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Am J Physiol Heart Circ Physiol 293: H1833-H1838, 2007. First published June 22, 2007; doi:10.1152/ajpheart.00488.2007
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Blockade of hypoxia-reoxygenation-mediated collagen type I expression and MMP activity by overexpression of TGF-beta1 delivered by AAV in mouse cardiomyocytes

Chang-Ping Hu,1,2,* Abhijit Dandapat,1,* Yong Liu,1 Paul L. Hermonat,1 and Jawahar L. Mehta1

1Division of Cardiovascular Medicine, Gene Therapy Program, Department of Internal Medicine, University of Arkansas for Medical Sciences and Department of Veterans Affairs Medical Center, Little Rock, Arkansas; and 2Department of Pharmacology, School of Pharmaceutical Sciences, Central South University, Changsha, China

Submitted 24 April 2007 ; accepted in final form 17 June 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Transforming growth factor (TGF)-beta1 is one of the most pleiotropic and multifunctional peptides known. While the cardioprotective effect of TGF-beta1 during ischemia is well known, the specific role of TGF-beta1 in altering the cardiac remodeling process remains unclear. This study was designed to examine the regulation of hypoxia-reoxygenation-mediated collagen type I expression and activity of matrix metalloproteinases (MMPs) by overexpression of TGF-beta1 in cultured HL-1 mouse cardiomyocytes. TGF-beta1 was overexpressed in cardiomyocytes by transfection with adeno-associated virus (AAV)/TGF-beta1Latent or with AAV/TGF-beta1ACT (active TGF-beta1). Twenty-four hours of hypoxia followed by 3 h of reoxygenation (H-R) markedly enhanced (pro)collagen type I expression and activity of MMPs concomitant with an increase in reactive oxygen species (ROS) release and LOX-1 expression. Overexpression of TGF-beta1 reduced these alterations induced by H-R. TGF-beta1 overexpression also blocked H-R-mediated p38 and p44/42 MAPK activation. Transfection with AAV/TGF-beta1ACT was superior to that with AAV/TGF-beta1Latent. These data for the first time demonstrate that H-R induces signals for cardiac remodeling in cardiomyocytes and TGF-beta1 can modulate, possibly via antioxidant mechanism, these signals. These findings contribute to further understanding of the role of TGF-beta1 in the cardiac remodeling process.

transforming growth factor-beta1; HL-1 adult murine cardiomyocytes; metalloproteinases

HEART FAILURE IS THE FINAL common pathway of various cardiovascular diseases, including myocardial ischemia. Alterations in extracellular matrix (ECM), including its structure and properties, are important pathological features of the failing heart; these pathological alterations are collectively known as cardiac remodeling. Coupled with a decrease in ECM degradation by proteinases, such as matrix metalloproteinases (MMPs) (11), ECM deposition leads to cardiac fibrosis, common to both systolic and diastolic (especially the latter) heart failure (34). Reactive fibrosis in the region remote from the area of myocardial infarct is clearly detrimental to ventricular function and contributes to adverse outcomes after myocardial infarction. Therefore, ECM is an important therapeutic target in the management of acute myocardial infarction and treatment of established heart failure.

Two major components of ECM are collagen types I and III, which play an important role in maintenance of structure and function of the heart (11, 34). Collagen type I is usually present in the form of thick fibers with high tensile strength. Therefore, collagen type I is considered a major determinant of myocardial stiffness (34). Besides fibroblasts (11, 34), cardiac myocytes are also an important source of collagen type I (21, 30).

Transforming growth factor (TGF)-beta1 is one of the most pleiotropic and multifunctional peptides known (23). It has potent effects on many different cell types and is involved in a wide variety of biological processes (23). The cellular actions of TGF-beta1 are dependent not only on the cell type, but also on its state of differentiation and the cytokine milieu (17). While TGF-beta1 stimulates fibroblast-like cell growth, enhances collagen synthesis, and suppresses collagen degradation (23), there are studies showing that this growth factor can also limit myocardial ischemia-reperfusion (I/R) injury in isolated rat cardiac myocytes and intact hearts (3, 16, 26, 36). The specific effect of TGF-beta1 on the cardiac remodeling process following I/R, however, remains unclear. TGF-beta1 is synthesized in cells as a precursor molecule, TGF-beta1Latent. Conversion from cysteine (Cys223/225) into serine (Ser223/225) in the TGF-beta1Latent molecule is associated with the formation of TGF-beta1ACT (1). It is the TGF-beta1ACT that appears to be functionally relevant in the process of I/R (36).

