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Cardiovascular Research 2002 53(2):304-312; doi:10.1016/S0008-6363(01)00448-5
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Copyright © 2001, European Society of Cardiology

Is angiotensin II a proliferative factor of cardiac fibroblasts?

Fatiha Bouzegrhane and Gaétan Thibault*

Laboratory of Cell Biology of Hypertension, Multidisciplinary Research Group in Hypertension of the Canadian Institutes of Health Research, Institut de recherches cliniques de Montréal (IRCM) and Université de Montréal, 110 Pine Avenue West, Montréal, Québec, Canada H2W 1R7

* Corresponding author. Tel.: +1-514-987-5613; fax: +1-514-987-5585 thibaug{at}ircm.qc.ca

Received 15 June 2001; accepted 24 August 2001


   Abstract
Top
 Abstract
1. Introduction
 2. In vivo effects...
 3. In vitro proliferation...
 4. How different are...
 5. Ang II augments...
 6. Ang II and...
 7. Is the cell...
 8. Conclusion
 References
 
Angiotensin II has been implicated as an important factor in cardiac remodeling, particularly in the development of pathological left ventricular hypertrophy. It is generally assumed that angiotensin II is able to alter the phenotype of cardiac myocytes and fibroblasts, and several experiments have suggested that this peptide can particularly affect the proliferation of cardiac fibroblasts. However, a review of the published results indicates that there is no evidence that angiotensin II can directly trigger mitogenesis through activation of the cyclin-dependent pathway. The observed proliferative effect might well be caused by stimulation of the synthesis of growth or inflammatory substances like platelet-derived growth factor and cytokines, by integrin activation due to secreted extracellular matrix proteins, or by a combination of these mechanisms. Angiotensin II thus appears to differentiate cardiac fibroblasts into a growth substance-secreting phenotype.

KEYWORDS Angiotensin; Connective tissue; Fibrosis; Growth factors; Remodeling


   1. Introduction
Top
 Abstract
 1. Introduction
2. In vivo effects...
 3. In vitro proliferation...
 4. How different are...
 5. Ang II augments...
 6. Ang II and...
 7. Is the cell...
 8. Conclusion
 References
 
Increased hemodynamic workload is well known to alter the structure and muscle content of the heart and blood vessels [1,2]. While this response is sometimes considered a normal adaptive and defense mechanism to decrease wall stress and maintain cardiac function, it may also result under pathological conditions in increased myocardial stiffness and ventricular diastolic dysfunction leading to chronic heart failure [3].

This structural impairment is characterized by two remodeling events: an increment of the myocardial mass with or without a disproportionate accumulation of fibrous tissue [4], which involve two major cell types of ventricular tissue. Cardiomyocytes, forming the contractile component of the myocardium, become hypertrophied increasing the cardiac mass. These cells may eventually die by necrosis or apoptosis [5]. The majority of the non-myocyte interstitial fraction, cardiac fibroblasts, present exaggerated growth and cause structural alteration of the cardiac interstitium [6].

The disproportionate involvement of the non-myocytes in the accumulation of extracellular matrix (ECM) is not univocally accompanied by myocardium hypertrophy [4,7]. In fact, fibrosis often appears in association either with the hypertrophied left ventricle, as seen with suprarenal aortic occlusion and unilateral renal ischemia, or with the hypertrophied right ventricle, as observed in pulmonary arterial banding. On the other hand, fibrosis does not develop in the hypertrophied ventricles following chronic volume overload, caused by anemia, compensated arteriovenous fistula, hyperthyroidism, exercise training and atrial septal defect, or pressure overload with infrarenal aortic banding. The observed diversity of response in these models indicates that specific stimuli are sometimes influencing the growth of cardiomyocytes and fibroblasts and the development of fibrosis in an independent fashion.

During cardiac remodeling, cardiac fibroblasts can undergo three important, but not necessarily associated, phenotypic changes: (1) they assume a myofibroblast character by expressing smooth muscle {alpha}-actin and containing contractile stress fibers [8], (2) they proliferate, and (3) they produce ECM components, including fibronectin, laminin and collagen I and III in the interstitium and around blood vessels [9]. As a consequence, fibrous material deposition, called reactive fibrosis, alters the highly organized matrix and the interconnections between cardiac myocytes and neighboring capillaries, impairing myocyte contractility, oxygenation, and metabolism [4].

