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Am J Physiol Heart Circ Physiol 294: H1675-H1684, 2008. First published February 22, 2008; doi:10.1152/ajpheart.91493.2007
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Activation of the intracellular renin-angiotensin system in cardiac fibroblasts by high glucose: role in extracellular matrix production

Vivek P. Singh, Kenneth M. Baker, and Rajesh Kumar

Division of Molecular Cardiology, Cardiovascular Research Institute, Texas A&M Health Science Center, College of Medicine, Scott & White and Central Texas Veterans Health Care System, Temple, Texas

Submitted 18 December 2007 ; accepted in final form 11 February 2008


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The occurrence of a functional intracellular renin-angiotensin system (RAS) has emerged as a new paradigm. Recently, we and others demonstrated intracellular synthesis of ANG II in cardiac myocytes and vascular smooth muscle cells that was dramatically stimulated in high glucose conditions. Cardiac fibroblasts significantly contribute to diabetes-induced diastolic dysfunction. The objective of the present study was to determine the existence of the intracellular RAS in cardiac fibroblasts and its role in extracellular matrix deposition. Neonatal rat ventricular fibroblasts were serum starved and exposed to isoproterenol or high glucose in the absence or presence of candesartan, which was used to prevent receptor-mediated uptake of ANG II. Under these conditions, an increase in ANG II levels in the cell lysate represented intracellular synthesis. Both isoproterenol and high glucose significantly increased intracellular ANG II levels. Confocal microscopy revealed perinuclear and nuclear distribution of intracellular ANG II. Consistent with intracellular synthesis, Western analysis showed increased intracellular levels of renin following stimulation with isoproterenol and high glucose. ANG II synthesis was catalyzed by renin and angiotensin-converting enzyme (ACE), but not chymase, as determined using specific inhibitors. High glucose resulted in increased transforming growth factor-β and collagen-1 synthesis by cardiac fibroblasts that was partially inhibited by candesartan but completely prevented by renin and ACE inhibitors. In conclusion, cardiac fibroblasts contain a functional intracellular RAS that participates in extracellular matrix formation in high glucose conditions, an observation that may be helpful in developing an appropriate therapeutic strategy in diabetic conditions.

angiotensin II; intracrine; hyperglycemia; renin; collagen; transforming growth factor-β

CARDIAC FIBROBLASTS CONSTITUTE one of the major cell types contributing to structural and functional properties of the heart (10). Cardiac fibroblasts produce extracellular matrix and secret growth factors and cytokines that are involved in the maintenance of myocardial function (23). There is a direct association between heart failure and cardiac fibroblast proliferation and deposition of extracellular matrix (8). Cardiac fibroblasts also interact dynamically with cardiac myocytes through mechanical, chemical, and electrical signals (5). ANG II is an important factor that induces cardiac myocyte hypertrophy, fibroblast proliferation, and collagen deposition. ANG II is produced by both types of cells and thus functions as an autocrine/paracrine factor in the heart (16, 28, 40).

Recently, we and others have described an intracrine role of ANG II in several cell types (3, 4, 13, 15, 20, 25). An intracrine mechanism supports the role of ANG II, acting from inside the cell, without binding to the plasma membrane AT1 receptor. For intracrine actions, ANG II should be either generated intracellularly or internalized by the cells. Local ANG II is presumed to be synthesized in the interstitial space from secreted angiotensinogen (AGT), through the actions of renin and angiotensin-converting enzyme (ACE). We recently reported that neonatal rat ventricular myocytes (NRVM) synthesize ANG II intracellularly; however, distribution of ANG II (intracellular or extracellular) is dependent on the nature of the activating stimulus (45). High glucose-induced ANG II is completely retained intracellularly and translocated to the nucleus, whereas isoproterenol-stimulated ANG II is largely secreted. The nature of the stimulus also determined the enzymes involved: chymase in high glucose- and ACE in isoproterenol-induced ANG II synthesis (45).