LOX-1 is a lectinlike receptor for oxidized low-density lipoprotein (ox-LDL) (24). LOX-1 is involved in the genesis of oxidant stress and inflammation during myocardial I/R (19). ox-LDL treatment enhances collagen formation in fibroblasts that can be blocked by LOX-1 antibody (14). It has been reported that TGF-beta1 can regulate LOX-1 expression in vascular endothelial cells, smooth muscle cells, and macrophages (10, 27).

The present study was conducted to examine three major hypotheses: 1) A brief period of hypoxia-reoxygenation (H-R) upregulates the signals for cardiac remodeling; 2) overexpression of TGF-beta1 reduces the signals for cardiac remodeling; and 3) TGF-beta1 overexpression can modulate LOX-1 and redox-sensitive signaling in cardiomyocytes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Generation of recombinant AAV/TGF-beta1ACT, AAV/TGF-beta1Latent, and AAV/Neo. Rat TGF-beta1 cDNA was generated as described previously (18). To obtain TGF-beta1ACT, site-directed mutagenesis of TGF-beta1 cDNA was performed with the GeneEditor in vitro site-directed mutagenesis system (Promega). Cysteine in positions 223 and 225 of TGF-beta1 mRNA was substituted with serine. The mutation resulted in TGF-beta1 protein in its biologically active form on secretion. Latent TGF-beta1 cDNA was acquired by amplification of high-fidelity PCR as described previously (31). Rat TGF-beta1 cDNA was used as template. The latent TGF-beta1 cDNA is from nt1247 to nt1585. The mutant and latent TGF-beta1 cDNA were verified by gene sequencing.

Latent or mutant rat TGF-beta1 cDNA was inserted into adeno-associated virus type 2 (AAV2) vector dl6-95, as described for other AAV vectors (18). Hereafter, the recombinant AAV vector will be referred to as AAV/TGF-beta1Latent or AAV/TGF-beta1ACT. The generation of AAV/Neo virus has been described previously (18). The virus stocks were generated and titered by dot blot hybridization (18). The titers were calculated to be ~1 x 1011 encapsidated genomes per milliliter.

Cell culture and AAV vector infection. HL-1 adult murine cardiomyocytes were a gift from Dr. W. C. Claycomb (Louisiana State University Health Science Center, New Orleans, LA) and were cultured (8). In brief, HL-1 cells were grown at 37°C under 5% CO2 in fibronectin-gelatin-coated flasks containing Claycomb medium (JRH Biosciences) supplemented with 10% fetal bovine serum (JRH Biosciences), 100 U/ml penicillin, 100 µg/ml streptomycin (Invitrogen), 2 mM L-glutamine (Invitrogen), and 0.1 mM norepinephrine (Sigma). To transfect the cultured cells, AAV vectors were added to cell culture dishes at a multiplicity of infection (MOI) of 104 and incubated with the cells for 72 h at 37°C in 5% CO2-95% air. The infection efficiency was evaluated by AAV/green fluorescent protein (GFP) expression with fluorescent microscopy.

Exposure of cardiomyocytes to hypoxia-reoxygenation. After 72 h of AAV vector (or culture medium only) transfection, myocytes were exposed to H-R and divided into the following groups: control: myocytes were continuously incubated in 95% air-5% CO2; H-R + Saline: myocytes were exposed to 24 h of hypoxia (95% N2 and 5% CO2, PO2 {approx} 30 mmHg) followed by 3 h of reoxygenation (95% air and 5% CO2); H-R + AAV/Neo: myocytes were transfected with AAV/Neo and then exposed to H-R; H-R + AAV/TGF-beta1ACT: myocytes were infected with AAV/TGF-beta1ACT and then exposed to H-R; H-R + AAV/TGF-beta1Latent: myocytes transfected with AAV/TGF-beta1Latent were exposed to H-R. This degree of H-R has been shown to result in myocyte injury, including apoptosis and necrosis (36). At the end of H-R, culture medium was collected for the determination of MMP activity, and myocytes were examined for measurement of reactive oxygen species (ROS) release and expression of specific proteins.

Measurement of MMP activity. MMP activity was measured by zymography (4). Briefly, the culture medium was subjected to electrophoresis in polyacrylamide gels containing 0.1% gelatin under nonreducing conditions. The gels were soaked in 2.5% Triton X-100 for 1 h and washed with water for 1 h. The gels were then incubated in a developing buffer containing 50 mM Tris, pH 7.4, 5 mM CaCl2, and 0.02% sodium azide overnight at 37°C. The gels were then stained with Coomassie blue for 1 h and photographed.