Cardiac myocyte phenotypic changes appear more closely related to ventricular loading, while the growth of cardiac fibroblasts appears to be regulated by several autocrine, paracrine, endocrine and mechanical factors [10–12]. The renin–angiotensin system (RAS) and its peptide product, angiotensin II (Ang II) acting through the angiotensin type 1 (AT1) receptor, have emerged as potent contributors of cardiac remodeling. Several results from the literature support a potential role of Ang II in cardiac myocyte and fibroblast growth, ECM protein production and adhesion to matrix proteins [13] (Fig. 1A). To date, remodeling has been largely ascribed to proliferation of non-myocytes. However, re-examination of the data indicates that the proliferative effects of Ang II on cardiac fibroblasts may not be as strong as initially suggested. In this review, we will firstly summarize results that were obtained in whole animals, suggesting that Ang II may potentially cause proliferation of fibroblasts in the myocardium. Secondly, we will review the in vitro experiments showing Ang II-induced fibroblast proliferation and discuss the points that Ang II may not fulfil the criteria of a true growth factor.


Figure 1
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  		Fig. 1 Schematic representation of the cellular effects of Ang II on cardiac fibroblasts. (A) Actual hypothesis. There is a direct link between activation of the AT1 receptor by Ang II and growth and proliferation of cardiac fibroblasts. (B) Revised hypothesis. Proliferation of cardiac fibroblasts is caused by Ang II-induced secretion of ECM proteins, growth factors and cytokines that then act on their respective receptors to activate the cyclin-CDK pathway. Transactivation of the growth factor receptors by Ang II is also mentioned as a possibility. CDK, cyclin-dependent kinase.

 

   2. In vivo effects of Ang II on cardiac fibroblast proliferation
Top
 Abstract
 1. Introduction
 2. In vivo effects...
3. In vitro proliferation...
 4. How different are...
 5. Ang II augments...
 6. Ang II and...
 7. Is the cell...
 8. Conclusion
 References
 
In vivo studies are essential to define the pathophysiological role of Ang II. However, there is little proof supporting a direct role of the growth effect of Ang II in experimental models of cardiac hypertrophy, compared to the number of in vitro studies. The main reason for this is that it is almost impossible to generate a model with physiological experimental conditions in which manipulation of the RAS would not induce a concomitant change in blood pressure. It is necessary to dissociate indirect effects, such as blood pressure and myocardium stretch, from direct effects of the peptide on cardiac cells in the development of hypertension-induced cardiac hypertrophy. Thus, precautions must be taken for correct interpretation of the results. Nevertheless, in vivo studies coupled with numerous in vitro findings, reported so far, represent substantial progress in elaborating the molecular and cellular mechanisms of Ang II-mediated cardiovascular diseases.

Ang II infusion in rats has been shown to induce cardiac hypertrophy via the AT1 receptor independently of its blood pressure-elevating effect. Chronic Ang II infusion (600–1000 ng/kg per min i.p.) in adult rats induces myocyte injury and, subsequently, wound healing [14]. In response to this injury, there is cardiac fibroblast reparative proliferation that peaks on day 2 of Ang II infusion, before any significant increase in blood pressure. In the same study, similar results were observed in a rat model of renovascular hypertension, in which endogenous stimulation of Ang II production occurred in the ischemic kidney by comparison with sham-operated rats or with hypertensive rats with infrarenal aortic constriction and no renal ischemia. The Ang II-stimulated DNA synthesis reflected a wound-healing process defined as fibroblast proliferation and replacement fibrosis of myocytolysis. Furthermore, in the renovascular hypertension rat model, both myocyte injury and fibroplasia were prevented with captopril, an angiotensin-converting enzyme (ACE) inhibitor, suggesting that Ang II can act directly on cardiac cells. Indeed, myocytolysis-stimulated fibroblast proliferation and collagen deposition correspond to a reparative process: myocytes are replaced by non-contractile cells [15].

