Angiotensin II in the lesional skin of systemic sclerosis patients contributes to tissue fibrosis via angiotensin II type 1 receptors

Authors


Abstract

Objective

Tissue fibrosis in systemic sclerosis (SSc) is attributed to excessive deposition of extracellular matrix components produced by fibroblasts in skin lesions. Angiotensin II (Ang II), a vasoconstrictive peptide, is reported to have profibrotic activity as a result of induction of the extracellular matrix. The aim of the present study was to examine the expression of Ang II and its type 1 (AT1) and type 2 (AT2) receptors in affected skin and dermal fibroblasts from patients with SSc and to study the role of Ang II in collagen production by SSc dermal fibroblasts.

Methods

Levels of Ang II in sera from SSc patients and normal subjects were measured by a solid-phase immobilized-epitope immunoassay. Expression of angiotensinogen (Angt) in the skin was evaluated by immunohistochemistry. Expression of Angt, AT1, and AT2 in cultured dermal fibroblasts was analyzed by reverse transcription–polymerase chain reaction and immunohistochemistry. Levels of type I procollagen produced by cultured dermal fibroblasts were measured by enzyme-linked immunosorbent assay.

Results

Serum Ang II levels in patients with diffuse cutaneous SSc were significantly higher than those in patients with limited cutaneous SSc and in healthy donors. Immunohistochemical and immunoblotting analyses showed that Angt was present in skin from SSc patients, but not in normal skin. Angt messenger RNA (mRNA) was expressed in fibroblasts from patients with diffuse cutaneous SSc who had high levels of serum Ang II, but not in normal fibroblasts. AT1 mRNA expression was found in both SSc and normal fibroblasts, whereas AT2 mRNA was found only in SSc fibroblasts. Exogenous Ang II augmented the production of type I procollagen and transforming growth factor β1 by cultured fibroblasts via activation of AT1.

Conclusion

Aberrant Ang II production may be involved in tissue fibrosis through excessive production of the extracellular matrix components in SSc dermal fibroblasts. This suggests that the use of AT1 receptor antagonists may be a novel strategy for the treatment of tissue fibrosis in SSc patients.

Systemic sclerosis (SSc; scleroderma) is an autoimmune disorder of unknown cause (1). Its clinical features include tissue fibrosis, endothelial damage, inflammation, and the formation of specific autoantibodies (1). Tissue fibrosis results from excessive deposition of extracellular matrix components produced by fibroblasts (2), which might be affected by several cytokines and growth factors (3–9). Most SSc patients experience Raynaud's phenomenon as their first symptom, either simultaneously with, or followed by, skin thickening. Raynaud's phenomenon is defined as vasospasm of the arteries or arterioles, with endothelial injuries resulting in a fixed blood vessel deficit. It has been reported that an important factor involved in the endothelial damage caused by SSc is endothelin 1, which regulates vasoconstriction as well as cell growth (10). This evidence suggests that soluble mediators that exhibit vasoconstrictive and profibrotic properties, such as endothelin 1, may play a crucial role in the pathogenesis of SSc.

Angiotensin II (Ang II) is a vasoactive peptide that induces vascular constriction and regulates the hemodynamics in organs such as the kidney and heart (11, 12). Angiotensinogen (Angt), the precursor of angiotensin, is synthesized primarily by the liver and is secreted into the circulation. The conversion of Angt to Ang II is dependent on enzymatic cleavage by renin and angiotensin-converting enzyme (ACE). Ang II also has profibrotic activity, which has a regulatory effect on cell growth and synthetic properties, mainly related to the production of extracellular matrix components (13–15). Ang II was recently implicated in the pathogenesis of experimental fibrosis in the kidney and heart (16–18), and inhibition of the angiotensin system prevented tissue fibrosis of the kidney and heart in animal models (19, 20). Treatment of scleroderma renal crisis with ACE inhibitors has been shown to dramatically improve prognosis (21). It has also been reported that treatment with an Ang II type 1 (AT1) receptor antagonist is effective in Raynaud's phenomenon and was shown to improve levels of N-terminal type I procollagen propeptide, the serum indicator of tissue fibrosis in SSc (22). Thus, it can be inferred that Ang II may be a good candidate for the soluble mediators that modulate tissue fibrosis in SSc. However, the role of the angiotensin system in SSc has yet to be determined.

The goal of the present study was to further assess the fibrotic potential of Ang II in SSc dermal fibroblasts. We first determined whether Ang II was increased in the blood and skin of SSc patients compared with normal healthy donors. In addition, we performed an in vitro analysis of the expression of Angt, AT1 receptor, and AT2 receptor in SSc dermal fibroblasts. Our findings suggest that the action of Ang II as an autocrine factor in skin tissues may contribute to the fibrosis in SSc.

