Aims Fibroblast growth factor-2 (FGF-2), implicated in paracrine induction of cardiac hypertrophy, is translated as high molecular weight (Hi-FGF-2) and low molecular weight (Lo-FGF-2) isoforms. Paracrine activities are assigned to Lo-FGF-2, whereas Hi-FGF-2 is presumed to have nuclear functions. In this work, we re-examined the latter presumption by asking whether: cardiac non-myocytes (CNMs) accumulate and export Hi-FGF-2 in response to pro-hypertrophic [angiotensin II (Ang II)] stimuli; an unconventional secretory pathway requiring activated caspase-1 affects Hi-FGF2 export; and secreted Hi-FGF-2 is pro-hypertrophic.
Methods and results Using neonatal rat heart-derived cultures and immunoblotting, we show that CNMs accumulated over 90% Hi-FGF-2, at levels at least five-fold higher than cardiomyocytes (CMs). Pro-hypertrophic agents (Ang II, endothelin-1, and isoproterenol) up-regulated CNM-associated Hi-FGF-2. The Ang II effect was mediated by Ang II receptor-1 but not Ang II receptor-2 as it was blocked by losartan but not PD123319. CNM-derived Hi-FGF-2 was detected in two extracellular pools: in conditioned medium from Ang II-stimulated CNMs and in association with the cell surface/matrix, eluted with a gentle 2 M NaCl wash of the cell monolayer. Conditioned medium from Ang II-treated CNMs increased neonatal CM size, an effect prevented by anti-FGF-2-neutralizing antibodies. The caspase-1 inhibitor YVAD prevented the Ang II-induced release of Hi-FGF-2 to both extracellular pools.
Conclusion CNMs are major producers of Hi-FGF-2, up-regulated by hypertrophic stimuli and exported to the extracellular environment by a mechanism requiring caspase-1 activity, suggesting a link to the innate immune response. Hi-FGF-2 is likely to promote paracrine induction of myocyte hypertrophy in vivo.
High molecular weight FGF-2 secretion
Pathological myocardial growth (cardiac hypertrophy) is associated with several cardiac diseases and is a risk factor for the development of heart failure.1 Understanding cell and molecular mechanisms of cardiomyocyte (CM) hypertrophy therefore can provide strategies for preventing or delaying its development and thus improve cardiac outcome. In this context, cardiac non-myocytes (CNMs), such as fibroblasts and in particular ‘activated’ fibroblasts (myofibroblasts), a major cardiac cell population, are believed to play a major role in the induction of hypertrophy and overall cardiac remodelling.2,3 CNMs respond to pro-hypertrophic stimuli such as angiotensin II (Ang II) and isoproterenol by synthesizing and secreting bioactive molecules including fibroblast growth factor-2 (FGF-2), which can then induce CM hypertrophy in a paracrine fashion.3,4
FGF-2 is a multifunctional protein, translated from the same mRNA as high molecular weight (>20 kDa; Hi-FGF-2) or low molecular weight (18 kDa; Lo-FGF-2) isoforms. Expression of FGF-2 isoforms varies accordingly to the cell type, tissue type, and developmental stage.5 There is some evidence that stress stimuli (oxidative stress and heat shock) promote preferential translation of the Hi-FGF-2 isoforms.6 Hi-FGF-2 is believed to localize preferentially to the cell nucleus and exert exclusively intracrine activities.5,7 As a consequence, the auto- or paracrine FGF-2 activities are presumed to represent the action of Lo-FGF-2,7 with a few exceptions.8
FGF-2 does not have a signal peptide for secretion and is exported by cells by a non-conventional mechanism.9 It can also be released to the environment due to cell injury.10 When FGF-2-deficient mice, lacking all FGF-2 isoforms, failed to develop pressure overload- and Ang II-induced hypertrophy, the presumption has been that Lo-FGF-2 (secreted by CNMs) was the pro-hypertrophic, paracrine-acting agent.4,11 Later studies using purified proteins showed that recombinant Hi-FGF-2 (but not Lo-FGF-2) induced CM hypertrophy in vivo and in vitro.12 Although these studies pointed to the possibility that endogenous Hi- rather than Lo-FGF-2 is pro-hypertrophic, there is no information as to whether Hi-FGF-2 can actually be externalized by cardiac cells.
In this work, we will present evidence that: (i) CNMs represent a major source of pro-hypertrophic Hi-FGF-2; (ii) Ang II (and other pro-hypertrophic agents such as endothelin-113 or isoproterenol14) up-regulates cell-associated Hi-FGF-2 in primary neonatal rat CNM cultures; (iii) Ang II receptor-1 (AT1) but not receptor-2 (AT2) mediates Hi-FGF-2 up-regulation, and (iv) Hi-FGF-2 is secreted to the extracellular environment by non-injured CNMs, by a process stimulated by Ang II and mediated by caspase-1 activity.
The investigation conforms with the Guide for the Care and Use of Laboratory Animals by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Approval was given by the Protocol Management and Review Committee of the University of Manitoba. This section summarizes key methods. A full description can be found in the Supplementary material online.
Primary cultures of rat neonatal CNMs or CMs were obtained as in Doble et al.15,16 Adult heart-derived CNMs as in Brilla et al.,17 and CMs as in Liu et al.18 All CNMs studies used cells at passage P2.