In another study, chronic infusion of Ang II (520 ng/kg per min s.c.) induces at day 3 a significant rise in blood pressure together with an acute fibrotic response within the heart. Ang II stimulates fibronectin, collagen type I and IV expression in association with the proliferation of interstitial fibroblasts by a direct action that could be prevented by losartan, an AT1 receptor antagonist [16].

Sun and Weber [17] showed that ACE was colocalized in cardiac infarcted scars with myofibroblasts, suggesting that local production of Ang II be involved in tissue repair. Recently, Higaki et al. [18] reported that in vivo transfection of a human ACE transgene in the rat myocardium resulted in a significant increase in cardiac ACE activity together with myocyte hypertrophy and increased collagen content. This indicates that, independently of systemic and hemodynamic factors, autocrine/paracrine Ang II can directly cause hypertrophy and fibrosis. In addition, a growing body of evidence from pharmacological investigations and clinical studies on the effects of ACE inhibitors supports the notion that Ang II may act as an endogenous growth factor for the myocardium. However, ACE inhibitors can also inhibit kinin and bradykinin catabolism. Therefore, some effects of these drugs might not have been solely due to Ang II inhibition [19].

Ang II may differentially regulate cell proliferation in the myocardium. McEvans et al. [20] assessed 5-bromo-2'-deoxy-uridine (BrdU) incorporation in the heart after a 2-week treatment with Ang II infusion (200 ng/kg per min). Concomitantly with a substantial elevation in blood pressure, proliferation was increased within medial vascular smooth muscle cells (VSMC) and in associated adventitial/interstitial fibroblasts of intramyocardial coronary blood vessels of the atria and ventricles. In contrast, proliferation of arteriolar endothelial cell was decreased. Concomitant treatment with losartan prevented the Ang II-induced fibroblast proliferation, implying participation of the AT1 receptor.

To further characterize the mode of action of Ang II in cardiac remodeling, an AT1 receptor chimeric mouse was generated by mixing embryonic stem cells harboring or not the AT1A receptor gene, utilizing the ROSA26 transgenic mouse strain [21]. In response to a chronic hypertensive Ang II infusion (1000 ng/kg per min s.c.), extensive proliferation of fibroblasts and fibrosis was observed, mostly in areas of the myocardium expressing the AT1 receptor. The authors concluded that the effects of Ang II were mediated both by systemic and local actions. The latter effect was mediated by the AT1 receptor on myocytes, implying that communication between cardiac myocytes and fibroblasts plays an important role in the process of Ang II-induced cardiac remodeling.

Campbell et al. [22] reported temporal changes in fibroblast proliferation in vivo, using proliferating cell nuclear antigen S-phase as a marker of cell proliferation. They demonstrated a direct proliferative effect of Ang II in two experimental models where increased circulating Ang II level was achieved by chronic Ang II infusion (150 ng/kg per min s.c.) or by unilateral renal ischemia. Clusters of proliferating cardiac fibroblasts were localized between days 2 and 4 at sites of myocyte necrosis and around the vessel walls. In comparison, this study also revealed that chronic aldosterone administration elicited its proliferative response until week 4. The authors concluded that Ang II and aldosterone induce cell proliferation and, ultimately, fibrosis by separate mechanisms. Interestingly in this study, the myocardium was also infiltrated by macrophages and lymphocytes in a time course similar to that of fibroblast proliferation, suggesting that cytokines may also participate in the reparative fibrotic process.

In that context, many in vivo studies reported an inflammatory response that operates both systemically and locally prior to fibroblast proliferation and subsequent perivascular fibrosis that may contribute to myocardial cell death and necrosis. This response can be considered as a defense reaction by the fact that inflammatory cytokines attract and activate fibroblasts, stimulate cell growth and proliferation and, thus, promote repair and healing. Myofibroblasts also play an important role in the inflammatory response, through secretion of cytokines and chemokines, and in wound repair leading to local fibrosis through production of ECM proteins such as collagen, glycoaminoglycans, tenascin and fibronectin [23]. In addition, Ang II itself can participate in several steps of the immune and inflammatory responses: it is a potent mediator of oxidative stress, it stimulates release of cytokines and growth-regulatory factors by mononuclear cells and regulates the recruitment of proinflammatory cells into the site of injury [24,25]. The inflammatory cells can also release enzymes, including ACE and chymases, that generate Ang II thus creating a positive feedback mechanism for local Ang II generation.