Measurement of ROS in cardiomyocytes. Intracellular ROS generation was measured with the use of the fluorescent signal 2',7'-dichlorodihydrofluorescein diacetate (H2DCF-DA, 10 µM; Cayman), a cell-permeant indicator for ROS. H2DCF-DA is nonfluorescent until the acetate groups are removed by intracellular ROS. The ROS-mediated fluorescence was observed under a fluorescent microscope (Nikon, Eclipse E600) with excitation set at 502 nm and emission set at 523 nm. Measurement of 2',7'-dichlorofluorescein (DCF) fluorescence intensity was performed by image processing with Image J 1.34 (National Institutes of Health). For each photograph the cellular and background average fluorescence values were obtained by tracing the shape of cells. Results are displayed in a ratiometric fashion normalized for the control condition.

Protein preparation and analysis by Western blot. Cardiomyocytes were lysed in iced lysis buffer and centrifuged at 4,000 rpm for 15 min at 4°C. The lysate proteins (50 µg/lane) were separated by SDS-PAGE (or 8% nondenatured PAGE for collagen I) and transferred to nitrocellulose membranes. After incubation in blocking solution (5% nonfat milk; Sigma), membranes were incubated with primary antibodies against mouse TGF-beta1 (v-, sc-146), procollagen I, collagen type I, LOX-1, mitogen-activated protein kinases (MAPKs; p38 and p44/42), or beta-actin (source of antibodies: Santa Cruz) for overnight at 4°C. Membranes were washed and then incubated with 1:4,000 dilution specific secondary antibodies (Amersham) for 1 h at room temperature, and the membranes were washed and detected with the ECL system (Amersham). The relative densities of protein bands were analyzed by Scan-gel-it, and the relative density of each protein band was normalized to that of beta-actin.

Statistical analysis. Data are expressed as means ± SE. All values were analyzed by using one-way ANOVA and the Newman-Keuls-Student t-test. The significance level was chosen as P < 0.05.


   RESULTS
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DISCUSSION
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AAV/GFP expression. In pilot experiments, myocytes were treated with AAV/GFP vectors (MOI 0–105) and incubated for 72 h at 37°C in 5% CO2-95% air. Since the most optimal (>90%) transfection was observed at an MOI of 104 (Fig. 1A), this concentration was used in all subsequent experiments with AAV.


Figure 1
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  		Fig. 1. A: Adeno-associated virus (AAV)/green fluorescent protein (GFP) expression with fluorescent microscopy. More than 90% of the cells showed GFP expression at a multiplicity of infection of 104. B: total transforming growth factor (TGF)-beta1 expression measured by Western blot analysis. Total TGF-beta1 expression (protein) was significantly increased in AAV/TGF-beta1ACT or AAV/TGF-beta1Latent-transfected cells. Hypoxia-reoxygenation (H-R) itself modestly enhanced the expression of TGF-beta1. AAV/Neo transfection had no effect on TGF-beta1 expression. C: effect of AAV/TGF-beta1 transfection itself on basal expression of specific proteins in the absence of H-R. AAV/TGF-beta1 transfection itself had no effect on the basal expressions of (pro)collagen I, LOX-1, and p38 and p44/42 mitogen-activated protein kinases (MAPKs). These data represent 3 separate experiments. p-p44/42 MAPK, p-p38 MAPK, phosphorylated p44/42 and p38 MAPKs.

 
Overexpression of TGF-beta1. After 72 h of AAV infection, cultured cardiomyocytes were exposed to H-R. At the end of H-R, the cells were harvested for Western blot analysis of total TGF-beta1. As shown in Fig. 1B, total TGF-beta1 expression was markedly increased in AAV/TGF-beta1ACT- or AAV/TGF-beta1Latent-transfected cells, indicating successful delivery of the transgene into cardiomyocytes. In keeping with a previous study (26), H-R itself induced the expression of TGF-beta1. As expected, AAV/TGFbeta1-overexpressing cells had a much greater expression of TGF-beta1. AAV/Neo had no effect on TGF-beta1 expression. We next examined whether AAV/TGF-beta1 transfection itself affects the basal expression of certain proteins. As shown in Fig. 1C, AAV/TGF-beta1 transfection itself had no effect on the basal expressions of (pro)collagen I, LOX-1, and p38 and p44/42 components of redox-sensitive MAPKs. We also found that AAV/TGF-beta1 transfection itself had no effect on the basal levels of ROS release and activity of MMP-2 and MMP-9 in the absence of H-R (data not shown).