MATERIALS AND METHODS

Reagents

Olmesartan (23), a selective AT1 receptor antagonist, was donated by Sankyo Pharmaceutical (Tokyo, Japan). Angiotensin II and PD 123,319 (a selective AT2 receptor antagonist) were purchased from Sigma-Aldrich (St. Louis, MO).

Patients

Serum samples were obtained from 27 patients with limited cutaneous SSc (lcSSc) and 36 patients with diffuse cutaneous SSc (dcSSc). All patients were Japanese citizens who met the SSc criteria of the American College of Rheumatology (formerly, the American Rheumatism Association) (24). The clinical subsets of dcSSc and lcSSc were defined according to the classification described by LeRoy et al (25). Skin thickening was evaluated using the modified Rodnan skin thickness score (26). SSc patients with a history of renal dysfunction, including scleroderma renal crisis and renal hypertension, were excluded from the study.

Serum samples were also obtained from 20 patients with rheumatoid arthritis (RA) and 20 healthy volunteer donors. These subjects served as controls. Table 1 summarizes the characteristics of the SSc patients and controls. Informed consent was obtained from all study subjects.

Table 1. Characteristics of the SSc Patients and Healthy Donors*
 SSc patientsRA patients (n = 20)Healthy controls (n = 20)
dcSSc (n = 36)lcSSc (n = 27)
  • *

    None of the systemic sclerosis (SSc) patients were taking high-dose prednisolone (>15 mg/day) at study entry. dcSSc = diffuse cutaneous SSc; lcSSc = limited cutaneous SSc; RA = rheumatoid arthritis.

Age, mean (range) years49 (20–76)55 (27–78)47 (28–66)43 (24–58)
Sex, no. of women/no. of men35/125/29/119/1
Disease duration, mean (range) years4.3 (0.7–12)5.6 (1–15)
Treatment at study entry, no. of patients    
 Prednisolone (<15 mg/day)12620
 Cyclophosphamide2000
 D-penicillamine181110
 Vasodilators362400

Fibroblast cultures

Fibroblasts were prepared by an explant method, as previously described (10). Skin samples were obtained by biopsy of affected areas of the forearm of 10 patients with dcSSc and of normal skin from the forearm of 5 healthy donors. Fibroblasts were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (FBS; Sigma-Aldrich) and penicillin/streptomycin (Invitrogen). Cells from the third to fifth passages were used in the present study.

Determination of serum Ang II levels

Serum samples from SSc patients and controls were collected after centrifugation of 10 ml of peripheral blood and were immediately preserved at –80°C until the time of Ang II measurement. Serum levels of Ang II were measured with a solid-phase immobilized-epitope immunoassay developed by SPI-BIO (Massy, France) (27). This kit contains 2 kinds of specific monoclonal anti–Ang II antibodies. After immunologic reaction with the first anti–Ang II antibody on 96-well plates, the trapped molecules in the sera were covalently linked to the plate with glutaraldehyde. After washing and a denaturing treatment, Ang II was reacted with the acetylcholinesterase-labeled anti–Ang II antibody used as tracer. An enzymatic substrate for acetylcholinesterase was then added to each well, and the absorbance was measured in 405 nm. Cross-reactivity with Ang I in this assay was <4%.

Immunohistochemical staining of angiotensins and AT1 and AT2 receptors

Skin biopsy tissues were immediately frozen in OCT compound using liquid nitrogen. Cryostat sections of frozen skin on glass slides were stained, and fibroblasts were cultured in 4-chamber glass slides. Skin sections were air-dried on glass slides and fixed with cold acetone at –20°C for 10 minutes. Cultured fibroblasts were fixed with 2% paraformaldehyde at 4°C for 30 minutes and rinsed extensively in phosphate buffered saline (PBS).

Skin sections and cultured fibroblasts were blocked with 2.5% horse serum (Vector, Burlingame, CA) in PBS for 10 minutes at room temperature, and then incubated with the first antibody or control IgG (Vector) for 60 minutes at room temperature. The following first antibodies were used: a goat polyclonal antibody against human angiotensins (Ang I/II [N-10]) (5 μg/ml; Santa Cruz Biotechnology, Santa Cruz, CA), which reacts with human Angt, Ang I, and Ang II; a rabbit polyclonal antibody against human Ang II (1:500 dilution; Peninsula Laboratories, San Carlos, CA), which does not cross-react with either Angt or Ang I; and rabbit polyclonal antibodies against human AT1 (5 μg/ml; Santa Cruz) and AT2 (5 μg/ml; Santa Cruz).

After extensive washing with PBS, biotin-labeled horse polyclonal anti-rabbit or anti-goat IgG antibody (Santa Cruz) was added, and incubation continued for 10 minutes. After washing with PBS, the slides were incubated for 5 minutes in streptavidin–peroxidase complex working solution (Santa Cruz). Streptavidin–peroxidase complexes were developed (brown color) with diaminobenzidine (Enhanced DAB Substrate kit; Pierce, Rockford, IL). Hematoxylin was used as a counterstain.