ANG II has a significant role in diabetes induced organ damage (12). In hyperglycemia, circulating ANG II levels are reduced, indicating that locally produced tissue ANG II is critically important (19). Intracellular retention of ANG II by cardiac myocytes, in high-glucose conditions, suggests that an intracrine mechanism might play a major role in diabetes-induced cardiac dysfunction (25). Because of ACE-independent synthesis and AT1-independent effects of intracellular ANG II, angiotensin receptor antagonists (ARBs) and ACE inhibitors would not block the intracellular renin-angiotensin system (RAS) of cardiac myocytes. Cardiac fibroblasts also express components of the RAS and contribute to the cardiac ANG II pool (40). However, the site of synthesis and distribution of ANG II in cardiac fibroblasts and the contribution of these cells to extracellular ANG II, for autocrine/paracrine actions, are not known. Given the therapeutic benefits of ARBs and ACE inhibitors, there is likelihood of cardiac fibroblasts being the major source of extracellular ANG II in diabetic conditions. In the case of intracellular synthesis of ANG II in cardiac fibroblasts, it would be important to determine the role, if any, of intracrine mechanisms in extracellular matrix deposition. In the present study, we demonstrate that cardiac fibroblasts synthesize ANG II intracellularly and extracellularly. Intracellular ANG II upregulates transforming growth factor-β (TGF-β) and collagen-1 production by cardiac fibroblasts. This study identifies significant differences in the regulation of the intracellular RAS by high glucose between cardiac fibroblasts and myocytes.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. All animal protocols were approved by the Institutional Animal Care and Use Committee of the Texas A&M Health Science Center. Neonatal rat ventricular fibroblasts were isolated from hearts of 0- to 2-day-old Sprague-Dawley rat pups by enzymatic dispersion of the ventricular tissue and differential plating, as previously described (40). Nonmyocytes were maintained for 2 days in DMEM-M199 3:1 medium containing 5.5 mM glucose and supplemented with 10% horse serum, 5% fetal calf serum, 0.1 mM ascorbic acid, 1 µg/ml transferrin, 10 ng/ml sodium selenite, 100 U/ml penicillin, and 100 µg/ml streptomycin. Confluent cells were passaged two times to yield cultures that were almost exclusively fibroblasts (>99% purity), as previously reported (27, 41). Culture medium was changed to serum free for 24 h, and cells were exposed to isoproterenol (10 µM) or high glucose (media containing 12.5, 17, and 25 mM glucose) for 24, 48, and 72 h. To correct for hyperosmolarity, we added equivalent amounts of mannitol to control sets of cells. To exclude internalization of ANG II through AT1 receptor-mediated endocytosis, we added candesartan (1 µM; AstraZeneca) to the culture medium 24 h before and during exposure to isoproterenol or high glucose.

ANG II measurement. ANG II was measured in the cell lysates and culture medium by quantitative, competitive ELISA, using a specific anti-ANG II antibody (Peninsula Labs), as previously described (3). Briefly, cells were washed with PBS, scraped in ice-cold 1 M acetic acid containing a protease inhibitor cocktail (Sigma), and lysed by sonication (29, 42). The lysate was sedimented at 20,000 g for 10 min, and the supernatant was dried in a vacufuge, followed by reconstitution in 1% acetic acid. The samples were applied to a conditioned DSC-18 column (Supelco), washed, and eluted with methanol. The eluted samples were dried and reconstituted in PBS for ELISA. For extracellular ANG II, culture medium was collected and protease inhibitor cocktail as added before filtration through Amicon Ultra-15 filters. The filtered medium was applied to DSC-18 columns, and ANG II was eluted as described for the cell lysate. Using this procedure, we have obtained >90% recovery of exogenously added ANG II. ELISA was performed on protein A- and anti-ANG II antibody-coated 96-well dishes. Competitive binding of synthetic biotinylated ANG II, in the presence of the extracted peptide, was detected with streptavidin-horseradish peroxidase conjugate. A standard curve, generated from binding of a constant amount of biotinylated ANG II with increasing concentrations of nonbiotinylated synthetic ANG II, was used to calculate the concentration of the peptide in the sample. The concentration of ANG II, in both the cell lysate and culture medium, is expressed per milligram of cellular protein. The specificity of the ANG II measurement by ELISA was validated by mass spectrometric analysis of immunoprecipitated ANG II from the cell lysates of NRVM and was reported previously (45).