In light of the in vivo investigations, the peripheral effects (elevation of blood pressure) are often a constant of Ang II infusion. Consequently the mechanical actions of Ang II can hardly be dissociated from a direct cellular mitogenic induction or from indirect growth-promoting secreted substances. Unresolved questions and controversies concerning the mechanisms by which Ang II exerts its effects on cardiac fibroblasts still remain.


   3. In vitro proliferation of cardiac fibroblasts by Ang II: neonatal versus adult cell
Top
 Abstract
 1. Introduction
 2. In vivo effects...
 3. In vitro proliferation...
4. How different are...
 5. Ang II augments...
 6. Ang II and...
 7. Is the cell...
 8. Conclusion
 References
 
Several results from the literature, reviewed in the previous section, indicate that there is a close relationship between differentiation and proliferation of mesenchymal cells in the myocardium and Ang II. They suggest that Ang II may act as a potent stimulator of fibroblast proliferation.

However, because in vivo experiments do not allow direct association between a causal agent and its consequence, many investigators have turned to in vitro models. For this purpose, cultures of cardiac fibroblasts were established. These cultures are relatively easy to prepare from the myocardium and grow free of contaminating cells. Two types of cardiac fibroblast cultures can be prepared, either from newborn or from adult animals. In the case of newborn animals, the heart ventricle is dissected and digested by a collagenase–trypsin mixture. At this stage of life, cardiac myocytes are still relatively undifferentiated, compared to their adult counterparts, and are fairly resistant to protease digestion. As a consequence, the digested myocardium of neonatal animals mainly contains a mixture of cardiac myocytes and fibroblasts, along with additional cells including VSMC, endothelial cells, neuronal cells and macrophages. A pre-plating step on plastic culture dishes, usually repeated twice, allows the attachment of fast adhering cells like fibroblasts and enriches the non-adherent preparation in cardiac myocytes. Pre-plated cells contain more than 90% neonatal cardiac fibroblasts. If greater purity is needed, it can be obtained by passaging the cells 1–3 times since cardiac myocytes will not tolerate this treatment. The reader is referred to the literature cited in Table 1 for further methodological details.


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  		Table 1 Effects of Ang II stimulation (10–10-10–6 mol/l) on cultured cardiac fibroblasts from newborn rats

 
During the preparation of cardiac fibroblasts from the adult heart, cardiomyocytes, which become Ca2+-intolerant in adulthood, will be lysed by the digestion process. As a consequence, the digestion mixture does not contain any viable cardiomyocytes and is plated directly on plastic culture dishes (1 heart/1500 cm2). Within 4 days, the cell culture is subconfluent with the cells occupying 60–90% of the plastic surface. Under these conditions, the adult fibroblast culture is greater than 95% pure. Occasionally, some isolated islets of endothelial cells or some nerve cells can be observed. As with neonatal cells, one or two passages can remove any contaminating cells.

These two cardiac fibroblast culture models have been used by several investigators to examine whether or not Ang II is able to induce cell proliferation. Among the two models, fibroblast culture from hearts of newborn rats was used most often (Table 1). Usually, rats were 1–3 days old, and fibroblasts were not passaged more than fourfold. Either subconfluent or confluent cultures were used. All cultures were first submitted for 24–48 h to a low-serum medium to synchronize the cells in the G0 phase of the cell cycle. The cells were stimulated with Ang II concentration between 10–8 and 10–6 mol/l for a period of 24–48 h. [3H]Thymidine incorporation was the most frequently-used assay assessing cell proliferation indirectly. But, as already discussed by Boulton and Hodgson [26], the incorporation of labeled thymidine, although rapid, may not be totally specific, and may generate false positive results. Incorporation of BrdU, measurement of DNA content and mitochondrial metabolic transformation of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) will also measure the extent of DNA synthesis or cell viability, which may not perfectly correlate with cell division and thus remain crude proliferative indices. Cell count is probably the most reliable assay that will present an exact picture of cell division in the dishes, although this technique is more laborious and less sensitive. In most of the recorded studies (14 out of 17), Ang II demonstrates its ability to increase the proliferation of cardiac fibroblasts of newborn rats, generally by a factor of 20–40% in a period of 24–48 h.

