Decorin Gene Delivery Inhibits Cardiac Fibrosis in Spontaneously Hypertensive Rats by Modulation of Transforming Growth Factor-β/Smad and p38 Mitogen-Activated Protein Kinase Signaling Pathways

Abstract

Fibrosis is the response of heart and other organs to injuries. Excessive fibrosis can cause organ dysfunction or even failure. Transforming-growth factor (TGF)-β is a cytokine that induces fibroblast proliferation and increases the synthesis of a number of extracellular matrix proteins including collagens. Decorin (DCN) is a natural antagonist of TGF-β. In the current study, we investigated the potential antifibrotic effects of DCN gene delivery by a recombinant adeno-associated viral (rAAV) vector to inhibit cardiac fibrosis in old, spontaneously hypertensive rats (SHRs), which develop severe cardiac and kidney fibrosis if without intervention. The rAAV-DCN vector was injected (at a dose of 1 × 10¹¹ vector genomes) via the tail vein into 5-month-old male SHRs, resulting in persistent, stable expression of DCN (up to 16 weeks). rAAV-DCN treatment significantly reduced collagen content and fibrosis in the heart and attenuated cardiomyocyte hypertrophy. Hemodynamics data at 16 weeks showed that DCN gene delivery induced a significant increase in left ventricular end-systolic pressure and maximal–minimal rate of pressure increase (±dp/dt _max), but a decrease in left ventricular end-diastolic pressure (p < 0.05), compared with those of control animals. The expression of TGF-β and α-smooth muscle actin, and the phosphorylation levels of Smad2 and p38 MAPK, were markedly reduced by rAAV-DCN treatment as compared with the controls. Thus, these results suggest that rAAV-mediated DCN overexpression led to the inhibition of hypertension-induced cardiac fibrosis and hypertrophy and improved cardiac function, and therefore may have therapeutic potential for organ fibrosis.

Introduction

H ypertension affects 15 to 20% of the adult population, leading to structural remodeling of the left ventricular (LV) myocardium and eventually heart failure. A growing body of evidence indicates that besides cardiomyocyte hypertrophy, cardiac fibrosis is one of the key pathological features of hypertensive heart disease and interferes with normal structure and function of the myocardium (Brilla et al., 1991; Weber and Brilla, 1991; Weber, 2000). Similarly, remodeling plays an important role in pathogenesis in many other cardiovascular diseases such as postinfarct myocardial remodeling, and hypertrophic or dilated cardiomyopathies. Cardiac fibrosis is the result of a variety of structural changes that occur after pathological stimuli to the cardiovascular system and is characterized by destruction of cardiomyocytes and a disproportionate accumulation of fibrillar collagen (Jugdutt, 2003). The mechanisms responsible for the transition of cardiac fibroblasts to myofibroblasts have been elucidated to be associated with cardiac fibrosis. Fibroblasts, which are called the proximal effector cells in this process, may then be activated to become myofibroblasts, which produce an excessive amount of collagens in response to inflammatory mediators or cytokines, such as transforming growth factor (TGF)-β (Lijnen et al., 2000). A role for TGF-β in cardiac fibrosis has become increasingly apparent in that it is a critical factor stimulating both myofibroblast formation and collagen production. It has been previously proven that overexpression of TGF-β can lead to interstitial fibrosis, hypertrophic growth of cardiac myocytes, and production of extracellular matrix (ECM) components (Ignotz and Massague, 1986; Eghbali et al., 1991a,b; Sigel et al., 1996).

With the emerging evidence that TGF-β is involved in the pathogenesis and progression of cardiac fibrosis, potential treatment with specific factors that may interrupt the activity of TGF-β will become more promising and attractive. One such proposed agent is decorin (DCN), a member of the small proteoglycan family characterized by core proteins with leucine-rich repeats (Hocking et al., 1998; Iozzo, 1999; Reed and Iozzo, 2002). Yamaguchi and colleagues reported that DCN can bind to and neutralize extracellular TGF-β, and thus may be a naturally occurring inhibitor that antagonizes TGF-β (Yamaguchi et al., 1990).