Western analysis. On the basis of the time course of ANG II synthesis, cardiac fibroblasts were exposed to high glucose (25 mM) or isoproterenol (10 µM) for 24 h, as described above. Equal amounts of cell lysate or culture medium proteins (30 µg) were separated on 4–20% SDS-polyacrylamide gels and transferred to nitrocellulose membranes as previously described (45). Blots were probed with anti-AGT (Swant), anti-renin (Swant and Dr. Tadashi Inagami, Vanderbilt University), anti-chymase (Abcam), or anti-ACE-1 antibody (Abcam). Equal protein loading was confirmed by anti-GAPDH or anti-actin antibody for cell lysates and Coomassie staining for culture medium. Protein bands were detected using anti-mouse and anti-rabbit secondary antibodies labeled with infrared dye 680/800 and the Odyssey Infrared Imaging System (LI-COR Biosciences).

Enzyme inhibition. To identify the enzymes involved in ANG II synthesis, we added specific inhibitors to the culture medium at the time of exposure to isoproterenol or high glucose. The following inhibitors were used: aliskiren (renin inhibitor from Novartis, 1–50 µM), benazeprilat (ACE inhibitor, 0.1–10 µM), and chymostatin (chymase inhibitor, 1–100 µM).

Confocal immunofluorescence microscopy. Cardiac fibroblasts grown on two-well, collagen-coated chamber slides, at a density of 0.3 x 105 cells/well, were exposed to isoproterenol or high glucose, and immunostaining was performed as described previously (45). Specificity of the anti-ANG II antibody was confirmed by preadsorption with 10-fold molar excess of synthetic ANG II. Images were acquired through z-axis scanning with a x60 objective on a three-dimensional confocal fluorescence microscope (Olympus Fluoview 300) and analyzed by three-dimensional rendering using the Volocity 4 software package (Improvision). To study internalization of ANG II by cardiac fibroblasts, we added Alexa Fluor 488-labeled ANG II (50 nM; Molecular Probes) to the culture medium at the time of exposure to isoproterenol and high glucose. After 24 h, cells were washed with PBS and analyzed for the presence of labeled ANG II by confocal microscopy, as described previously (45).

TGF-β and collagen-1 analysis. Cardiac fibroblasts were grown in high glucose (25 mM)-containing medium in the absence or presence of candesartan (1 µM), aliskiren (10 µM), or benazeprilat (10 µM) for 24 h. In a control experiment, ANG II (100 nM) was added to the culture medium in the absence or presence of candesartan. TGF-β and collagen-1 were analyzed in cell lysates and culture medium by Western analysis, using anti-TGF-β (Santa Cruz) and anti-collagen-1 antibodies (Santa Cruz).

Statistical analysis. Values are means ± SE. ANOVA with Tukey's post hoc test was used for statistical analysis. P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Our recent demonstration of intracellular synthesis and intracrine effects of ANG II in cardiac myocytes has revealed a novel, intracellular aspect of the RAS that might have a significant role in cardiac pathophysiology (45). Cardiac fibroblasts represent the major nonmuscle cells in the heart, which synthesize and respond to ANG II, with significant implications in cardiac pathophysiology. In this report, we present evidence of an intracellular RAS in cardiac fibroblasts that differs significantly from that reported in cardiac myocytes.

To demonstrate de novo synthesis of ANG II, we grew cardiac fibroblasts in serum free medium and stimulated the RAS with high glucose or isoproterenol. The AT1 receptor antagonist candesartan was added to the culture medium to prevent cellular uptake of ANG II, which was confirmed by adding Alexa Fluor 488-labeled ANG II (see Supplemental Fig. 1S). (Supplemental data for this article is available online at the American Journal of Physiology-Heart and Circulatory Physiology website.) Under these conditions, an increase in ANG II levels in the cell lysate was considered to be due to intracellular synthesis. An increase in culture medium ANG II could be due to extracellular synthesis or release of intracellularly synthesized ANG II.

Isoproterenol and high glucose increase both intracellular and extracellular synthesis of ANG II. Exposure of cardiac fibroblasts to isoproterenol (10 µM) for 24 h resulted in an increase in ANG II levels in the cell lysate (41 ± 6%) and culture medium (139 ± 8%) (Fig. 1, A and B). Similarly, high glucose also increased ANG II levels in both cell lysate and the culture medium (59 ± 4 and 92 ± 4%, respectively) (Fig. 1, C and D). Significantly increased intracellular levels of ANG II were also observed in the presence of candesartan following exposure to isoproterenol and high glucose (73 ± 8 and 80 ± 7%, respectively), confirming intracellular synthesis rather than uptake. Mannitol, used as a control for osmolarity, did not have any significant effect on ANG II concentrations. The increase in extracellular ANG II in response to high glucose was in contrast to cardiac myocytes, where only intracellular levels were elevated (45).