Fewer proliferation experiments have been carried out on adult cardiac fibroblasts (Table 2). Cultured fibroblasts were generated from 7- to 10-week-old rats and were used between the 1st and the 10th passage. Numerous successive passages of primary cultured cardiac fibroblasts may selectively modify their phenotypes and consequently affect their responses. All cultures were deprived of serum for at least 24 h before stimulation. The fibroblasts were incubated with 10–11–10–3 mol/l Ang II for a period varying from 24 h to 9 days. Four studies out of six could not detect any increase in proliferation as investigated by [3H]thymidine incorporation or cell count. However, the state of cell density seems to greatly affect the proliferative response of adult fibroblasts to Ang II. In this regard, the article of Simm and Diez [27] is particularly interesting. These authors observed that confluent cultures (above 1x105 cells/cm2) of rat adult cardiac fibroblasts did not respond at all to 10–7 mol/l Ang II, whereas proliferation can occur with lower density cultures (below 2x104 cells/cm2). However, this positive response took at least 40 h of incubation to be recorded. Even more interesting, this growth response can be completely blocked by the addition of anti-platelet-derived growth factor (PDGF)-AA antibodies. As concluded by these authors, the Ang II-dependent cell proliferation of adult cardiac fibroblasts was mainly due to cell density-dependent expression of PDGF-AA. Cell–cell contact is a characteristic of high density cultures and contact inhibition may result in failure to induce response.


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  		Table 2 Effects of Ang II stimulation (10–11-10–3 mol/l) on cultured cardiac fibroblasts from adult rats

 
Cultures of cardiac fibroblasts from adult human and rabbit myocardia have also been used to verify the proliferative action of Ang II. As indicated in Table 3, all studies, except 1, found that Ang II had a positive proliferative effect. In three cases, incubation was longer than 48 h, suggesting the autocrine/paracrine action of an unidentified growth factor. Mutagenic substances are expected to induce DNA replication within a period shorter than 24 h after their addition [28]. In addition, it must also be specified that human and rabbit cardiac fibroblasts seem to possess an atypical angiotensin receptor that, in some cases, could not be blocked with the AT1 antagonist losartan and could be stimulated by angiotensin IV [29–31].


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  		Table 3 Effects of Ang II stimulation (10–10–10–6 mol/l) on cultured cardiac fibroblasts from other species

 
The results presented in Table 1 suggest that Ang II possesses some mitogenic action and induces proliferation of neonatal cardiac fibroblasts mainly through the AT1 receptor. However, no experiments have been performed on these cells to test whether Ang II can also stimulate the synthesis of a proliferative factor that can act through autocrine/paracrine mechanisms. In contrast, Ang II-induced proliferation of adult cardiac fibroblasts seems to be more questionable. Although in vivo experiments in adult animal models, with either AT1 antagonists or ACE inhibitors, suggest a role for Ang II in the growth of these cells, in vitro experiments do not seem to supply evidence for such a role. Ang II can effectively increase the synthesis of pro-fibrotic substances, like collagen and transforming growth factor (TGF)-β1, in adult cardiac fibroblasts (Table 2). It can also partially differentiate these cells into myofibroblasts as evaluated by an increased synthesis of integrins, actinin, actin and its organization into myofilaments [32,33]. But there is no evidence that the peptide has a direct action on the cell cycle. This, however, does not exclude the possibility that Ang II may induce a growth factor, which will then turn on the cell division machinery, as described by Simm and Diez [27].