AAV/TGF-beta1 transfection and collagen type I expression and MMP activity induced by H-R. H-R significantly increased the expression of procollagen I, collagen type I, and activity of MMP-2 and MMP-9 (vs. cells kept under control conditions) (P < 0.01, Fig. 2). Both TGF-beta1Latent and TGF-beta1ACT had a dramatic inhibitory effect on H-R-mediated upregulation of the expression of collagen type I and procollagen I and the activity of MMPs. TGF-beta1ACT was superior to TGF-beta1Latent in this regard.


Figure 2
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  		Fig. 2. Effect of transfection of AAV/ TGF-beta1ACT or AAV/TGF-beta1Latent on (pro)collagen type I expression (A) and activity of matrix metalloproteinase (MMP)-2 and -9 (B) induced by H-R. Both TGF-beta1ACT and TGF-beta1Latent inhibit H-R-mediated (pro)collagen type I expression (A) and activity of MMP-2 and -9 (B) induced by H-R. TGF-beta1ACT is more potent than TGF-beta1Latent in this process. n = 5. **P < 0.01 vs. Control; ++P < 0.01 vs. H-R + AAV/Neo; #P < 0.05, ##P < 0.01 vs. H-R + AAV/TGF-beta1Latent. AU, arbitrary units.

 
It is of note that AAV/Neo transfection had no effect on H-R-mediated (pro)collagen type I expression and activity of MMPs [P = nonsignificant (NS) vs. H-R + Saline], indicating that the effects of TGF-beta1ACT or TGF-beta1Latent were not mediated by the mere presence of AAV.

AAV/TGF-beta1 transfection and redox-sensitive signaling during H-R. H-R markedly increased DCF fluorescence, reflecting intracellular ROS level. Both AAV/TGF-beta1ACT and AAV/ TGF-beta1Latent transfection significantly reduced DCF fluorescence despite H-R (P < 0.01; Fig. 3). It has been shown that both I/R (19) and ROS (28) increase LOX-1 expression, and LOX-1 activation itself stimulates the formation of ROS (9). In keeping with these studies, H-R in cardiomyocytes induced LOX-1 expression (P < 0.01 vs. control), which was inhibited by the overexpression of TGF-beta1 (Fig. 4).


Figure 3
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  		Fig. 3. Effect of transfection of AAV/TGF-beta1ACT or AAV/TGF-beta1Latent on oxidative stress in HL-1 cardiomyocytes induced by 24 h of hypoxia and 3 h of reoxygenation (H-R). Left: representative 2',7'-dichlorofluorescein fluorescence images indicating intracellular reactive oxygen species (ROS) during H-R. Right: summary of data expressed as % of control fluorescence. n = 5. **P < 0.01 vs. Control; ++P < 0.01 vs. H-R + AAV/Neo; ##P < 0.01 vs. H-R + AAV/TGF-beta1Latent.

 

Figure 4
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  		Fig. 4. Effect of transfection of AAV/TGF-beta1ACT or AAV/TGF-beta1Latent on LOX-1 expression induced by H-R. Both TGF-beta1ACT and TGF-beta1Latent inhibit H-R-mediated LOX-1 expression. TGF-beta1ACT is more potent than TGF-beta1Latent in inhibition of LOX-1. n = 5. **P < 0.01 vs. Control; ++P < 0.01 vs. H-R + AAV/Neo; #P < 0.05 vs. H-R + AAV/TGF-beta1Latent.

 
We next measured the expression of p38 and p44/42 MAPKs. As shown in Fig. 5, the protein expression of p38 as well as p44/42 MAPKs was not altered during H-R. AAV/TGF-beta1 transfection before H-R reduced the phosphorylation of both p38 and p44/42 MAPKs (P < 0.01). AAV/Neo transfection had no effect. Again, TGF-beta1ACT was superior to TGF-beta1Latent (P < 0.05).


Figure 5
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  		Fig. 5. Effect of transfection of AAV/TGF-beta1ACT or AAV/TGF-beta1Latent on activation of p38 MAPK (A) and p44/42 MAPK (B) induced by H-R. Both TGF-beta1ACT and TGF-beta1Latent inhibit H-R-mediated activation of MAPKs. TGF-beta1ACT is more potent than TGF-beta1Latent in inhibition of activation of MAPKs. n = 5. **P < 0.01 vs. Control; ++P < 0.01 vs. H-R + AAV/Neo; #P < 0.05 vs. H-R + AAV/TGF-beta1Latent.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This study confirmed all three of our hypotheses: 1) a brief period of H-R upregulates the signals for cardiac remodeling; 2) overexpression of TGF-beta1 reduces the signals for cardiac remodeling; and 3) TGF-beta1 overexpression can modulate LOX-1 and redox-sensitive signaling in cardiac myocytes. In addition, we demonstrate that AAV2 is a very efficient vector for transfection of mouse cardiomyocytes with TGF-beta1. Finally, TGF-beta1ACT is superior to TGF-beta1Latent with regard to modulation of H-R-mediated cardiac remodeling pathways.