Figure 1
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  		Fig. 1. Isoproterenol (Iso) and high glucose (HG) stimulate intra- and extracellular ANG II synthesis in neonatal rat ventricular fibroblasts. Cardiac fibroblasts were grown in serum-free normal glucose (NG) medium [5.5 mM glucose (Cont)] for 24 h and exposed to 10 µM Iso, 25 mM glucose (HG), or 5.5 mM glucose + 19.5 mM mannitol (NG + M) in the absence or presence of 1 µM candesartan. A quantitative competitive ELISA was used to measure ANG II in cell lysates (A and C) and culture medium (B and D). Values are means ± SE from 3 independent experiments performed in duplicate. *P < 0.05 vs. Cont (A and B) or NG (C and D).

 
Dose response and time course of high glucose-stimulated ANG II synthesis. To study high glucose-induced ANG II synthesis in more detail, we incubated cardiac fibroblasts in media with different glucose concentrations (5.5, 12.5, 17, and 25 mM) for 24 h. In control cells, mannitol was added to equalize osmolarity. The increase in ANG II synthesis reached significant levels with glucose concentrations of 25 mM (Fig. 2, A and B). A time course study showed a significant increase at 24 h following exposure to 25 mM glucose (Fig. 2, C and D), which declined steadily with longer incubation, likely due to slowing cellular metabolism in serum-free medium.


Figure 2
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  		Fig. 2. Time and dose dependency of HG-stimulated intracellular ANG II synthesis. A and B: cardiac fibroblasts grown in culture medium containing 5.5–25 mM glucose for 24 h. Mannitol was used as a control for hyperosmolarity. C and D: cardiac fibroblasts grown in 5.5 or 25 mM glucose medium for 12–72 h. ANG II was measured in cell lysates (A and C) and culture medium (B and D) using a quantitative competitive ELISA. Values are means ± SE from 3 separate experiments performed in duplicate. *P < 0.05 vs. mannitol (A and B) or NG (C and D).

 
Intracellular localization of ANG II in cardiac fibroblasts. To determine intracellular localization of ANG II, we stained cardiac fibroblasts grown in medium containing normal glucose (5.5 mM), high glucose (25 mM), or isoproterenol (10 µM) for 24 h with anti-ANG II antibody, followed by detection with confocal fluorescence microscopy. A punctate cytoplasmic staining was observed in both isoproterenol- and high glucose-stimulated cells (Fig. 3). Three-dimensional analysis using Volocity 4 software demonstrated significant perinuclear staining with small amounts of ANG II inside the nucleus.


Figure 3
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  		Fig. 3. Immunofluorocytometric localization of intracellular ANG II by confocal microscopy. Cardiac fibroblasts were grown in 5.5 mM glucose (NG; a–d) or 25 mM glucose medium (HG; i–l) or exposed to 10 µM Iso (e–h) in the presence of 1 µM candesartan for 24 h. Cells were costained for nuclei (blue; a, e, and i) and ANG II (green; b, f, and j). Cells were visualized using z-axis optical sectioning through a x60 objective in a confocal microscope. Images, shown in different channels, represent the same z-axis plane. Images in c, g, and k show ANG II and stained nuclei merged together. Images in d, h, and i show a 3-dimensional rendering (using the Volocity 4 software package) of the indicated cells in c, g, and k. Staining for ANG II is very faint in cells grown in 5.5 mM glucose and more intense in cells grown in 25 mM glucose or exposed to Iso. ANG II is mainly present in the perinuclear region with small amounts in the nucleus of cells exposed to HG. Identical ANG II staining was observed in the absence of candesartan. DAPI, 6-diamidino-2-phenylindole.