Western blotting

Proteins were extracted from skin tissues and cultured dermal fibroblasts after homogenization with NE-PER cytoplasmic extraction reagents (Pierce) containing protease inhibitors (2 μg/ml of aprotinin, 2 μg/ml of leupeptin, and 0.75 mM phenylmethylsulfonyl fluoride; final concentrations in the lysis buffer). The protein samples were resolved in sodium dodecyl sulfate–12.5% polyacrylamide gels under reducing conditions, according to the method described by Laemmli (28), and electrophoretically transferred to a polyvinylidene difluoride membrane, according to the method described by Towbin et al (29). The membrane was incubated with anti-human angiotensins (Angt, Ang I, and Ang II) for 2 hours at room temperature. After extensive washing with PBS, the membrane was incubated with horseradish peroxidase–conjugated secondary antibody (anti-goat IgG antibody) for 1 hour. Blots were exposed to x-ray film, and bands were detected by an enhanced chemiluminescence technique using a Western blot ECL-Plus system (Amersham Pharmacia Biotech, Piscataway, NJ).

Reverse transcription–polymerase chain reaction (RT-PCR)

Fibroblasts derived from 6 dcSSc patients and 5 healthy controls were allowed to reach confluency in 100-mm dishes. The medium was changed to serum-free medium (catalog no. QBSF-51; Sigma-Aldrich) and cultured in RNAzol (Biotecx, Houston, TX) for 72 hours to collect total RNA. One microgram of total RNA from each sample was reverse-transcribed to complementary DNA using Superscript II (Invitrogen). Two microliters of each RT sample was then subjected to PCR in a volume of 50 μl containing 1.5 mM MgCl2, a pair of specific primers (final concentration 500 nM), and 2.5 units of Taq polymerase (Perkin Elmer, Norwalk, CT). Thirty-five cycles of PCR were performed, with denaturing at 94°C for 45 seconds, annealing at 56°C for 45 seconds, and extension at 72°C for 1 minute 30 seconds, using a GeneAmp 9600 PCR instrument (Perkin Elmer). The PCR products (10-μl aliquots) were subjected to electrophoresis on ethidium bromide–stained 2% agarose gels and visualized under ultraviolet light. Amplification of the same RNA with β-actin primers confirmed that equal amounts of RNA were reverse-transcribed.

The sequences of the specific primers were as follows: for Angt, 5′-ACTACAGCAGAAGGGTATGCGG-3′ (sense) and 5′-TTGGAGCAGGTATGAAGGTGGG-3′ (antisense); for AT1, 5′-GCATTAGACAGATGACGGCTGC-3′ (sense) and 5′-TCTTACGGGCATTGTTTTGGC-3′ (antisense); for AT2, 5′-AAATATGCCCAATGGTCAGC-3′ (sense) and 5′-AAGGAAGTGCCAGGTCAATG-3′ (antisense); for cathepsin D, 5′-CCTCGTTTGACATCCACT-3′ (sense) and 5′-AGGGTGGACACCTTCTCA-3′ (antisense); and for β-actin, 5′-AAGAGAGGCATCCTCACCCT-3′ (sense) and 5′-TACATGGCTGGGGTGTTGAA-3′ (antisense).

Production of type I procollagen and transforming growth factor β1 (TGFβ1) by cultured dermal fibroblasts from SSc patients and healthy controls

Dermal fibroblasts were derived from 6 patients with dcSSc who had high levels of Ang II and from 5 healthy control subjects. Cultured fibroblasts were prepared at a density of 4 × 104 cells/well of 24-well culture plates in DMEM plus 10% FBS. After culturing for 24 hours, media were removed, and the cells were cultured in serum-free medium (QBSF-51) containing various concentrations of Ang II, olmesartan, and PD 123,319 for 24, 48, 72, and 120 hours. Concentrations of type I procollagen and TGFβ1 in the fibroblast supernatants were measured by commercial enzyme-linked immunosorbent assay kits from Takara Shuzo (Otsu, Japan) and R&D Systems (Minneapolis, MN), respectively.

Statistical analysis

Data are presented as the mean ± SD. Student's paired t-test was used to analyze changes in Ang II levels in sera and culture supernatants. Analysis of variance was used to analyze differences between groups of SSc patients or healthy individuals. P values less than 0.05 were considered statistically significant.

RESULTS

Elevated serum levels of Ang II in patients with SSc.

