensin II-induced hypertrophy of cardiac myocytes and hyperplasia of cardiac fibroblasts. Circ Res. 1993; 73: 413–423.[Abstract/Free Full Text]
  27. Weber KT, Sun Y, Katwa LC. Myofibroblasts and local angiotensin II in rat cardiac tissue repair. Int J Biochem Cell Biol. 1997; 29: 31–42.[CrossRef][Medline] [Order article via Infotrieve]
  28. Yu W, Woessner JF Jr. Heparin-enhanced zymographic detection of matrilysin and collagenases. Analytical Biochem. 2001; 293: 38–42.[CrossRef][Medline] [Order article via Infotrieve]
  29. Watanabe H, Shimizu T, Nishihira J, Abe R, Nakayama T, Taniguchi M, Sabe H, Ishibashi T, Shimizu H. Ultraviolet A-induced production of matrix metalloproteinase-1 is mediated by macrophage migration inhibitory factor (MIF) in human dermal fibroblasts. J Biol Chem. 2004; 279: 1676–1683.[Abstract/Free Full Text]
  30. Brilla CG, Zhou G, Matsubara L, Weber KT. Collagen metabolism in cultured adult rat cardiac fibroblasts: response to angiotensin II and aldosterone. J Mol Cell Cardiol. 1994; 26: 809–820.[CrossRef][Medline] [Order article via Infotrieve]
  31. Peng J, Gurantz D, Tran V, Cowling RT, Greenberg BH. Tumor necrosis factor-alpha-induced AT1 receptor upregulation enhances angiotensin II-mediated cardiac fibroblast responses that favor fibrosis. Circ Res. 2002; 91: 1119–1126.[Abstract/Free Full Text]
  32. Seeger H, Lippert C, Wallwiener D, Mueck AO. Valsartan and candesartan can inhibit deteriorating effects of angiotensin II on coronary endothelial function. J Renin Angiotensin Aldosterone Syst. 2001; 2: 141–143.[Abstract/Free Full Text]
  33. Rouet-Benzineb P, Gontero B, Dreyfus P, Lafuma C. Angiotensin II induces nuclear factor-kappa B activation in cultured neonatal rat cardiomyocytes through protein kinase C signaling pathway. J Mol Cell Cardiol. 2000; 32: 1767–1778.[CrossRef][Medline] [Order article via Infotrieve]
  34. Bergman MR, Cheng S, Honbo N, Piacentini L, Karliner JS, Lovett DH. A functional activating protein 1 (AP-1) site regulates matrix metalloproteinase 2 (MMP-2) transcription by cardiac cells through interactions with JunB-Fra1 and JunB-FosB heterodimers. Biochem J. 2003; 369: 485–496.[CrossRef][Medline] [Order article via Infotrieve]
  35. Zafari AM, Ushio-Fukai M, Akers M, Yin Q, Shah A, Harrison DG, Taylor WR, Griendling KK. Role of NADH/NADPH oxidase-derived H2O2 in angiotensin II-induced vascular hypertrophy. Hypertension. 1998; 32: 488–495.[Abstract/Free Full Text]
  36. Reckelhoff JF, Romero JC. Role of oxidative stress in angiotensin-induced hypertension. Am J Physiol. 2003; 284: R893–R912.
  37. Harrison DG, Cai H, Landmesser U, Griendling KK. Interactions of angiotensin II with NAD(P)H oxidase, oxidant stress and cardiovascular disease. J Renin-Angiot-Aldost Sys. 2003; 4: 51–61.[Abstract/Free Full Text]
  38. Sano M, Fukuda K, Sato T, Kawaguchi H, Suematsu M, Matsuda S, Ogawa S. ERK and p38 MAPK, but not NF-{kappa}B, are critically involved in reactive oxygen species–mediated induction of IL-6 by angiotensin II in cardiac fibroblasts. Circ Res. 2001; 89: 661–669.[Abstract/Free Full Text]
  39. Dobrian AD, Schriver SD, Khraibi AA, Prewitt RL. Pioglitazone prevents hypertension and reduces oxidative stress in diet-induced obesity. Hypertension. 2004; 43: 48–56.[Abstract/Free Full Text]
  40. Inoue I, Katayama S. The possible therapeutic actions of peroxisome proliferator-activated receptor {alpha} (PPAR {alpha}) agonists, PPAR {gamma} agonists, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, angiotensin converting enzyme (ACE) inhibitors and calcium (Ca)-antagonists on vascular endothelial cells. Current drug targets-cardiovascular & hematological disorders. 2004; 4: 35–52.
  41. Finch T, Baldwin AS Jr. Mechanistic aspects of NF-kappa B regulation: the emerging role of phosphorylation and proteolysis. Immunity. 1995; 3: 263–272.[CrossRef][Medline] [Order article via Infotrieve]
  42. Verma IM, Stevenson JK, Schwartz EM, Van Antwerp D, Miyamoto S. Rel/NF-kappa B/I kappa B family: intimate tales of association and dissociation. Genes Dev. 1995; 9: 2723–2735.[Free Full Text]
  43. Storz P, Toker A. NF-kappaB signaling–an alternate pathway for oxidative stress responses. Cell Cycle. 2003; 2: 9–10.[Medline] [Order article via Infotrieve]
  44. Hattori Y, Akimoto K, Kasai K. The effects of thiazolidinediones on vascular smooth muscle cell activation by angiotensin II. Biochem Biophys Res Commun. 2000; 273: 1144–1149.[CrossRef][Medline] [Order article via Infotrieve]
  45. Schenk H, Klein M, Erdbrugger W, Droge W, Schulze-Osthoff K. Distinct effects of thioredoxin and antioxidants on the activation of transcription factors NF-kappa B and AP-1. Proc Natl Acad Sci U S A. 1994; 9: 1672–1676.