 
Increase in intracellular renin levels following exposure to isoproterenol and high glucose. Intracellular generation of ANG II would require the presence of RAS components intracellularly and regulation by the synthesis promoting stimuli. To determine the levels of AGT, renin, ACE, and chymase, we subjected cell lysate and the culture medium of cardiac fibroblasts to Western blot analysis using respective antibodies. Renin protein levels were significantly increased in both the cell lysate and culture medium following exposure to high glucose and isoproterenol (Figs. 4 and 5, respectively), corroborating the observed increase in ANG II levels. These results were confirmed using a second, well-characterized renin antibody obtained from Dr. Tadashi Inagami (data not shown). AGT levels were significantly increased only in the culture medium, whereas ACE and chymase levels in the cell lysate were not affected. The latter two enzymes were not detectable in the medium (data not shown). These results suggested that the renin-catalyzed rate-limiting step of AGT to ANG I conversion controlled intracellular generation of ANG II.


Figure 4
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  		Fig. 4. Western analysis of renin-angiotensin system (RAS) components in cardiac fibroblasts after exposure to HG. Cardiac fibroblasts were grown in 5.5 (NG) or 25 mM glucose medium (HG) for 24 h. Cell lysates (A, C, E, and F) and culture medium (B and D) were analyzed by Western blotting for changes in the level of angiotensinogen (AGT; A and B), renin (C and D), angiotensin-converting enzyme (ACE; E), and chymase (F) using specific antibodies. ACE and chymase were not detectable in the medium (not shown). Equal protein loading was confirmed by GAPDH or actin detection in cell lysates and by Coomassie staining of medium (not shown). Densitometric data were obtained from 3 independent experiments designated as set 1, set 2, and set 3. Blot image with the position of molecular weight markers is shown. Values are means ± SE. *P < 0.05 vs. NG.

 

Figure 5
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  		Fig. 5. Western analysis of RAS components in cardiac fibroblasts after exposure to Iso. Cardiac fibroblasts were exposed to 10 µM Iso for 24 h. Cell lysates (A, C, E, and F) and culture medium (B and D) were analyzed by Western blot for changes in the levels of AGT (A and B), renin (C and D), ACE (E), and chymase (F) using specific antibodies. ACE and chymase were not detectable in the medium (not shown). Equal protein loading was confirmed by GAPDH or actin detection in cell lysates and by Coomassie staining of medium (not shown). Densitometric data were obtained from 3 independent experiments designated as set 1, set 2, and set 3. Blot image with the position of molecular weight markers is shown. Values are means ± SE. *P < 0.05 vs. Cont.

 
Enzymes involved in cardiac fibroblast ANG II synthesis. To further elucidate the synthesis pathway, we measured ANG II levels in cells exposed to isoproterenol or high glucose in the presence of specific inhibitors of renin (aliskiren), ACE (benazeprilat), and chymase (chymostatin). Aliskiren, a very potent and specific inhibitor of human renin (IC50 = 0.6 nM) also has been shown to inhibit rat renin (IC50 = 80 nM) (52, 53). The inhibitors need to enter the cells to block intracellular ANG II synthesis. Cardiac myocytes effectively internalized aliskiren, resulting in intracellular concentrations about threefold higher than those in the medium 24 and 48 h following addition (data not shown). Benazeprilat is at the top of the relative rank order of tissue potency of the ACE inhibitors, suggesting efficient membrane permeability (17). Chymostatin was solubilized in DMSO, which enhances cell penetration of the drug (55). Thus the above inhibitors were suitable to study the enzymes involved in intracellular ANG II synthesis. As shown in Fig. 6, both isoproterenol- and high glucose-induced ANG II generation was completely inhibited by aliskiren and benazeprilat in the cell lysate as well as culture medium. Chymostatin did not have any effect on ANG II generation in cardiac fibroblasts (data not shown), unlike that in NRVM, where intracellular synthesis was catalyzed by chymase (45). The observed small effect of renin and ACE inhibitors on basal ANG II levels is likely due to the very low level of ANG II-forming activity in unstimulated cells.


Figure 6
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  		Fig. 6. Enzymes involved in Iso and HG-induced ANG II synthesis. Cardiac fibroblasts were exposed to Iso (A and B) or 25 mM glucose (C and D) in the presence of 50 µM aliskiren (a renin inhibitor) and 10 µM benazeprilat (an ACE inhibitor). ANG II was measured in cell lysates (A and C) and culture medium (B and D) using a quantitative competitive ELISA. Data from lower inhibitor concentrations that did not completely block the stimulated increase in ANG II synthesis are not shown. Values are means ± SE from 3 separate experiments performed in duplicate. *P < 0.05 vs. Cont (A and B) or NG (C and D) without inhibitor.