As shown in Figure 1, serum Ang II levels were significantly higher in dcSSc patients than in normal controls (mean ± SD 38.4 ± 52.6 versus 8.0 ± 4.0 pg/ml; P < 0.02). There was no significant difference between lcSSc patients and normal controls. Notably, the dcSSc patient group comprised 2 subgroups with high and low concentrations of serum Ang II. The clinical features of the 11 dcSSc patients with serum Ang II concentrations that were higher than the mean values in the entire group of dcSSc patients are shown in Table 2. All of these patients were admitted to our hospital and treated with immunosuppressants (i.e., corticosteroids, cyclophosphamide). The systolic and diastolic blood pressure of these 11 dcSSc patients were within normal limits (systolic <140 mm Hg and diastolic <90 mm Hg) even though the circulating Ang II levels were elevated.

Figure 1.

Serum angiotensin II (Ang II) levels in diffuse cutaneous systemic sclerosis (dcSSc) patients, limited cutaneous SSc (lcSSc) patients, normal healthy control (NC) subjects, and rheumatoid arthritis (RA) patients. Concentrations of Ang II were determined by solid-phase immobilized-epitope immunoassay. NS = not significant.

Table 2. Clinical Features of the dcSSc Patients with High Serum Levels of Ang II*
PatientAge/sexComplicationsTreatmentCRP, mg/dlAng II, pg/mlMRSS
  • *

    Serum levels of angiotensin II (Ang II) were measured by solid-phase immobilized-epitope immunoassay. Patients with diffuse cutaneous systemic sclerosis (dcSSc) who had high levels of Ang II were admitted to the hospital and treated with prednisolone (Pred.; >40 mg/day) or cyclophosphamide (CYC; 500 mg/month intravenously or 25–50 mg/day orally). Levels of C-reactive protein (CRP) and the degree of skin thickening (determined according to the modified Rodnan skin thickness score [MRSS; maximum 51]) were determined at the time of hospital admission. ILD = interstitial lung disease; CM = cardiomyopathy; ER = esophageal reflux; PH = pulmonary hypertension; M = myositis.

dcSSc148/FILD, CM, ERPred., CYC0.217026
dcSSc248/FILD, CMPred., CYC0.39722
dcSSc357/FILD, ERPred.0.29927
dcSSc420/FILD, CM, ER, PHPred., CYC2.914028
dcSSc530/FILD, CMPred.4.123022
dcSSc629/FILD, M, ERPred.1.65020
dcSSc763/FILDCYC0.86526
dcSSc859/FILDCYC0.17834
dcSSc946/MILDCYC0.34622
dcSSc1052/FILDCYC4.34220
dcSSc1133/FILDPred.0.38926

To explore the effect of corticosteroid administration on the induction of Ang II, we measured serum concentrations of Ang II in 20 RA patients who were receiving prednisolone (Figure 1). Serum Ang II levels in the RA patients were similar to those in the healthy donors (12.1 ± 5.7 versus 8.0 ± 4.0 pg/ml).

Immunohistochemical staining for expression of angiotensins in cryostat sections of skin.

To clarify the mechanism inducing the elevation of circulating Ang II levels, we sought to determine whether increased serum Ang II levels reflected aberrant production of angiotensins (Angt, Ang I, and Ang II) in the fibrotic skin of dcSSc patients. We examined the accumulation of angiotensins by immunohistochemistry in skin derived from 3 dcSSc patients with high levels of serum Ang II, 2 dcSSc patients with low levels of serum Ang II, 2 lcSSc patients, and 3 normal controls.

Staining for angiotensins was detected consistently in skin sections from all dcSSc patients with high levels of serum Ang II, but not in skin sections from lcSSc patients or healthy controls. Very faint staining was detected in skin from dcSSc patients with low levels of Ang II. Representative results from 2 dcSSc patients with high serum Ang II levels, 1 dcSSc patient with a low serum Ang II level, 1 lcSSc patient, and 2 normal controls are shown in Figure 2. Immunostaining with anti–Ang II antibody was also positive in skin samples from all SSc patients (data not shown).

Figure 2.

Immunohistochemistry of angiotensins in skin sections from patients with dcSSc and lcSSc and from normal controls. Frozen skin sections on glass slides were incubated with antibody to human angiotensins (angiotensinogen, Ang I, and Ang II), and then incubated with biotin-labeled horse polyclonal anti-goat IgG antibody and streptavidin–peroxidase complex working solution. Positive signals (brown staining) were developed with diaminobenzidine substrate; hematoxylin was used as counterstain. Shown are results in patients dcSSc1 (A) and dcSSc2 (B), who had high serum levels of Ang II (Table 2), a dcSSc patient with low serum Ang II (C), a patient with lcSSc (D), and 2 normal control subjects (E and F). (Original magnification × 100.) See Figure 1 for definitions.

Expression of Angt and cathepsin D mRNA in cultured dermal fibroblasts.