   4. How different are neonatal from adult cardiac fibroblasts?
Top
 Abstract
 1. Introduction
 2. In vivo effects...
 3. In vitro proliferation...
 4. How different are...
5. Ang II augments...
 6. Ang II and...
 7. Is the cell...
 8. Conclusion
 References
 
For reasons of simplicity and reproducibility, the culture of myocytes from newborn rat hearts was, and is still, used as a model of choice to examine the behavior of these cells under different experimental conditions. Isolation of neonatal cardiomyocytes requires two pre-plating steps to remove cardiac fibroblasts. Such fibroblasts thus represent a byproduct of cardiomyocyte preparation, and have been used as a convenient source of cardiac fibroblasts for in vitro experiments.

However, it is now well-documented that neonatal cardiac myocytes are clearly distinct from adult myocytes. Neonatal cardiomyocytes represent partially differentiated cells. After birth and within 1–2 weeks, neonatal cardiomyocytes progressively lose the expression of so-called ‘fetal genes’ and adopt an adult phenotype [34]. Accordingly, the response of neonatal cardiac myocytes to any given stimulus may be quite distinct from that of adult cells. A typical example is probably the expression of natriuretic peptides: neonatal ventricular myocytes synthesize and secrete both atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). After birth, the cells rapidly lose this capacity, and adult ventricular myocytes, under normal conditions, only express a minute amount of these two peptides. However, under cardiac failure conditions, ‘fetal genes’, including both natriuretic peptide genes, are re-expressed at high level [35].

No direct comparisons have ever been made between neonatal and adult cardiac fibroblasts. Although data are lacking, it is conceivable that, considering their environment and maturation of the heart after birth, neonatal cardiac fibroblasts may differ and respond in a dissimilar way than adult cells.


   5. Ang II augments the synthesis and secretion of autocrine/paracrine substances
Top
 Abstract
 1. Introduction
 2. In vivo effects...
 3. In vitro proliferation...
 4. How different are...
 5. Ang II augments...
6. Ang II and...
 7. Is the cell...
 8. Conclusion
 References
 
One plausible reason that may explain why Ang II has been variably reported to increase the proliferation of adult fibroblasts is the secretion of growth substances. Under certain experimental conditions (number of passages, cell density, length of stimulation), Ang II may or may not trigger the synthesis of several factors. Indeed, there are many examples in the literature demonstrating that Ang II stimulates the secretion of several hormones by cardiac fibroblasts, as presented in Table 1: endothelin-1, TGF-β1, and PDGF-AA. Each of these agents can potentially act as a proliferative factor. In addition, Ang II augments the synthesis of extracellular matrix (ECM) proteins, like collagen, fibronectin, laminin and osteopontin, and decreases the secretion of ECM-degrading enzymes, the matrix metalloproteases. In coordination with their respective adhesion receptors, the integrins, ECM proteins can notably modify the growth behavior of cardiac fibroblasts. In that context, the work of Ashizawa et al. [36] is particularly informative. These authors incubated neonatal cardiac fibroblasts in the presence of Ang II. In parallel to the increase in DNA synthesis, they observed induction of synthesis and secretion of osteopontin. Osteopontin is an ECM protein containing an Arg–Gly–Asp (RGD) motif and is able to interact with RGD-dependent integrins, like {alpha}vβ3. Its presence has been associated with vascular remodeling and restenosis. In that particular experiment, an antibody against osteopontin as well as RGD peptide were both able to completely block Ang II-stimulated DNA synthesis. This indicates that the Ang II proliferative effect was mediated indirectly via integrin binding and activation of specific integrin signaling pathways. It is also possible that integrins act in concert with growth factor receptors at focal clusters to maximize cell-anchorage-dependent growth, as already described [37]. These results thus suggest that, even in neonatal cardiac fibroblasts, the proliferative action of Ang II may not be direct but mediated by other substances (Fig. 1B).