 
Intracellular ANG II enhances TGF-β and collagen-1 production in cardiac fibroblasts. Intracellular ANG II has biological effects in several cells, such as cardiac myocytes, vascular smooth muscle (VSMC), and Chinese hamster ovary cells (3, 4, 18). To determine whether the increase in intracellular ANG II levels has physiological significance in cardiac fibroblasts, we determined TGF-β and collagen-1 production in cells exposed to high glucose. To exclude the effects of extracellular ANG II, we added candesartan to the culture medium. To confirm that any change in TGF-β or collagen was due to increased intracellular ANG II and not to high glucose, we blocked ANG II synthesis with aliskiren and benazeprilat. As shown in Fig. 7, candesartan inhibited collagen-1 production only partially in both the cell lysate and culture medium. In a control experiment, extracellular ANG II-induced collagen-1 synthesis was completely inhibited by candesartan. In contrast, aliskiren and benazeprilat, which block ANG II generation both extra- and intracellularly, completely prevented high glucose-induced collagen-1 synthesis. A similar effect of these agents on TGF-β production was also observed (Fig. 8). These results demonstrate that intracellular ANG II is involved in TGF-β and collagen-I synthesis in cardiac fibroblasts. These observations reinforce the concept of only partial efficacy of angiotensin receptor antagonists in blocking the cardiac RAS.


Figure 7
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  		Fig. 7. Intracellular ANG II regulates HG-induced collagen-1 synthesis in cardiac fibroblasts. Cardiac fibroblasts were grown in 5.5 (NG) or 25 mM glucose medium (HG) for 24 h in the absence or presence of 1 µM candesartan, 50 µM aliskiren, or 10 µM benazeprilat (A and B). In a control experiment, cells grown in NG were exposed to 100 nM ANG II in the culture medium, in the absence or presence of 1 µM candesartan, for 24 h (C and D). Collagen-1 expression was measured by Western analysis in the cell lysate (A and C) and culture medium (B and D). Equal protein loading was confirmed by GAPDH detection in cell lysates and by Coomassie staining of medium (not shown). Densitometric data were obtained from 3 separate experiments. Values are means ± SE. *P < 0.05 vs. NG (A and B) or Cont (C and D). Representative blots of these experiments are presented as Supplemental Fig. 2S.

 

Figure 8
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  		Fig. 8. Intracellular ANG II regulates HG-induced transforming growth factor-β (TGF-β) expression in cardiac fibroblasts. Cardiac fibroblasts were grown in 5.5 (NG) or 25 mM glucose medium (HG) for 24 h in the absence or presence of 1 µM candesartan, 50 µM aliskiren, or 10 µM benazeprilat (A and B). In a control experiment, cells grown in NG were exposed to 100 nM ANG II in the culture medium, in the absence or presence of 1 µM candesartan, for 24 h (C and D). TGF-β expression was measured by Western analysis in the cell lysate (A and C) and culture medium (B and D). Equal protein loading was confirmed by GAPDH detection in cell lysates and by Coomassie staining of medium (not shown). Densitometric data were obtained from 3 separate experiments. Values are means ± SE. *P < 0.05 vs. NG (A and B) or Cont (C and D). Representative blots of these experiments are presented as Supplemental Fig. 3S.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present investigation, we have identified an intracellular RAS in neonatal cardiac fibroblasts. We have demonstrated intracellular synthesis, localization, and enzymatic pathways of ANG II generation. For the first time, we report involvement of intracellular ANG II in TGF-β and collagen-1 production by cardiac fibroblasts. Interestingly, the composition and regulation of the intracellular RAS in cardiac fibroblasts differs from cardiac myocytes, which may have significant pathophysiological implications.