We next examined the expression of Angt mRNA in cultured fibroblasts from 6 dcSSc patients (3 with high levels of serum Ang II and 3 with low levels) and 5 normal controls. Angt mRNA was constitutively expressed in all fibroblast lines derived from the 6 dcSSc patients, but the specific bands in 2 dcSSc patients were very weak (Figure 3). In contrast, Angt mRNA expression was not detected in the normal controls (Figure 3).

Figure 3.

Expression of mRNA for angiotensinogen (Angt), angiotensin II (Ang II) type 1 (AT1) and type 2 (AT2) receptors, and cathepsin D (CathD) in cultured dermal fibroblasts from 6 patients with diffuse cutaneous systemic sclerosis (dcSSc) and 5 healthy controls. One microgram of total RNA from each sample was reverse-transcribed to cDNA, and aliquots were subjected to 35 cycles of polymerase chain reaction (PCR). The PCR products (10-μl aliquots) were electrophoresed in ethidium bromide–stained 2% agarose gels and visualized under ultraviolet light. Amplification of the same RNA with β-actin primers confirmed that equal amounts of RNA were reverse-transcribed. Shown are results in normal controls (lanes 1–5), dcSSc patients with low serum levels of Ang II (lanes 6–8), and dcSSc patients with high serum levels of Ang II (lanes 9–11). Molecular weight markers are shown in lane M.

Cathepsin D is an aspartyl protease that cleaves Angt to form Ang I. We examined the expression of cathepsin D mRNA in cultured fibroblasts from the same subjects. Fibroblasts from all 6 dcSSc patients showed a strong band of cathepsin D mRNA (Figure 3). Fibroblasts from 3 of the normal subjects showed a weak band of cathepsin D, and those from the other 2 normal subjects did not show an obvious band with this PCR method (Figure 3).

Immunohistochemical staining for expression of angiotensins in cultured dermal fibroblasts.

To examine whether cultured fibroblasts express the proteins of angiotensins (Angt, Ang I, and Ang II), immunohistochemical studies using antibody against human angiotensins were performed in cultured fibroblasts derived from 3 dcSSc patients with high levels of serum Ang II, 3 dcSSc patients with low levels of serum Ang II, and 3 normal controls. Angiotensins were detected in cultured fibroblasts derived from the dcSSc patients with high levels of serum Ang II, but were not detected in either the normal controls or the dcSSc patients with low levels of serum Ang II. Representative results in the dcSSc patients with high levels of serum Ang II and in normal controls are shown in Figures 4A and 4B. In contrast, immunohistochemical staining using antibody specific for Ang II was negative in all dcSSc fibroblasts (data not shown).

Figure 4.

Immunohistochemistry of A and B, angiotensins (Angt, Ang I, and Ang II), C and D, AT1 receptors, and E and F, AT2 receptors in cultured dermal fibroblasts. Fibroblasts from 3 dcSSc patients with high levels of serum Ang II (A, C, and E) and 3 healthy donors (B, D, and F) were cultured in 4-chamber slides. Cells (80% confluency) were fixed with 2% paraformaldehyde in phosphate buffered saline, incubated with the 3 kinds of antibodies against angiotensins, AT1 receptor, and AT2 receptor, respectively, then incubated with biotin-labeled horse polyclonal anti-goat IgG antibody, and finally, incubated in streptavidin–peroxidase complex working solution. Positive signals (brown staining) were developed with diaminobenzidine substrate; hematoxylin was used as counterstain. (Magnification × 200.) See Figure 3 for definitions.

Western blot analysis of angiotensins in skin and cultured dermal fibroblasts.

To examine the molecular weights of angiotensins (Angt, Ang I, and Ang II) expressed in skin and cultured fibroblasts of dcSSc patients with high levels of serum Ang II, immunoblotting was performed using proteins extracted from the skin and cultured fibroblasts. We identified a specific immunoreactive band at ∼56 kd in both skin and cultured fibroblasts (Figure 5). Similar experiments were performed with cultured fibroblasts from dcSSc patients who had low levels of serum Ang II and from normal healthy donors. An immunoreactive band was not identified in cell lysates from any fibroblast lines from these subjects (data not shown).

Figure 5.

Western blotting of angiotensins (Angt, Ang I, and Ang II) in skin and cultured dermal fibroblast (Fb) from a patient with dcSSc and high serum levels of Ang II. Proteins from lysates of skin and cultured dermal fibroblasts were subjected to electrophoresis in a sodium dodecyl sulfate–12.5% polyacrylamide gel, transferred to a polyvinylidene difluoride membrane, and the membrane was immunoblotted with antibody against human angiotensins. See Figure 3 for other definitions.

AT1 and AT2 receptor mRNA expression and protein levels in cultured dermal fibroblasts.