Previous studies demonstrated that transfection of DCN cDNA into skeletal muscle in order to deliver decorin into the circulation, or systemic administration of decorin, alleviates excess renal extracellular matrix accumulation and improves proteinuria in experimental glomerulonephritis in the rat (Border et al., 1992; Isaka et al., 1996). In addition, in the myocardial infarction rat model, a wider distribution of collagen fibril sizes with less organized and loose packing in mature scar of decorin-null mice has been documented (Weis et al., 2005). Furthermore, intratracheal microinjection of a recombinant adenovirus containing DCN cDNA resulted in overexpression of the exogenous decorin gene in airway epithelium. It was found that decorin can antagonize bioactive TGF-β during lung growth and differentiation (ZHao et al., 1999). In vitro, when exogenous DCN was added, cultured human cardiac fibroblasts exposed to TGF-β demonstrated decreased collagen production (Jahanyar et al., 2007). Thus, these studies have identified DCN as an important regulator and therapeutic candidate for inhibiting fibrosis.

The recombinant adeno-associated viral (rAAV) vector possesses several unique advantages over other vectors, including long-term transgene expression and stable transduction, the ability to infect both dividing and nondividing cells, nonpathogenicity, and low vector toxicity (Sun et al., 2000). The establishment of packaging and improvement of packaging capacity, and also replication systems independent of adenoviral helper, make rAAV an appealing vector for long-term gene therapy (Xiao et al., 1998; Duan et al., 2000). It has also been reported that AAV serotype 8 more efficiently delivers genes to muscle and heart. In the present study, we used rAAV-8 (Gao et al., 2002; Wang et al., 2005). Therefore, it is critical to evaluate the role of rAAV-DCN in a model of cardiac fibrosis in spontaneously hypertensive rats (SHRs) that has proven fidelity and predictability for therapeutic effects in human disease. The objective of the current study was to investigate whether long-term and stable expression of DCN could not only prevent cardiac fibrosis associated with hypertension but also achieve the therapeutic effects by blocking fibrosis-related signaling pathways.

Materials and Methods

Construction and preparation of rAAV

The rAAV vector backbone plasmid (pXXUF1) carrying the green fluorescent protein (GFP) reporter gene, the adenoviral helper plasmid pHelper, and the packaging plasmid pXX8 have been described previously (Xiao et al., 1998). A 1080-base pair human decorin (DCN) cDNA fragment (NotI/NotI) containing the open reading frame was subcloned into pXXUF1 downstream of the cytomegalovirus (CMV) promoter to produce the plasmid pUF1-DCN. The vectors for rAAV-DCN and rAAV-GFP were prepared by a triple-plasmid cotransfection method in 293 cell lines, as described previously (Xiao et al., 1998). The rAAV was aliquoted and stored at −80°C for vector delivery. To determine the titer of rAAV vectors, 5 μl of the rAAV was digested with 5 units of DNase I at 37°C for 1 hr. After DNase inactivation, 2 × proteinase K buffer and 100 μg of proteinase K were added and incubated with the rAAV solution for 1 hr at 37°C. The reaction was then extracted with an equal volume of phenol–chloroform–isoamyl alcohol. Finally, dot-blot hybridization with a ³²P-labeled CMV promoter DNA probe was used to determine the titer of rAAV-DCN and rAAV-GFP.

Animal treatment and gene delivery

Twenty-one male SHRs, 4 to 5 months old, weighing 260–290 g, were purchased from the Wei Tong Li Hua Animal Center (Beijing, China). Experimental protocols were consistent with National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Chinese Academy of Sciences (Beijing, China). All animals were housed at 25 ± 3°C and 50 ± 20% humidity, with 12-hr light/dark cycles, and were allowed free access to normal rat chow (9% fat, 20% protein, 53% starch, 5% fiber) plus water. Anesthesia for surgical procedures was accomplished by intraperitoneal injection of pentobarbital (50 mg/kg body weight). The animals were then divided randomly into three groups (n = 7 for each group). The control group received 1 ml of saline solution intravenously. Rats in the GFP-treated group (rAAV-GFP) and the DCN-treated group (rAAV-DCN) were injected with a single dose of rAAV-GFP or rAAV-DCN (about 1 × 10¹¹ vector genomes [VG] per rat in 1 ml of saline solution), respectively, via the tail vein. The rats were then kept warm until they recovered. Animals were killed 16 weeks after rAAV administration under pentobarbital anesthesia (50 mg/kg body weight), and after blood had been drained the heart, lung, kidney, aorta, and liver were harvested, frozen in liquid nitrogen, and stored at −80°C. A portion of the organs was fixed with neutralized formalin for histological analysis.