An intracellular RAS is defined by the presence of all RAS components and generation of ANG II intracellularly, coupled with biological effects of ANG II from an intracellular location. Such a system seemed inconceivable based on known properties of conventional RAS components (AGT, renin, and ACE) that direct them to secretion or membrane localization. However, given the alternative ANG II-generating mechanisms (such as via cathepsin D and chymase) (21, 26, 31) and rerouting of AGT and renin to intracellular locations (before or after secretion) under certain conditions (30, 34, 43), the possibility of an intracellular RAS existed. Recently, we convincingly demonstrated intracellular ANG II synthesis, localization, and a synthetic pathway in NRVM (45). Earlier, we and others had reported that intracellular ANG II has multiple biological effects in several types of cells (3, 4, 13, 15, 20, 25). It is generally believed that the local RAS has a more significant role than the circulating system in cardiac pathophysiology (36). Significantly, it has been suggested that intracellular ANG II might be a major regulator of the local RAS (24, 37). Considering the pathophysiological significance of the intracellular RAS, as discussed in recent reviews (25, 35, 38), it was important to investigate it in major cell types.

Other than cardiac myocytes, fibroblasts are the major cells of heart that are RAS positive and involved in cardiac structure and function (5, 7, 40). Exposure of cardiac fibroblasts to isoproterenol and high glucose increased intracellular levels of renin, suggesting that enhanced intracellular ANG II production was likely due to activation of the rate-limiting step catalyzed by renin. The latter is supported by complete attenuation of de novo intracellular ANG II synthesis by aliskiren, a specific renin inhibitor. Although there is controversy about renin expression, we and other investigators have previously demonstrated both renin mRNA and protein in cardiac cells, including fibroblasts (6, 16, 28, 40). Similarly, benazeprilat completely blocked the increase in ANG II synthesis. The presence of ACE in the intracellular milieu and participation in ANG II generation had been earlier reported by others and us (14, 16). In addition, N-domain isoforms of ACE have been detected intracellularly, including in the nucleus, in renal mesangial cells. N-domain ACE colocalized with ANG II, suggesting participation in intracellular ANG II synthesis (9, 39). Whether one or both of the steps of AGT-to-ANG II conversion occurred in the secretory vesicles (due to cosorting of AGT, renin, and ACE) or in the cytosol remains to be determined. The significant observation is that at least a part of the newly synthesized ANG II is retained intracellularly and translocated to perinuclear and nuclear regions, where it is likely to have a functional role.

Recently, other investigators have reported intracellular synthesis of ANG II in VSMC and renal mesangial cells (26, 44, 50). From these and our studies, it appears that the intracellular RAS differs in composition and regulation in different cells. For example, high glucose-induced intracellular ANG II synthesis is catalyzed by renin and chymase in cardiac myocytes (45), renin and ACE in cardiac fibroblasts (this study), cathepsin D and chymase in VSMC (26), and renin and chymase or ACE in renal mesangial cells (44, 50). High glucose seems to be a major common factor for activation of the intracellular RAS in all cell types studied thus far, although other stimuli also result in intracellular ANG II synthesis, as we observed in cardiac myocytes and fibroblasts with isoproterenol. Furthermore, the nature of the stimulus determines the intracellular synthesis pathway in cardiac myocytes, with chymase being important with high glucose and ACE by sympathetic stimulation (45). However, cardiac fibroblasts utilize ACE for ANG I-to-ANG II conversion under both conditions. The involvement of ACE or chymase might be determined by the site of ANG II synthesis, i.e., secretory vesicles or other cytosolic compartments, which may be further influenced differently in various cells, by different stimuli. High glucose appears to translocate ANG II precursors from the secretory apparatus to other cytosolic sites in cardiac myocytes, as was evident by an observed lack of increase in AGT and renin in the extracellular medium, although not in fibroblasts (45).

The mechanism of activation and differential translocation of RAS components in cardiac myocytes and fibroblasts might be related to differences in the metabolism of glucose by these two cell types. In cardiac myocytes, excessive glucose leads to overactivity of the hexosamine biosynthesis and polyol pathways, which influence protein glycosylation and intracellular calcium, both of which determine transport of proteins inside and outside of the cells (11, 32, 33). The metabolism of glucose in cardiac fibroblasts, under hyperglycemic conditions, has not been investigated.