   6. Ang II and the cell cycle
Top
 Abstract
 1. Introduction
 2. In vivo effects...
 3. In vitro proliferation...
 4. How different are...
 5. Ang II augments...
 6. Ang II and...
7. Is the cell...
 8. Conclusion
 References
 
Cell division is a complex mechanism that requires passage through different activation phases, from a basal quiescent state (G0) to mitosis (M) and including DNA replication (S). Growth factors are able to reintroduce the cells from the quiescent G0 phase into the G1 phase of the cycle up to the restriction point (R) after which cells are committed to enter the S phase with concurrent mitogenesis [28]. Unfortunately at present, no studies have examined the activation state of cyclin/cyclin-dependent kinase pathway after Ang II stimulation in cardiac fibroblasts. Such studies should be conducted under a reasonable time frame (within 24 h) after Ang II addition to avoid any possible artifacts of secreted growth substances. Lessons from VSCM can certainly be useful in this context. Indeed, Ang II can cause VSCM proliferation, but through the indirect synthesis of several growth factors [38,39]. Further investigations have demonstrated that, in contrast to PDGF-BB, Ang II was unable to either activate cellular events of G1/S transition [40,41] or to induce formation of the cdc2/cyclin B complex in S/M transition [42]. In addition, recent reports, reviewed by Saito and Berck [43], suggest that a direct transactivation of growth factor tyrosine kinase receptors by Ang II is possible in VSMC. Whether or not similar patterns of response are possible in cardiac fibroblasts remains to be investigated.


   7. Is the cell culture model a good model?
Top
 Abstract
 1. Introduction
 2. In vivo effects...
 3. In vitro proliferation...
 4. How different are...
 5. Ang II augments...
 6. Ang II and...
 7. Is the cell...
8. Conclusion
 References
 
Results to date may thus suggest that, even in vivo, the actions of Ang II on the proliferation of fibroblasts in the myocardium are mediated through autocrine/paracrine factors. However, this assertion must be viewed with caution. Fibroblasts in vivo are in a different environment than fibroblasts grown on plastic, and may therefore present different behavior. Several parameters in vivo are absent from cells grown in an artificial medium. Cells in tissue are surrounded by a three-dimensional matrix and are bathed in an extracellular medium rich in proteins and hormones. The proximity of other fibroblasts and of other cell types is difficult to reproduce and experiment in vitro. Recently, attempts have been made to study the interaction of cardiac fibroblasts embedded in a collagen gel [36]. This may partially reproduce the in vivo three-dimensional structure of tissue, and it is expected that fibroblasts will present a different morphology than that observed on culture dishes. No experiments have yet been conducted to further characterize the proliferative response of these fibroblasts to Ang II in a three-dimensional matrix.


   8. Conclusion
Top
 Abstract
 1. Introduction
 2. In vivo effects...
 3. In vitro proliferation...
 4. How different are...
 5. Ang II augments...
 6. Ang II and...
 7. Is the cell...
 8. Conclusion
References
 
Results in the literature indicate that Ang II can increase DNA synthesis in neonatal cardiac fibroblasts. However, so far, no systematic studies have been performed to determine the magnitude of these effects on the cell cycle progression compared to those of a true mitogenic factor like PDGF. In addition, there is some evidence that ECM proteins and integrins might be the mediators of the Ang II response.

In adult cardiac fibroblasts, Ang II-induced proliferation is even more conflictual. Only few studies were able to show [3H]thymidine incorporation, and in positive investigations, secreted growth factors may well be involved in that response.

Questions still linger about the Ang II-induced proliferation of fibroblasts in the adult myocardium. Is Ang II driving cell division or is it engaging the fibroblast into a growth factor-secreting phenotype? So far, recent results suggest that the second hypothesis is the most plausible.

Time for primary review 26 days.


   Acknowledgements
 
This work was supported by research grants from the Canadian Institutes of Health Research and from the Natural Science and Engineering Research Council of Canada. F.B. is a fellow of the Heart and Stroke Foundation of Canada.


   References
Top
 Abstract
 1. Introduction
 2. In vivo effects...
 3. In vitro proliferation...
 4. How different are...
 5. Ang II augments...
 6. Ang II and...
 7. Is the cell...
 8. Conclusion
 References
 

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