Presently there are three main viral vectors in use for gene therapy protocols: adenoviruses, retroviruses, and AAV. Among them, AAV-based vectors are naturally capable of efficient and stable gene delivery (13). In fact, AAV is clearly the best virus to use for long-term gene transduction and expression (35). Furthermore, neither wild-type nor recombinant AAV infection has been found to be associated with any significant pathology. A number of investigators have shown efficient transfection of genes in smooth muscle cells and fibroblasts with AAV (29, 37). In our previous studies (18, 20), we induced upregulation of IL-10 and TGF-beta1ACT genes by using AAV2 by tail vein injection and showed sustained gene upregulation in vascular tissues of LDL receptor-knockout mice. The present study for the first time shows that mouse cardiomyocytes avidly take up AAV2.

The upregulation of (pro)collagen I in mouse cardiomyocytes following a brief period of H-R is a relatively novel observation. It is traditionally thought that the signals for cardiac remodeling appear late after ischemia. In previous studies, Kossmehl and colleagues (15) documented the release of carboxy-terminal propeptide of type I procollagen (PICP) in the perfusion fluid of porcine hearts as early as 2 h after coronary artery occlusion. This finding strongly hints that collagen synthesis had already commenced at this time. They also demonstrated release of fibronectin and osteopontin in porcine hearts after I/R, which are key players in cardiac remodeling along with collagen (15). In earlier in vitro studies, we found that a brief exposure of cardiac fibroblasts to H-R results in enhanced collagen I synthesis (7). Takino et al. (33) in a recent study showed release of PICP in patients with myocardial infarction soon after deployment of reperfusion strategy. Release of PICP peaked at 2–3 wk after myocardial infarction and correlated with cardiac relaxation abnormality. Thus our present observations of increased collagen signals early after H-R are in concordance with several previous studies (7, 15, 33). The novelty of our study relates to the fact that cardiomyocytes also represent a potent source of collagen (21, 30).

The present study showed that resting mouse cardiomyocytes express modest signals for (pro)collagen type I as well as activity of their regulatory MMPs. H-R increased the signals for (pro)collagen type I as well as activity of MMPs by two- to threefold. We believe that the increase in the activity of MMPs is an autoregulatory response to (pro)collagen type I signal as in cardiac fibroblasts (4, 7). It is possible that MMPs contribute to cardiomyocyte injury, because previous studies have demonstrated limitation of cardiomyocyte injury by inhibitors of MMPs (3).

The intracellular signal for collagen expression appears to be release of ROS and activation of proinflammatory and prooxidant MAPKs, both p38 and p44/42. Again, this phenomenon appears to be akin to that in rat cardiac fibroblasts (4, 7). In hearts exposed to I/R, there is strong evidence for the release of ROS during the early phase of reperfusion and subsequent activation of MAPKs and redox-sensitive transcription factors (22). Previous in vitro studies have also documented that inhibition of ROS itself inhibits activation of MAPKs and reduces cardiomyocyte injury (5).

We observed a marked upregulation of LOX-1 in cardiomyocytes after H-R. This observation is supported by previous in vitro and in vivo studies (19, 25). LOX-1 is upregulated by ROS and can itself upregulate ROS generation in a positive feedback fashion (9, 28). It is of note that cytokine TNF-{alpha} that is released during H-R can also induce LOX-1 expression (25). LOX-1 activation in endothelial cells and fibroblasts enhances redox-sensitive signals, including activation of MAPKs and NF-{kappa}B (6).

TGF-beta1 is a pluripotent cytokine that has been shown to reduce H-R-mediated injury in rat cardiomyocytes as well as in intact rat hearts (3, 16, 26, 36). We showed previously (36) that while TGF-beta1Latent is upregulated during I/R, the conversion of TGF-beta1Latent to its active form is reduced. The present study demonstrates that overexpression of this cytokine reduces the signals for cardiac remodeling in mouse cardiomyocytes exposed to H-R. Furthermore, both TGF-beta1Latent and TGF-beta1ACT markedly attenuated H-R-associated intercellular signals, including ROS generation, LOX-1 expression, and activation of MAPKs. Most importantly, this study utilizing AAV-mediated overexpression strategy clearly demonstrates that TGF-beta1ACT is much more potent than TGF-beta1Latent in modulating signals for cardiac remodeling. TGF-beta1 is generally thought to be profibrotic in the heart. Nevertheless, arguments can be formulated for both pro- and antifibrotic roles for TGF-beta1 (12). For example, TGF-beta1 may act primarily as a mitogen and as an inducer of cell migration and ECM synthesis. It is also possible that TGF-beta1 acts as a cytostatic and immunosuppressive agent, preventing fibroblast division and decreasing tissue inflammation. These activities would prevent or mitigate fibrous tissue formation. Furthermore, the limitation of I/R injury during the acute phase per se may abrogate the signals for long-term fibrosis. Obviously, studies need to be performed to examine the idea that cardiac remodeling in ischemic hearts is attenuated by overexpression of TGF-beta1ACT over the long term. Such studies are currently under way in our laboratory.