Plasma and urine collection

Twenty-four-hour urine samples were collected in metabolic cages with 500 μl of toluene to prevent decay and stored at −80°C before the urinary microalbumin concentration were measured. Plasma was collected by centrifuging blood and then stored at −80°C for DCN level determination.

Whole heart and left ventricular weight determination

Sixteen weeks after DCN gene delivery, the animals were killed under pentobarbital anesthesia and their hearts were obtained and weighed. The left ventricle was weighed followed by careful dissection of the right ventricular free wall and both atria. The interventricular septum was included in the left ventricle for the weight measurement. Ratios of left ventricle to whole heart were then calculated.

Protein extraction and Western blots

Protein concentration was measured by the Bradford method with bovine serum albumin as standard. Western blotting was performed to detect DCN expression levels in heart, kidney, and liver. Briefly, samples containing 20 μg of total protein were loaded in 10% sodium dodecyl sulfate (SDS)–polyacrylamide gels, and transferred onto polyvinylidene difluoride (PVDF) membranes after electrophoresis. After incubation in blocking buffer (5% nonfat dry milk in Tris-buffered saline [pH 7.6], 0.1% Tween 20) (TBST), the membranes were incubated with anti-DCN antibody (1:500 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) or anti-β-actin antibody (1:3000 dilution; Sigma-Aldrich, St. Louis, MO) overnight at 4°C. The membranes were then washed three times with TBST and incubated at room temperature for 1 hr with horseradish peroxidase-labeled anti-rabbit or anti-mouse IgG solution (1:8000 dilution; New England BioLabs, Ipswich, MA) in the blocking solution. Immunoreactive bands were visualized with an enhanced chemiluminescence (ECL) detection system (GE Healthcare Life Sciences, Piscataway, NJ). Cell proliferation and fibroblast differentiation within the LV was examined by Western blot analysis, using monoclonal antibodies to α-smooth muscle actin (α-SMA; Santa Cruz Biotechnology). Western blotting of the housekeeping protein β-actin (Sigma-Aldrich) was also performed to demonstrate equal loading of the protein samples. For heart samples, proteins were extracted, quantified, and separated by SDS–polyacrylamide gel electrophoresis (PAGE) as described previously to identify the effects of rAAV-DCN treatment on heart protein expression. Western blots were probed with antibodies against TGF-β, phosphorylated Smad2 (p-Smad2), Smad2, and Smad6 (Santa Cruz Biotechnology) as well as against phosphorylated p38 mitogen-activated protein kinase (p-p38 MAPK), p38 MAPK, p-Akt, and Akt (Santa Cruz Biotechnology) and β-actin (Sigma-Aldrich).

Human DCN mRNA analysis by reverse transcription-polymerase chain reaction

Total RNA was extracted from fresh rat tissues with TRIzol (GIBCO/Invitrogen, Carlsbad, CA), according to the manufacturer's instructions. Reverse transcription-polymerase chain reaction (RT-PCR) analysis specific for human DCN and rat β-actin (DCN primers, 5′-ACCGCGACTTCGAGCCCTC-3′, 5′-CATTCTCATGGGCACGCAGC-3′; and β-actin primer, 5′-GGAGAAGATGACCCAGATC-3′, 5′-GATCTTCATGAGGTAGTCAG-3′) was then performed with the RT-PCR kit (Takara Biotechnology, Otsu, Shiga, Japan), according to the manufacturer's instructions.

Determination of DCN levels by enzyme-linked immunosorbent assay

Plasma DCN levels were determined by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions (Adlitteram Diagnostic Laboratories, ADL). The plates were read at 490 nm with an ELx800 ELISA reader (Bio-Tek instruments, Winooski, Vermont) and DCN levels were calculated and expressed as nanograms per milliliter.

Urinary microalbumin measurement

Urinary microalbumin was measured by ELISA according to the manufacturer's instructions. (Adlitteram Diagnostic laboratories, ADL). The plates were read at 490 nm on an ELx800 absorbance microplate reader (Bio-Tek instruments, Winooski, VT) and urinary microalbumin levels were calculated and expressed as milligrams per liter.

Hemodynamics analysis

Rats were anesthetized by intraperitoneal administration of pentobarbital at 50 mg/kg body weight. A catheter tip manometer (Millar Instruments, Houston, TX) was induced into the left ventricle (LV) from the right carotid artery via the aortic arch in order to assess LV systolic and diastolic function after the 4-month treatment period. Contractility and relaxation were assessed on the basis of LV end-systolic pressure (LVESP), the maximal–minimal rate of pressure increase (dp/dt _max), and LV end-diastolic pressure (LVEDP). At the end of cardiac functional assessment, organs were harvested for further analysis.