This article has been cited by other articles:


Home page
 The Annals of PharmacotherapyHome page
U. Khanderia, R. Pop-Busui, and K. A Eagle
Thiazolidinediones in Type 2 Diabetes: A Cardiology Perspective
Ann. Pharmacother., October 1, 2008; 42(10): 1466 - 1474.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
C. Hu, A. Dandapat, L. Sun, J. Chen, M. R. Marwali, F. Romeo, T. Sawamura, and J. L. Mehta
LOX-1 deletion decreases collagen accumulation in atherosclerotic plaque in low-density lipoprotein receptor knockout mice fed a high-cholesterol diet
Cardiovasc Res, July 15, 2008; 79(2): 287 - 293.
[Abstract] [Full Text] [PDF]

Home page
 Exp PhysiolHome page
C.-H. Pan, C.-H. Wen, and C.-S. Lin
Interplay of angiotensin II and angiotensin(1-7) in the regulation of matrix metalloproteinases of human cardiocytes
Exp Physiol, May 1, 2008; 93(5): 599 - 612.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
L. L. Shang, S. Sanyal, A. E. Pfahnl, Z. Jiao, J. Allen, H. Liu, and S. C. Dudley Jr.
NF-{kappa}B-dependent transcriptional regulation of the cardiac scn5a sodium channel by angiotensin II
Am J Physiol Cell Physiol, January 1, 2008; 294(1): C372 - C379.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
C. Hu, A. Dandapat, J. Chen, Y. Fujita, N. Inoue, Y. Kawase, K.-i. Jishage, H. Suzuki, T. Sawamura, and J. L. Mehta
LOX-1 deletion alters signals of myocardial remodeling immediately after ischemia-reperfusion
Cardiovasc Res, November 1, 2007; 76(2): 292 - 302.
[Abstract] [Full Text] [PDF]

Home page
 HypertensionHome page
C. Hu, A. Dandapat, and J. L Mehta
Angiotensin II Induces Capillary Formation From Endothelial Cells Via the LOX-1 Dependent Redox-Sensitive Pathway
Hypertension, November 1, 2007; 50(5): 952 - 957.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
Y. C. Chan and P. S. Leung
Angiotensin II Type 1 Receptor-Dependent Nuclear Factor-{kappa}B Activation-Mediated Proinflammatory Actions in a Rat Model of Obstructive Acute Pancreatitis
J. Pharmacol. Exp. Ther., October 1, 2007; 323(1): 10 - 18.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. Rev.Home page
F. G. Spinale
Myocardial Matrix Remodeling and the Matrix Metalloproteinases: Influence on Cardiac Form and Function
Physiol Rev, October 1, 2007; 87(4): 1285 - 1342.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
N. A. Turner, R. S. Mughal, P. Warburton, D. J. O'Regan, S. G. Ball, and K. E. Porter
Mechanism of TNF{alpha}-induced IL-1{alpha}, IL-1{beta} and IL-6 expression in human cardiac fibroblasts: Effects of statins and thiazolidinediones
Cardiovasc Res, October 1, 2007; 76(1): 81 - 90.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
B. E.J. Teunissen, P. J.H. Smeets, P. H.M. Willemsen, L. J. De Windt, G. J. Van der Vusse, and M. Van Bilsen
Activation of PPAR{delta} inhibits cardiac fibroblast proliferation and the transdifferentiation into myofibroblasts
Cardiovasc Res, August 1, 2007; 75(3): 519 - 529.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
H. K. Graham and A. W. Trafford
Spatial disruption and enhanced degradation of collagen with the transition from compensated ventricular hypertrophy to symptomatic congestive heart failure
Am J Physiol Heart Circ Physiol, March 1, 2007; 292(3): H1364 - H1372.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
V. Subramanian, P. Krishnamurthy, K. Singh, and M. Singh
Lack of osteopontin improves cardiac function in streptozotocin-induced diabetic mice
Am J Physiol Heart Circ Physiol, January 1, 2007; 292(1): H673 - H683.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
J. Chen and J. L. Mehta
Angiotensin II-mediated oxidative stress and procollagen-1 expression in cardiac fibroblasts: blockade by pravastatin and pioglitazone
Am J Physiol Heart Circ Physiol, October 1, 2006; 291(4): H1738 - H1745.
[Abstract] [Full Text] [PDF]

Home page
 FASEB J.Home page
S. Johar, A. C. Cave, A. Narayanapanicker, D. J. Grieve, and A. M. Shah
Aldosterone mediates angiotensin II-induced interstitial cardiac fibrosis via a Nox2-containing NADPH oxidase
FASEB J, July 1, 2006; 20(9): 1546 - 1548.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
A. M. Deschamps and F. G. Spinale
Pathways of matrix metalloproteinase induction in heart failure: Bioactive molecules and transcriptional regulation
Cardiovasc Res, February 15, 2006; 69(3): 666 - 676.
[Abstract] [Full Text] [PDF]