Figure 6 reflects our thinking on the pathways of procollagen I and collagen type I expression during H-R. We believe that ROS generation and LOX-1 expression are intertwined in a positive feedback fashion. LOX-1 activation stimulates MAPKs and related transcription factors, leading to upregulation of procollagen I and collagen type I expression. Increase in the activity of MMPs is an autoregulatory response to increased collagen formation. The critical role of LOX-1 in this process is supported by earlier observations that a specific antibody to LOX-1 blocks the expression of collagen I as well as MMPs (24, 14). TGF-beta1ACT acts as a protective cytokine by inhibiting ROS generation and LOX-1 expression/activation.


Figure 6
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  		Fig. 6. Hypothesized pathways of H-R-mediated upregulation of the expression of (pro)collagen type I and MMPs. H-R causes release of ROS that upregulate LOX-1; LOX-1 induces further ROS generation. LOX-1 causes activation of MAPKs followed by transcription of redox-sensitive transcription factor(s) that induce the gene for (pro)collagen. MMPs are activated as autoregulatory response to collagen synthesis. TGF-beta1 blocks ROS generation and LOX-1 expression with inhibition of downstream signals.

 
The mechanism of the effects of TGF-beta1 is diverse and has been reviewed in several recent publications (2, 12, 23); it involves activation of Smads and other signals including p38 MAPK, p44/42 MAPK, JNK, and TGF-beta-activated kinase 1 (TAK1) (2). It has also been reported that regulation of membrane type 1 (MT1)-MMP/MMP-2 in cardiac fibroblasts by TGF-beta1 involves furin-convertase (32). Whether any or all of these mechanisms are involved in the effects described in the present study is not known. Certainly, we have shown that overexpression of TGF-beta1 in mouse cardiomyocytes blocks H-R-mediated collagen type I expression and MMP activity mediated through ROS release and p38 and p44/42 MAPK activation.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
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This study was supported by a grant from the Department of Veterans Affairs and the national American Heart Association.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. L. Mehta, Cardiovascular Medicine, Univ. of Arkansas for Medical Sciences, 4301 West Markham St. Slot 532, Little Rock, AR 72205 (e-mail: mehtajl{at}uams.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* C.-P. Hu and A. Dandapat contributed equally to this work. Back


   REFERENCES
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 MATERIALS AND METHODS
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 DISCUSSION
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 REFERENCES
 