Analysis of morphology and collagen deposition

Portions of rat heart and kidney were maintained in zinc formalin solution (10% zinc dichromate, 4% formaldehyde, 0.9% NaCl), embedded in paraffin, sectioned into 4-μm-thick slices, and stained with Sirius red (collagen stains red with Sirius red staining), as described previously (Dobrzynski et al., 2000). All sections were assessed by observers who were blinded to the experiment. Cardiomyocyte diameter and percentage of interstitial collagen content of the heart and kidney were observed microscopically and analyzed with an HAIPS pathological Imagic analysis system developed by the Ultrastructural Pathology Department of Tongji Hospital (Wuhan, China). The distance across the myocardial cell at its narrowest plane across the nucleus was measured in 75 cells from each LV (25 from the epicardium, 25 from the mesocardium, and 25 from the endocardium), and the average diameter was calculated. Red staining was expressed as a percentage of the total tissue area within a field to quantify ECM.

Statistical analysis

The statistical significance of differences between the various groups was determined with SAS software (SAS Institute, Cary, NC). All data are expressed as means ± SE, and differences were considered significant at p < 0.05.

Results

rAAV-DCN-mediated long-term in vivo expression of decorin

To evaluate expression of the rAAV-mediated DCN gene, proteins were prepared from harvested rat heart, liver, and kidney 16 weeks after vector administration and Western blotting were performed. Results showed abundant DCN protein expression in heart, liver, and kidney in rAAV-DCN-treated SHRs compared with rAAV-GFP-treated SHRs (Fig. 1A). RT-PCR was carried out with primers specific for the human DCN gene. Results showed that human DCN mRNA was detected in rAAV-DCN-treated rats but not in controls (Fig. 1B). Furthermore, we examined plasma DCN levels in SHRs by ELISA at various time points after gene delivery. The plasma DCN level in rAAV-DCN-treated rats reached 23.1 ± 1.5 ng/ml, significantly higher than in controls (4.8 ± 0.7 ng/ml in controls; p < 0.05) at week 2 and remained at similar levels throughout the entire experimental period (Fig. 1C). These results suggest that rAAV-mediated gene delivery led to efficient and stable expression of the DCN gene in vivo.

FIG. 1.

Recombinant adeno-associated virus serotype 8 (rAAV8)-mediated long-term decorin (DCN) expression in spontaneously hypertensive rats (SHRs). (A) Representative Western blot showing that DCN protein expression is increased in heart, kidney, and liver of SHRs receiving rAAV-DCN rather than rAAV-GFP treatment. Experiments were repeated three times. (B) RT-PCR of human-specific DCN mRNA, showing that an abundant amount of DCN mRNA was detected in rAAV-DCN-treated rats but not in the controls. (C) Histogram showing plasma DCN level measured by ELISA at various time points after gene delivery. Data are expressed as means ± SE (*p < 0.05 vs. rAAV-GFP group; n = 7).

Improvement of hemodynamics in rAAV-DCN-treated rats

Hemodynamics parameters were monitored with a catheter tip manometer advanced from the right carotid artery via the aortic arch into the left ventricle. We measured left ventricular end-systolic pressure (LVESP), left ventricular end-diastolic pressure (LVEDP), and the maximal–minimal rate of left ventricular pressure increase (±dp/dt _max) to evaluate both systolic and diastolic functions. Under baseline conditions, differences in hemodynamics were observed between the rAAV-DCN and rAAV-GFP groups (Fig. 2). The cardiac function index in SHRs was markedly deteriorated compared with normal control rats. With rAAV-mediated DCN gene administration, there were significant increases in LVESP (Fig. 2A) and +dp/dt _max (Fig. 2B) (p < 0.05), but decreases in LVEDP (Fig. 2C) (p < 0.05) and enhancement of −dp/dt _max (Fig. 2D) (p < 0.05), compared with controls. These data indicate an improvement in systolic and diastolic functions after rAAV-DCN treatment.

FIG. 2.

Improvement of cardiac hemodynamics functions. Hemodynamics analysis was performed with a cardiac catheter tip manometer advanced from the right carotid artery via the aortic arch into the left ventricle at 16 weeks. Results show that rAAV-DCN delivery improves systolic (A and B) and diastolic functions (C and D). Data are expressed as means ± SE (*p < 0.05 vs. saline and rAAV-GFP groups; n = 7).