An RT-PCR using specific primers for AT1 and AT2 was performed to detect mRNA expression in cultured fibroblasts. As shown in Figure 3, both AT1 and AT2 mRNA were constitutively expressed in SSc fibroblasts, whereas only AT1 mRNA was expressed in normal fibroblasts. Immunostaining for AT1 and AT2 was performed using cultured fibroblasts from 3 dcSSc patients with high levels of serum Ang II and 3 dcSSc patients with low levels, and 3 normal subjects. Both AT1 and AT2 were constitutively expressed in all lines of dcSSc fibroblasts irrespective of the levels of serum Ang II. In contrast, normal fibroblasts expressed only AT1, which was consistent with the RT-PCR results. Representative results are shown in Figures 4C–F.

Effect of exogenous Ang II on type I procollagen and TGFβ1 production in cultured dermal fibroblasts.

To assess the effect of exogenous Ang II on the production of extracellular matrix components in fibroblasts, levels of procollagen (type I procollagen C-peptide) were measured in cultured fibroblasts using serum-free media with Ang II. Procollagen levels were significantly higher in SSc fibroblasts than in normal fibroblasts that had been cultured for 72 hours without Ang II (mean ± SD 340.0 ± 99.6 ng/105 cells versus 204.0 ± 36.5 ng/105 cells; P < 0.05). Procollagen levels in culture supernatants of fibroblasts were measured at 24, 48, 72, and 120 hours after the addition of 10–6M Ang II. Procollagen production was gradually increased in a time-dependent manner, and reached a plateau at 72 hours (Figure 6A). Dose-response curves for Ang II–induced procollagen production showed an increase in procollagen for up to 72 hours in a dose-dependent manner in both SSc and normal fibroblasts (Figure 6B). Stimulation with >0.1 μM Ang II caused a significant increase in procollagen production (Figure 6B).

Figure 6.

Effect of angiotensin II (Ang II) on type I procollagen production in cultured dermal fibroblasts from 6 patients with diffuse cutaneous systemic sclerosis and high serum levels of Ang II (SSc) and from 5 normal controls (NC). Fibroblasts were cultured in 24-well culture plates in serum-free media containing various concentrations of Ang II. Procollagen in the culture supernatants was measured by enzyme-linked immunosorbent assay. A, Procollagen production by fibroblasts cultured with 1 μM Ang II for the times shown. B, Procollagen production by fibroblasts cultured for 72 hours with the concentrations of Ang II shown. = P < 0.05; ∗∗ = P < 0.01; ∗∗∗ = P < 0.001 versus levels without Ang II.

To explore the precise role of angiotensin receptors in procollagen production by fibroblasts, we examined the effects of specific AT1 and AT2 receptor antagonists on Ang II–induced procollagen production in fibroblasts. Dose-response curves for olmesartan, an AT1 receptor antagonist, indicated that at doses >0.1 μM, olmesartan exhibited an inhibitory effect on Ang II–induced procollagen production (Figure 7). Subsequent experiments were performed with 1 μM Ang II and 1 μM AT1 receptor antagonist incubated for 72 hours. In SSc and normal fibroblasts, increased production of procollagen by Ang II was abolished by the AT1 receptor antagonist (olmesartan at 1 μM), whereas the AT2 receptor antagonist (PD 123,319 at 10 μM) had no effect on procollagen production (Figure 8A). Interestingly, procollagen production in SSc fibroblasts that had been cultured in serum-free media containing both Ang II (1 μM) and AT1 receptor antagonist (1 μM) was significantly lower than spontaneous procollagen production in SSc fibroblasts (P < 0.05), but this was not seen in normal fibroblasts (Figure 8A).

Figure 7.

Inhibitory effect of olmesartan (OL), an angiotensin II (Ang II) type 1 (AT1) receptor antagonist, on procollagen production in cultured dermal fibroblasts from 3 patients with diffuse cutaneous systemic sclerosis and high serum levels of Ang II (SSc) and from 3 normal controls (NC). Fibroblasts were cultured in 24-well culture plates in serum-free media containing 1 μM Ang II and various concentrations of olmesartan. After 72 hours, the supernatants were collected, and procollagen concentrations were measured by enzyme-linked immunosorbent assay. Procollagen production was significantly elevated in Ang II–stimulated SSc and normal fibroblasts (P < 0.01 by Student's paired t-test). = P < 0.05; ∗∗ = P < 0.01 versus levels in the presence of 1 μM Ang II and in the absence of AT1.

Figure 8.

Functional analyses of angiotensin II (Ang II) type 1 (AT1) and type 2 (AT2) receptors on cultured dermal fibroblasts from 6 patients with diffuse cutaneous systemic sclerosis and high levels of serum Ang II (SSc) and 5 normal controls (NC). Fibroblasts were cultured in 24-well culture plates with serum-free media containing 1 μM Ang II, 1 μM olmesartan (OL; an AT1 receptor antagonist), and 10 μM PD 123,319 (PD; an AT2 receptor antagonist). Production of A, procollagen and B, transforming growth factor β (TGFβ) in the supernatants was measured by enzyme-linked immunosorbent assays. = P < 0.05; ∗∗ = P < 0.01; ∗∗∗ = P < 0.001 versus levels without stimuli.