  1. Blobe G, Schiemann W, Lodish H. Role of transforming growth factor beta in human disease. N Engl J Med 342: 1350–1358, 2000.[Free Full Text]
  2. Bujak M, Frangogiannis NG. The role of TGF-beta signaling in myocardial infarction and cardiac remodeling. Cardiovasc Res 74: 184–195, 2007.[Abstract/Free Full Text]
  3. Chen H, Li D, Saldeen T, Mehta JL. TGF-beta1 attenuates myocardial ischemia-reperfusion injury via inhibition of upregulation of MMP-1. Am J Physiol Heart Circ Physiol 284: H1612–H1617, 2003.[Abstract/Free Full Text]
  4. Chen J, Mehta JL. Angiotensin II-mediated oxidative stress and procollagen-1 expression in cardiac fibroblasts: blockade by pravastatin and pioglitazone. Am J Physiol Heart Circ Physiol 291: H1738–H1745, 2006.[Abstract/Free Full Text]
  5. Chen JX, Zeng H, Tuo QH, Yu H, Meyrick B, Aschner JL. NADPH oxidase modulates myocardial Akt, ERK1/2 activation, and angiogenesis after hypoxia-reoxygenation. Am J Physiol Heart Circ Physiol 292: H1664–H1674, 2007.[Abstract/Free Full Text]
  6. Chen K, Chen J, Liu Y, Xie J, Li D, Sawamura T, Hermonat PL, Mehta JL. Adhesion molecule expression in fibroblasts: alteration in fibroblast biology after transfection with LOX-1 plasmids. Hypertension 46: 622–627, 2005.[Abstract/Free Full Text]
  7. Chen K, Li D, Zhang X, Hermonat PL, Mehta JL. Anoxia-reoxygenation stimulates collagen type-I and MMP-1 expression in cardiac fibroblasts: modulation by the PPAR-gamma ligand pioglitazone. J Cardiovasc Pharmacol 44: 682–687, 2004.[CrossRef][Web of Science][Medline]
  8. Claycomb WC, Lanson NA, Stallworth BS, Egeland DB, Delcarpio JB, Bahinski A, Izzo NJ. HL-1 cells: a cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte. Proc Natl Acad Sci USA 95: 2979–2984, 1998.[Abstract/Free Full Text]
  9. Cominacini L, Pasini AF, Garbin U, Davoli A, Tosetti ML, Campagnola M, Rigoni A, Pastorino AM, Lo Cascio V, Sawamura T. Oxidized low density lipoprotein (ox-LDL) binding to ox-LDL receptor-1 in endothelial cells induces the activation of NF-{kappa}B through an increased production of intracellular reactive oxygen species. J Biol Chem 275: 12633–12638, 2000.[Abstract/Free Full Text]
  10. Draude G, Lorenz RL. TGF-beta1 downregulates CD36 and scavenger receptor A but upregulates LOX-1 in human macrophages. Am J Physiol Heart Circ Physiol 278: H1042–H1048, 2000.[Abstract/Free Full Text]
  11. Heeneman S, Cleutjens JP, Faber BC, Creemers EE, van Suylen RJ, Lutgens E, Cleutjens KB, Daemen MJ. The dynamic extracellular matrix: intervention strategies during heart failure and atherosclerosis. J Pathol 200: 516–525, 2003.[CrossRef][Web of Science][Medline]
  12. Hermonat PL, Li D, Yang B, Mehta JL. Mechanism of action and delivery possibilities for TGFbeta1 in the treatment of myocardial ischemia. Cardiovasc Res 74: 235–243, 2007.[Abstract/Free Full Text]
  13. Hermonat PL, Muzyczka N. Use of adeno-associated virus as a mammalian DNA cloning vector: transduction of neomycin resistance into mammalian tissue culture cells. Proc Natl Acad Sci USA 81: 6466–6470, 1984.[Abstract/Free Full Text]
  14. Joseph J, Ranganathan S, Mehta JL. Low density lipoproteins modulate collagen metabolism in fibroblasts. J Cardiovasc Pharmacol Ther 8: 161–166, 2003.[Abstract/Free Full Text]
  15. Kossmehl P, Schonberger J, Shakibaei M, Faramarzi S, Kurth E, Habighorst B, von Bauer R, Wehland M, Kreutz R, Infanger M, Schulze-Tanzil G, Paul M, Grimm D. Increase of fibronectin and osteopontin in porcine hearts following ischemia and reperfusion. J Mol Med 83: 626–637, 2005.[CrossRef][Web of Science][Medline]
  16. Lefer A, Ma X, Weyrich A, Scalia R. Mechanism of the cardioprotective effect of transforming growth factor beta1 in feline myocardial ischemia and reperfusion. Proc Natl Acad Sci USA 90: 1018–1022, 1993.[Abstract/Free Full Text]
  17. Letterio JJ, Roberts AB. Regulation of immune responses by TGF-beta. Annu Rev Immunol 16: 137–161, 1998.[CrossRef][Web of Science][Medline]
  18. Li D, Liu Y, Chen J, Velchala N, Amani F, Nemarkommula A, Chen K, Rayaz H, Zhang D, Liu H, Sinha AK, Romeo F, Hermonat PL, Mehta JL. Suppression of atherogenesis by delivery of TGFbeta1ACT using adeno-associated virus type 2 in LDLR knockout mice. Biochem Biophys Res Commun 344: 701–707, 2006.[CrossRef][Web of Science][Medline]