Attenuation of cardiovascular remodeling and cardiac hypertrophy by rAAV-DCN treatment

Myocardial hypertrophy is an independent predictor of cardiovascular events in clinical practice, and of a considerable portion of heart injury induced by hypertension. In the present study, we analyzed the ratio of left ventricular weight to whole heart weight in order to identify the effects of rAAV-DCN on cardiac hypertrophy. rAAV-DCN delivery significantly attenuated the ratio of left ventricular weight to whole heart weight compared with control rats (Fig. 3A). In addition, DCN gene delivery also reduced cardiomyocyte size compared with control rats injected with saline or rAAV-GFP (Fig. 3B). These data suggested that rAAV-DCN treatment slightly, but significantly, attenuated hypertension-induced left ventricular hypertrophy.

FIG. 3.

Inhibition of cardiac hypertrophy in rats after rAAV-DCN treatment. (A) Comparison of the left ventricular weight–heart weight (LVW/HW) ratio shows that rAAV-DCN-treated rats had a lower LVW/HW ratio than did the control rats. (B) The cardiomyocyte diameter is reduced in rAAV-DCN-treated rats compared with control rats receiving rAAV-GFP or saline. Data are expressed as means ± SE (*p < 0.05 vs. saline and rAAV-GFP groups; n = 7).

Protective effects of DCN on heart histology

To analyze the antifibrotic effects of rAAV-DCN, we inspected the extent of heart damage by Sirius red staining. The red-staining field indicated the percentage of collagen per cardiac tissue area and also provided an index of fibrosis. Myocardial structures were deranged and an obvious amount of red-stained area (collagen) could be seen in the left ventricular section of the heart of the control SHRs, representing notable fibrosis in the myocardium. In contrast, rAAV-DCN treatment significantly attenuated hypertension-induced heart damage by alleviating cardiomyocyte hypertrophy, derangement of structures, ECM deposition, and cardiac fibrosis in transverse and longitudinal sections. These data suggested that administration of rAAV-DCN reduced ECM accumulation compared with control animals (Fig. 4).

FIG. 4.

Inhibition of fibrosis and collagen deposition in SHR heart and kidney, as indicated by Sirius red staining. (A–C) Micrographs of Sirius red-stained hearts of SHRs receiving (A) saline, (B) rAAV-GFP, or (C) rAAV-DCN. The red color shows fibrosis and collagen deposition in the heart. (D) Sirius red-stained sections were analyzed with the HAIPS pathological Imagic analysis system to determine the percentage of extracellular matrix/collagen per surface area. (E–G) Micrographs of Sirius red-stained kidneys of SHRs receiving (E) saline, (F) rAAV-GFP, or (G) rAAV-DCN. (H) The percentage of extracellular matrix/collagen per surface area. ECM was markedly reduced both in heart and kidney of rAAV-DCN-treated SHRs compared with the control groups. Data are expressed as means ± SE (*p < 0.05 vs. saline and rAAV-GFP groups; n = 7).

Protective effects of long-term expression of DCN on kidney

Renal cortex and medulla were stained with Sirius red to determine collagen deposition. More intense and diverse red staining in glomerular and medulla vascular areas was found in control SHRs than in DCN-treated SHRs (Fig. 4), suggesting that rAAV-DCN treatment significantly attenuated renal fibrosis induced by hypertension.

Reduction of albumin concentration in urine

Urinary albumin level analysis showed that long-term rAAV-DCN treatment induced a significant decrease in urinary microalbumin level. Sixteen weeks after gene delivery, the urinary albumin level fell to 0.147 ± 0.021 mg/liter, significantly lower than in controls (0.267 ± 0.022 and 0.259 ± 0.017 mg/liter in control SHRs receiving saline and rAAV-GFP, respectively) (Fig. 5), which further suggested that DCN gene delivery can attenuate renal dysfunction in SHRs.

FIG. 5.

Urine microalbumin levels as measured by ELISA. Data are expressed as means ± SE (*p < 0.05 vs. saline and rAAV-GFP groups; n = 7).

Effect of DCN on cardiac fibroblast function

Differentiation of fibroblasts was analyzed by Western blotting of α-SMA expression. Results showed that rAAV-DCN treatment significantly reduced α-SMA expression in heart (Fig. 6A and B), consistent with its ability to inhibit ECM accumulation in heart, indicating that rAAV-DCN treatment inhibited differentiation of fibroblasts in the heart of hypertensive rats.