TGFβ1 production was also significantly increased by the addition of Ang II (1 μM) in SSc and normal fibroblasts (Figure 8B). Moreover, our results revealed that Ang II–induced TGFβ1 production was mediated by the signal through the AT1 receptor, but that stimulation of both Ang II and AT1 receptor antagonist did not affect the basal levels of TGFβ1 by SSc and normal fibroblasts (Figure 8B).

DISCUSSION

The fact that a subset of patients with dcSSc had high serum levels of Ang II is one of the major new findings of this study. SSc patients with high serum levels of Ang II were those who were in the early stage of the disease (<1 year from initial symptoms), had diffuse cutaneous lesions, and had involvement of internal organs. These findings suggest that Ang II may be partly involved in the pathogenesis of dcSSc.

The conventional first step in the generation of circulating Ang II is the cleavage of Ang I from Angt by renin, a renal acid proteinase (11, 12). It is well established that plasma levels of renin are increased in SSc patients with scleroderma renal crisis (30). However, the plasma renin activity in the SSc patients with high serum Ang II levels was within normal limits (data not shown). We speculated that the increase in serum Ang II levels in dcSSc patients may result from an aberrant production of Angt and may not be associated with increased levels of Angt-cleaving enzymes. As we expected, our results showed that the production of angiotensins (Angt, Ang I, and Ang II) was up-regulated in skin and cultured dermal fibroblasts derived from dcSSc patients with high levels of serum Ang II. In contrast, angiotensins were very weakly expressed or were not expressed in skin and cultured dermal fibroblasts from dcSSc patients with low levels of serum Ang II or from normal donors.

Two antibodies, each recognizing a different epitope, were used to detect Angt, Ang I, and Ang II in the present study. Immunohistochemical and immunoblotting studies found that Angt (56 kd) and Ang II were located in the fibrous tissues of SSc skin, and that Angt was produced in cultured dermal fibroblasts from SSc patients. The mature form of Angt consists of 452 amino acids, and its calculated molecular weight is 50 kd (31). Angt contains at least 2 potential sites for N-linked glycosylation, and different glycosylation patterns are apparently responsible for size variations in the circulating Angt (31). It has been reported that human lung myofibroblasts produce 58-kd Angt (32) and that human adipose cells produce 61-kd Angt (33). We considered that Angt in the skin of SSc patients may be a slightly glycosylated form of ∼56 kd. Although no immunoreactive band corresponding to Ang II (∼1.0 kd) was observed in SSc skin tissues, a peptide of this small size could not be detected by our blotting technique. It is possible that increased levels of serum Ang II may be mainly attributable to overproduction of Angt in the fibroblasts of fibrotic skin.

The classic pathway of Ang II synthesis is regulated by circulating renin and ACE (11, 12). Several studies, however, have identified the alternative pathway of local Ang II formation in numerous tissues distinct from the liver or kidney, implying that various tissues have the ability to synthesize Ang II independently of circulating renin and ACE (33–38). In the pathway of tissue Ang II synthesis, several angiotensin-cleaving enzymes, as well as renin and ACE, have been identified; these enzymes are cathepsin D, cathepsin G, tonin, and chymase (33, 34, 39, 40). In tissues, cathepsin D and cathepsin G can cleave Ang I from Angt (similar to the action of renin), and tonin and chymase can cleave Ang II from Ang I (similar to the action of ACE). Increased Ang II levels may be associated with an increase in tissue levels of angiotensin-cleaving enzymes as well as Angt in the skin of dcSSc patients with high serum levels of Ang II. Expression of mRNA for renin, ACE, and cathepsin G was not detected by RT-PCR in cultured dermal fibroblasts from SSc patients and normal controls (data not shown), whereas cathepsin D mRNA was expressed constitutively. The expression of cathepsin D mRNA was markedly higher in SSc dermal fibroblasts than in normal dermal fibroblasts. Proteolytic cleavage of renin is a rate-limiting step in the generation of circulating Ang II, while cathepsin D in fibroblasts appears to play this role within connective tissues.

We further examined whether the formation of Ang II from Angt was generated in cultured dermal fibroblasts from SSc patients. The concentrations of Ang II in culture supernatants of SSc fibroblasts were below the limits of detection by the solid-phase immobilized-epitope immunoassay method we used (data not shown). Moreover, AT1 receptor antagonist did not affect basal levels of procollagen and TGFβ produced by SSc fibroblasts, indicating no autocrine effect of Ang II in SSc fibroblasts in vitro. These findings suggest that the formation of Ang II is not generated in cultured SSc fibroblasts in serum-free media. In contrast, we found that the formation of Ang II is generated in skin tissues in vivo, which strongly suggests that the expression of tissue ACE or tissue chymase as well as cathepsin D may be maintained in the skin of SSc patients. Thus, the local expression of Angt and angiotensin-cleaving enzymes may play a crucial role in Ang II formation in SSc.