  19. Li D, Williams V, Liu L, Chen H, Sawamura T, Antakli T, Mehta JL. LOX-1 inhibition in myocardial ischemia-reperfusion injury: modulation of MMP-1 and inflammation. Am J Physiol Heart Circ Physiol 283: H1795–H1801, 2002.[Abstract/Free Full Text]
  20. Liu Y, Li D, Chen J, Xie J, Bandyopadhyay S, Zhang D, Nemarkommula AR, Liu H, Mehta JL, Hermonat PL. Inhibition of atherogenesis in LDLR knockout mice by systemic delivery of adeno-associated virus type 2-hIL-10. Atherosclerosis 188: 19–27, 2006.[CrossRef][Web of Science][Medline]
  21. Madani S, De Girolamo S, Munoz DM, Li RK, Sweeney G. Direct effects of leptin on size and extracellular matrix components of human pediatric ventricular myocytes. Cardiovasc Res 69: 716–725, 2006.[Abstract/Free Full Text]
  22. McCubrey JA, Lahair MM, Franklin RA. Reactive oxygen species-induced activation of the MAP kinase signaling pathways. Antioxid Redox Signal 8: 1775–1789, 2006.[CrossRef][Web of Science][Medline]
  23. Mehta JL, Attramadal H. The TGFbeta superfamily in cardiovascular biology. Cardiovasc Res 74: 181–183, 2007.[Free Full Text]
  24. Mehta JL, Chen J, Hermonat PL, Romeo F, Novelli G. Lectin-like, oxidized low-density lipoprotein receptor-1 (LOX-1): a critical player in the development of atherosclerosis and related disorders. Cardiovasc Res 69: 36–45, 2006.[Abstract/Free Full Text]
  25. 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 23: 2203–2208, 2003.[Abstract/Free Full Text]
  26. Mehta J, Li D, Chen H. Protection of myocytes from hypoxia-reoxygenation injury by nitric oxide is mediated by modulation of TGFbeta1. Circulation 105: 2206–2211, 2002.[Abstract/Free Full Text]
  27. Minami M, Kume N, Kataoka H, Morimoto M, Hayashida K, Sawamura T, Masaki T, Kita T. Transforming growth factor-beta1 increases the expression of lectin-like oxidized low-density lipoprotein receptor-1. Biochem Biophys Res Commun 272: 357–361, 2000.[CrossRef][Web of Science][Medline]
  28. Nagase M, Ando K, Nagase T, Kaname S, Sawamura T, Fujita T. Redox-sensitive regulation of LOX-1 gene expression in vascular endothelium. Biochem Biophys Res Commun 281: 720–725, 2001.[CrossRef][Web of Science][Medline]
  29. Nykanen AI, Pajusola K, Krebs R, Keranen MA, Raisky O, Koskinen PK, Alitalo K, Lemstrom KB. Common protective and diverse smooth muscle cell effects of AAV-mediated angiopoietin-1 and -2 expression in rat cardiac allograft vasculopathy. Circ Res 98: 1373–1380, 2006.[Abstract/Free Full Text]
  30. Parkes JG, Liu Y, Sirna JB, Templeton DM. Changes in gene expression with iron loading and chelation in cardiac myocytes and non-myocytic fibroblasts. J Mol Cell Cardiol 32: 233–246, 2000.[CrossRef][Web of Science][Medline]
  31. Qian SW, Kondaiah P, Roberts AB, Sporn MB. cDNA cloning by PCR of rat transforming growth factor beta-1. Nucleic Acids Res 18: 3059, 1990.[Free Full Text]
  32. Stawowy P, Margeta C, Kallisch H, Seidah NG, Chretien M, Fleck E, Graf K. Regulation of matrix metalloproteinase MT1-MMP/MMP-2 in cardiac fibroblasts by TGF-beta1 involves furin-convertase. Cardiovasc Res 63: 87–97, 2004.[Abstract/Free Full Text]
  33. Takino T, Nakamura M, Hiramori K. Circulating levels of carboxy terminal propeptide of type I pro-collagen and left ventricular remodeling after myocardial infarction. Cardiology 91: 81–86, 1999.[CrossRef][Web of Science][Medline]
  34. Weber KT. Fibrosis, a common pathway to organ failure: angiotensin II and tissue repair. Semin Nephrol 17: 467–491, 1997.[Web of Science][Medline]
  35. Xiao X, Li J, Samulski RJ. Efficient long-term gene transfer into muscle tissue of immunocompetent mice by adeno-associated virus vector. J Virol 70: 8098–8108, 1996.[Abstract]
  36. Yang BC, Zander D, Mehta JL. Hypoxia-reoxygenation-induced apoptosis in cultured adult rat myocytes and the protective effect of platelets and transforming growth factor-beta1. J Pharmacol Exp Ther 291: 733–738, 1999.[Abstract/Free Full Text]
  37. Zhao W, Zhong L, Wu J, Chen L, Qing K, Weigel-Kelley KA, Larsen SH, Shou W, Warrington KH Jr, Srivastava A. Role of cellular FKBP52 protein in intracellular trafficking of recombinant adeno-associated virus 2 vectors. Virology 353: 283–293, 2006.[CrossRef][Web of Science][Medline]



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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.
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