FIG. 6.

Decrease in α-SMA expression after rAAV-DCN treatment. Representative Western blots and densitometric quantification of at least three repeats for each experiment show the effects of rAAV-mediated DCN gene therapy on the expression of α-SMA. Blots were scanned, and relative expression levels were normalized to total β-actin. Data are expressed as means ± SE (*p < 0.05 vs. saline and rAAV-GFP groups; n = 7).

Inhibition of TGF-β/Smad signaling pathway by decorin

TGF-β binds to membrane receptors and transmits signals via the Smad-dependent transduction pathway. This process leads to activation of the cascade of signaling transduction events involving intracellular mediator Smad proteins. To investigate the signaling mechanisms through which DCN attenuates cardiac fibrosis, we evaluated the expression of TGF-β. Western blotting showed that TGF-β expression was significantly decreased in hearts from rAAV-DCN-treated SHRs compared with controls (Fig. 7A and B). Also, we evaluated the expression of Smad2 and Smad6. Results showed that expression of phosphorylated Smad2 was significantly decreased in hearts from rAAV-DCN-treated SHRs compared with controls (Fig. 7C and D). In contrast, rAAV-DCN treatment increased Smad6 expression compared with controls (Fig. 7E and F), suggesting that DCN overexpression significantly inhibited activation of the TGF-β/Smad signaling pathway.

FIG. 7.

Downregulation of TGF-β and its signaling by rAAV-DCN treatment. Representative Western blots and densitometric quantification of at least three repeats for each experiment show the effects of rAAV-mediated DCN gene therapy on TGF-β/Smad signaling pathways. (A and B) Effects of DCN gene therapy on the expression of TGF-β in heart. (C and D) Effects of DCN gene therapy on the phosphorylation of Smad2 in heart. (E and F) Effects of DCN gene therapy on the expression of Smad6 in heart. Blots were scanned, and relative expression levels were normalized to total Smad2 (T-Smad2) or β-actin. Data are expressed as means ± SE (*p < 0.05 vs. saline and rAAV-GFP groups; n = 7).

Effect of DCN on activation of p38 MAPK and Akt signaling pathway

The p38 mitogen-activated protein kinases (MAPKs) are important signaling molecules activated during fibrosis. Phosphorylated p38 MAPK (p-p38 MAPK) levels in rAAV-DCN-treated SHRs were markedly reduced in heart relative to control rats (Fig. 8A and B). The expression of Akt was also assessed in the hearts of SHRs. Levels of Akt phosphorylated at position Thr-308 were determined by immunoblotting with a phosphospecific anti-Akt antibody. A significant increase in p-Akt was noted in heart of SHRs treated with rAAV-DCN compared with control animals (Fig. 8C and D). There were no changes in total Akt levels between the groups. Together, these data indicate that the antifibrotic effect in rAAV-DCN-treated SHRs was associated with partial activation of the Akt signaling pathway.

FIG. 8.

Reduction of p38 MAPK phosphorylation and enhancement of AKT phosphorylation by rAAV-DCN treatment. Representative Western blots and densitometric quantification of at least three repeats for each experiment showing the effects of rAAV-mediated DCN gene therapy on activation of p38 MAPK (A and B) and p308-Akt (C and D). Effects of DCN gene therapy on the expression of phosphorylation of p38 MAPK and p308-Akt in heart. Data are expressed as mean ± SE (*p < 0.05 vs. saline and rAAV-GFP groups; n = 7).

Discussion

The present study identifies effective intravenous delivery of an rAAV-based vector carrying the DCN cDNA to old SHRs and examines the effects on heart function, collagen deposition, as well as cardiomyocyte hypertrophy. We found that a single administration of rAAV-DCN resulted in stable and long-term expression of DCN, prevented cardiac fibrosis and hypertrophy, and improved heart function. In addition, large areas of intense focal fibrosis as well as an increased albumin level in urine, a marker of kidney injuries and an independent predictor of cardiovascular diseases, were observed in the control groups, whereas rAAV-mediated DCN gene therapy greatly attenuated all these changes, indicating that DCN protected renal function in the SHRs and that the latter may in turn lead to improved cardiac function. DCN gene therapy may also downregulate signal transduction pathways associated with fibrosis in old SHRs, including reduced levels of phosphorylated Smad2 and elevated levels of Smad6, suggesting inhibition of the TGF-β/Smad pathway in heart tissue. Furthermore, rAAV-DCN treatment attenuated activation of p38 MAPK in SHRs. All these data indicate that rAAV-mediated DCN overexpression protects against cardiac and renal fibrosis in the old SHR model.