An important component of the pathological alterations observed in diabetic cardiomyopathy is the accumulation of extracellular matrix (ECM) proteins, in particular collagens (2, 49). Most of the understanding of collagen production by high glucose comes from studies on ECM-producing cells in the kidneys. In cultured human mesangial cells, a high glucose-induced increase in TGF-β1, fibronectin, and type IV collagen were partially blocked by candesartan, suggesting involvement of ANG II (22). A few reports on an increase in collagen synthesis and TGF-β1 expression by high glucose in cardiac fibroblasts have been published; however, the mechanism(s) is (are) not known (1, 46). Endothelin-1 was not involved, and treatment with antioxidant vitamin E did not have any effect on ECM accumulation. However, high glucose-induced upregulation of AT1 receptor was attributed to an increase in collagen synthesis in cultured neonatal cardiac fibroblasts (1, 46). In the present study, we observed that inhibition of high glucose-induced extracellular ANG II by the AT1 antagonist candesartan only partially blocked collagen-1 and TGF-β expression, whereas inhibition of ANG II synthesis completely prevented the high glucose effects. These observations suggest that intracellular ANG II, in addition to extracellular ANG II, mediates high glucose-induced matrix deposition by cardiac fibroblasts. The latter observations also suggest that inhibitors of ANG II synthesis may provide better therapeutic benefits than AT1 antagonists in the treatment of diabetic cardiac fibrosis. In fact, it could be inferred from a recent meta-analysis of clinical trials that the cardiovascular events that are largely blood pressure-dependent, such as stroke, are benefited similarly by ACE inhibitors and ARBs; however, the events that involve tissue remodeling, such as coronary heart disease, may benefit more from an ACE inhibitor than ARBs (48).

Clinical perspective. With an increasing prevalence of diabetes, it is imperative to enhance our understanding of the pathology of myocardial fibrosis to develop better pharmacological interventions. Inhibition of the RAS by ARBs and ACE inhibitors has provided significant therapeutic benefits; however, pharmacological outcomes may be even more impressive from complete inhibition of the RAS (47, 51). The difference could be related to the intracellular RAS, which may not be amenable to current therapies, as depicted in Fig. 9. Cardiac myocytes and fibroblasts respond differently to high-glucose conditions, with the former robustly activating only the intracellular RAS, whereas the latter contributes to both intra- and extracellular ANG II generation. ARBs only inhibit effects of extracellular ANG II, as is evident from this and our previous studies (3, 4), and ACE inhibitors would block ANG II synthesis only in cardiac fibroblasts. These observations suggest that ACE inhibitors or ARBs, when used alone, might not inhibit the cardiac RAS completely in diabetic conditions. The beneficial effects of ACE inhibition in AT1a knockout mice on left ventricular remodeling following myocardial infarction may be related to inhibition of intracellular ANG II synthesis in cardiac fibroblasts (54). The current study provides mechanistic data on the cardiac RAS at the cellular level and may lead to more appropriate therapeutic regimens in the treatment of cardiac diseases.


Figure 9
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  		Fig. 9. Schematic representation of cardiac intracellular RAS in hyperglycemia and putative clinical relevance. Cardiac myocytes and fibroblasts respond to hyperglycemic conditions by activating the local RAS. Although the cardiac myocyte response results in a robust increase in intracellular ANG II, fibroblasts contribute to both intra- and extracellular ANG II. Intracellular ANG II synthesis in cardiac myocytes is both renin and chymase dependent, whereas it is catalyzed by renin and ACE in fibroblasts. Intracellular ANG II causes cellular hypertrophy and increases gene expression in cardiac myocytes and results in TGF-β expression and collagen synthesis in fibroblasts. The actions of intracellular ANG II are not blocked by angiotensin receptor antagonists (ARBs). These observations suggest that ARBs would only block the effects of extracellular ANG II produced by cardiac fibroblasts, whereas ACE inhibitors would block ANG II synthesis by fibroblasts. Intracellular ANG II in cardiac myocytes would not be amenable to any of these agents, suggesting only partial efficacy of these drugs in hyperglycemic conditions. A renin inhibitor may provide better therapeutic benefits in conditions where the intracellular RAS is activated.

 

   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by an American Heart Association, Texas Affiliate, Beginning Grant-in-Aid (to R. Kumar).


   ACKNOWLEDGMENTS
 
We thank Texas A&M Health Science Center Microscopy Imaging Center for providing confocal microscopy services.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. Kumar, Cardiovascular Research Institute, Texas A&M HSC, College of Medicine, 1901 South 1st St., Bldg. 205, Temple, TX 76504 (e-mail: kumar{at}medicine.tamhsc.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.


   REFERENCES
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 DISCUSSION
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