The development of nonpeptide antagonists of Ang II receptors clearly showed the biologic distinctions between the 2 specific receptors AT1 and AT2 (41–45). Most of the known biologic effects of Ang II are mediated by the AT1 receptor subtype, which is a G-protein–linked receptor that activates protein kinase C through the formation of diacylglycerol and hydrolysis of phosphatidylinositol. Our findings indicate that dermal fibroblasts derived from healthy donors spontaneously express AT1 alone. The activation of AT1 by exogenous Ang II enhanced procollagen production in normal fibroblasts, indicating that Ang II exhibits profibrotic effects in human dermal fibroblasts. This phenomenon may be a common function of human fibroblasts, as previously reported in cardiac and renal fibroblasts (16–18).

This study represents the first demonstration that the AT2 receptor is spontaneously expressed in human dermal fibroblasts at the protein and transcription levels. AT2 receptor expression was not detected on the cell surface of normal dermal fibroblasts, but was present spontaneously on dermal fibroblasts derived from SSc patients. Many previous studies have shown that the activation of AT2 receptors inhibits the AT1 receptor–mediated functions of Ang II (46–49), although this phenomenon has been a somewhat controversial subject (50). Our experiments using selective AT1 and AT2 receptor antagonists indicated that the AT2 receptor–mediated signal of Ang II inhibited procollagen production in cultured SSc fibroblasts, which appeared to antagonize the AT1 receptor–mediated function. These findings indicate that collagen production by SSc fibroblasts could be attenuated as a result of AT2 receptor–mediated signal transduction. However, it is obvious from our observations and from previous reports (2, 3, 7) that SSc fibroblasts spontaneously produce higher amounts of collagen than do normal fibroblasts. This discrepancy might be explained by the differences between the biologic properties of AT1 and AT2 receptors (i.e., the numbers of receptors on the fibroblast; Ang II–binding affinity). Because activation of the AT2 receptor exhibited a biologic function against the effect of the AT1 receptor on procollagen production, we infer that overexpression of AT2 receptors could be a consequence of biologic feedback against the profibrotic effect mediated by spontaneous AT1 receptor activation in SSc fibroblasts in vivo.

Another important AT1 receptor–mediated event is the induction of TGFβ (51–53). Our findings revealed that AT1 receptor activation by Ang II induced TGFβ1 protein in cultured dermal fibroblasts. TGFβ stimulates gene expression of extracellular matrix components such as collagen and fibronectin (54). In the lesional skin of SSc patients, the expression of mRNA for TGFβ has been shown to be elevated, indicating an important role of TGFβ in the tissue fibrosis of SSc (55). Moreover, the response to TGFβ was recently shown to be enhanced in SSc fibroblasts compared with normal fibroblasts by the presence of increased receptors for TGFβ on SSc fibroblasts (56). Although TGFβ may be implicated in the pathogenesis of SSc, the mechanisms underlying the overproduction of TGFβ in vivo have yet to be clarified.

Because we found spontaneous AT1 receptor–mediated activation of fibroblasts in SSc, we speculated that increased TGFβ production in vivo may result from the aberrant expression of angiotensins and AT1 receptors in the skin of SSc patients, especially dermal fibroblasts. AT2 receptor activation in SSc dermal fibroblasts did not attenuate the basal levels of TGFβ1 production; this was different from the effect of AT2 receptor activation on procollagen production of SSc fibroblasts. These findings suggest different pathways of AT2 receptor signal transduction in procollagen and TGFβ1 production by SSc fibroblasts. In addition, Ang II has been shown to directly increase nuclear accumulation of phosphorylated Smad2 protein in cardiac fibroblasts, independent of TGFβ stimulation (57). Since Smad2 is an important nuclear factor in the transcription of the procollagen gene, it is possible that Ang II induces procollagen mRNA through the pathway for direct activation of Smad2 protein in dermal fibroblasts.

In conclusion, the present study revealed that Ang II is a key factor in the induction of tissue fibrosis in SSc, and that the formation of Ang II is attributed to the aberrant production of Angt by dermal fibroblasts in SSc patients. Moreover, we demonstrated that a selective AT1 receptor antagonism may be more effective in inhibiting collagen production in SSc fibroblasts than is the abolishment of Ang II formation because of the unique expression of AT2 receptors on SSc fibroblasts. Thus, administration of AT1 receptor antagonists may be a useful strategy for the treatment of tissue fibrosis in patients with dcSSc who have high levels of serum Ang II.

Acknowledgements

We gratefully thank Sankyo Pharmaceutical Company for the gift of olmesartan.

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