A fundamental characteristic of hypertensive cardiac remodeling is myocardial stiffness, which is associated with fibrosis, and altered contractile and relaxation properties. Fibrous tissue accumulation is a feature of the structural remodeling of the myocardium seen in hypertensive heart disease (Weber et al., 1991). The collagen accumulation will increase myocardial stiffness that leads to diastolic dysfunction and ultimately systolic dysfunction. Some reports showed that diastolic and systolic dysfunction, based on increased wall thickness, chamber stiffness, and impaired relaxation, was found in the 9- to 10-month-old SHR model (Pfeffer et al., 1979; Nishimura et al., 1985; Brilla et al., 1991), which was consistent with our findings. In addition, myocardial hypertrophy and fibrosis were observed in SHRs from 3 to 6 months of age (Pfeffer et al., 1979; Mukherjee and Sen, 1990). As a result, for our pilot study we chose SHRs that were about 4–5 months of age and had been identified with existing cardiac fibrosis. We found that DCN gene therapy did not result in reduction of animal blood pressure; however, it induced significant improvement in cardiac function, including changes in hemodynamic parameters, LVESP, ± dp/dt _max, and LVEDP, 4 months after rAAV-DCN administration. These data indicated that the regression of fibrosis after rAAV-DCN treatment did not correlate with a reduction in blood pressure.

It was found that SHRs had elevated levels of TGF-β, which directly stimulates cardiac fibroblast proliferation and differentiation into activated myofibroblasts (Petrov et al., 2002). The myofibroblasts, which express α-SMA as a hallmark and produce large amounts of collagens, are important contributors to the development of fibrosis (Eghbali, 1992). The present study found that DCN gene therapy in SHRs resulted in a significant reduction in the level of α-SMA, suggesting reduced fibroblast transformation into myofibroblasts in the heart by rAAV-DCN treatment.

Myofibroblast formation is controlled by growth factors, cytokines, and mechanical stimuli (Tomasek et al., 2002). Key hormones and cytokines for this transition are angiotensin II (ANG II), endothelin-1, TGF-β₁, insulin-like growth factor (IGF), and tumor necrosis factor TNF-α. ANG II exerts its effects directly through the ANG II type 1 receptor and indirectly through induction of TGF-β₁ (Weber et al., 1999). TGF-β₁ is a critical factor because it stimulates both myofibroblast formation and collagen production. Increased TGF-β₁ gene expression has been demonstrated in the hearts of SHRs during the transition from stable hypertrophy to failure (Boluyt et al., 1994). In vitro, TGF-β₁ induces the production of ECM components including fibrillar collagen, fibronectin, and proteoglycans in cardiac fibroblasts (Eghbali et al., 1991a,b). It also stimulates fibroblast proliferation and phenotypic conversion to myofibroblasts (Tomasek et al., 2002). It has been reported that neutralization of TGF-β by anti-TGF-β antibody attenuates kidney hypertrophy and extracellular matrix gene expression in streptozotocin (STZ)-induced diabetic mice (Sharma et al., 1996). High glucose caused cellular hypertrophy in wild-type cells and increased fibronectin expression, but failed to induce hypertrophy in cells from the kidneys of TGF-β₁ knockout mice (Chen et al., 2004). Overexpression of TGF-β₁ in transgenic mice results in cardiac hypertrophy characterized by both interstitial fibrosis and hypertrophic growth of cardiac myocytes (Rosenkranz et al., 2002). These suggest that TGF-β₁ plays a crucial role in the myocardial remodeling process, particularly in cardiac fibrosis. Hence, blockade of the TGF-β₁ receptor is highly desirable in the prevention and treatment of hypertensive heart disease.

As the prototypic member of an expanding family of small leucine-rich proteoglycans, decorin has attracted considerable attention primarily because of its emergence as a novel antifibrotic therapy. DCN can bind multiple cytokines and affect their activity, such as TGF-β and TNF-α (Tufvesson and Westergren-Thorsson, 2002), fibroblast growth factor, and connective tissue growth factor (Chen et al., 2000). The core protein of decorin binds and neutralizes extracellular TGF-β, thus antagonizing the profibrotic and prosclerotic effects of this cytokine (Border et al., 1992). rAAV vectors were identified to drive efficient and prolonged transgene expression in vivo. In the present study, DCN expression in SHRs reached high levels from rAAV8 vector in the liver, heart, and kidney. Because DCN is a secreted protein, elevated serum levels could exert systemic therapeutic effects. Although the precise mechanism by which DCN inhibits tissue fibrosis is not fully understood, its interaction with TGF-β₁ has been well documented to play a crucial role in mediating its antifibrotic action. The present study showed that DCN gene delivery could attenuate activated TGF-β signaling in the heart of SHRs, further suggesting that inhibition of TGF-β₁ significantly contributed to antifibrotic effects.

The Smad proteins are essential components of the intracellular signaling pathway activated by TGF-β₁, and participate in TGF-β₁-induced fibrosis (Massague and Chen, 2000). TGF-β₁ signals are transduced by transmembrane type II and type I receptors (Piek et al., 1999; Massague, 2000). Once its receptor is activated, a cascade of signaling transduction involving Smad proteins is initiated (ten Dijke et al., 2000). Smad2 or Smad3 is phosphorylated and then binds to Smad4, a common partner for all of the receptor-activated Smads, forming a Smad complex that moves into the nucleus and interacts with various transcription factors to regulate the transcription of many genes. During this process, Smad6 and Smad7 inhibit the phosphorylation of Smad2 or Smad3, thus inhibiting TGF-β signaling. Although the expression of Smad2, Smad3, Smad4, and TGF-β was elevated in the chronic phase of myocardial infarct scar healing (Hao et al., 1999), it is not clear whether the Smad pathway is involved in cardiac fibrosis resulting from hypertension. In this regard, we examined Smad2 phosphorylation and Smad6 in the heart in SHRs. We found that expression of phosphorylated Smad2 was significantly decreased in heart samples from rAAV-DCN-treated SHRs compared with controls. On the other hand, rAAV-DCN treatment increased Smad6 expression.

Evidence has suggested that TGF-β may also stimulate other downstream pathways involving p38 MAPKs (Hanafusa et al., 1999) or the TGF-β₁–TAK1 (TGF-β-activated kinase-1)–p38 MAPK pathway (Matsumoto-Ida et al., 2006). In the present study we found that rAAV-DCN treatment reduced the level of p38 MAPK phosphorylation compared with controls, and therefore contributed to attenuation of hypertension-induced myocardial hypertrophy. How the two pathways interact has been addressed in only a few cases (Engel et al., 1999; Hanafusa et al., 1999; Watanabe et al., 2001; Yu et al., 2002; Matsumoto-Ida et al., 2006; Wang, 2007). Akt isoforms are phosphorylated at the T-loop (Thr-308 in protein kinase B [PKB]-α) by 3-phosphoinositide-dependent protein kinase-1 (PDK1) and this phosphorylation appears to be crucial for activation of Akt (Bevan, 2001). Research shows that stimulation of physiological hypertrophy involves the signaling mediated by Akt/protein kinase B (Wakatsuki et al., 2004). It has also been reported that decorin can bind both IGF-I and its receptor. This interaction leads to Akt phosphorylation at Thr-308 in endothelial cells (Schonherr et al., 2005). In this regard, we evaluated Akt phosphorylation at Thr-308 in the heart of SHRs. We found that Akt phosphorylation at Thr-308 was increased by DCN gene therapy compared with controls. These data suggest that the inhibition of fibrosis by DCN gene therapy could be attributed, at least in part, to enhanced activation of Akt phosphorylation.

In summary, we have found that rAAV-DCN treatment improved heart function, reduced remodeling and fibrosis in heart and kidney, and alleviated pathologic changes in old SHRs. A mechanism study indicated that DCN gene therapy upregulated Smad6 and downregulated TGF-β₁ and attenuated its downstream signaling and p38 MAPK in old SHR heart. No detectable adverse effects were found in rAAV-injected animals. These results provide strong support for the potential application of rAAV-DCN as a safe and effective therapeutic approach in treating cardiac and renal fibrosis in patients with hypertension.

Footnotes

Acknowledgments

This work was supported by grants from National 973 projects (nos. 2007CB512004 and 2006CB503801) and by an NSFC grant (no. 30871068). Drs. Wen Yan and Peihua Wang contributed equally to this work.

Author Disclosure Statement

The authors declare no conflict of interest.

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