Rat recombinant histidine-tagged HisHi- and HisLo-FGF-2 were obtained as in Jiang et al.12,19 Losartan (Merck Frost), PD123319 (Tocris Bioscience), and YVAD (Ac-YVAD-CMK; Alexis Biochemicals) were used, respectively, at 10−6, 10−6, and 10−5 M.
Monoclonal anti-FGF-2 antibodies for western blot detection (#05-118, clone bFM-2, Millipore) and for activity neutralization (#05-117, clone bFM1, Cedarlane) have been validated in previous studies.20–23 Immunofluorescence of cardiac tissue sections was done as in Anderson et al.22
2.4 Cell treatments
Confluent CNMs were placed for 24 h in Ham's F-10 medium supplemented with 0.5% foetal bovine serum (FBS) and 10 µg/mL each of insulin, transferrin, and selenium; 20 µg/mL ascorbic acid; and 0.2% bovine serum albumin (BSA). Cells were then subjected to various treatments for 24 h unless stated otherwise.
Cell surface area of neonatal CMs was determined as in Jiang et al.12
2.6 High salt wash
After aspiration of conditioned medium, CNMs were gently washed with 2 mL (per 100 mm dish) of 2 M NaCl in 10 mM Tris–HCl, pH 7.2,24 supplemented with 0.5% BSA. High salt or phosphate-buffered saline (PBS) -‘washed’ cells were scraped and sonicated into SDS/PAGE sample buffer supplemented with protease inhibitor cocktail (PIC: 1 mM PMSF, 5 µg/mL pepstatin, and 5 µg/mL leupeptin).
2.7 Heparin–sepharose fractions
Conditioned media (pooled; 30–60 mL, as indicated), or the high salt washes (pooled; 6 mL), were either brought up to or diluted down to 0.5 M NaCl in the presence of 10 mM Tris–HCl (pH 7.2) with PIC. Each pooled sample was incubated with a heparin–sepharose CL-6B slurry (100 µL unpacked beads) and left at room temperature for 2 h with gentle agitation. Sepharose beads were pelleted and washed with PIC-supplemented PBS. Washed pellets were boiled in 35 µL of 2× SDS/PAGE sample buffer to elute heparin-bound proteins which were analysed by western blotting.
2.8 Western blotting
Cell lysates (50 µg protein/lane) or proteins eluted from heparin–sepharose beads were analysed in 15% SDS–PAGE gels, followed by western blotting for FGF-2 as in Sun et al.20 and Anderson et al.22 Blots were re-probed with anti-GAPDH, pan-actin, and β-tubulin antibodies or stained for Ponceau Red. Antigen–antibody complexes were detected by chemiluminescence.
2.9 Statistical analysis
Densitometric quantitation of each western blot band was done using Quantity One 1-D Analysis Software, connected to a GS-800 Calibrated Densitometer. Values were expressed as means ± SEM; n = 3 (for western blot analyses). One-way analysis of variance (ANOVA) followed by the Tukey–Kramer multiple comparisons test was used for comparing differences among multiple groups, with GraphPad InStat 3.0. Two-way ANOVA followed by the Holm–Sidak comparison test (SigmaStat 3.5) was also used (Figures 3C and 5B). Differences among groups were defined as significant at P < 0.05.
The work described here refers to results obtained from neonatal rat heart-derived non-myocyte (CNMs and fibroblasts) and myocyte (CM) cultures, unless specifically stated otherwise.
3.1 FGF-2 isoforms in cardiac cells
CNMs accumulated predominantly (93% of total) Hi-FGF-2, composed of 22 and 23 kDa immunoreactive bands, products of translation of the rat mRNA from CUG sites;5,7 18 kDa Lo-FGF-2, translated from the AUG start codon, was also detected (Figure 1A). As can be seen in Supplementary material online, Figure S1, the antibodies used strongly recognize our positive controls (recombinant Hi- and Lo-FGF-2) and detect no other band in total CNM lysates.
Expression of Hi-FGF-2 by neonatal heart-derived non-myocytes (CNMs) and cardiomyocytes (CMs). (A) Representative western blot probed for FGF-2 and corresponding quantitative data for relative Hi- and Lo-FGF-2 levels (as indicated, in black or white columns, respectively) in lysates (50 µg/lane) from CNMs and CMs (n = 3). Brackets point to comparisons between groups. ***P < 0.001; NS, non-significant. (B) Representative (n = 2) western blot images showing appropriate expression of cell-specific markers in CMs and CNMs, as indicated. TnT, cardiac troponin T; EDA-FN, extra-domain A fibronectin; myosin, striated muscle myosin. Reactivity for β-tubulin is used to indicate even protein loading.
As shown in Figure1A, CNMs accumulated significantly higher (approximately five-fold) levels of Hi-FGF-2 compared with CMs, but there was no significant difference between their corresponding Lo-FGF-2 levels. The distinct identity of CNM vs. CM cultures, as judged by expression of cell-type-specific markers, is illustrated in Figure1B. CMs, but not CNMs, express cardiac myofibrillar proteins troponin-T (TnT) and striated myosin, whereas CNMs (but not CMs) express extra-domain A (EDA)-fibronectin. Vimentin was more prominently expressed by CNMs but was also present in immature CMs, as expected.25
Lysates from adult heart CNMs, but not isolated adult CMs, elicited a strong anti-FGF-2 signal, composed mainly of Hi-FGF-2 (Figure 2A). In adult heart (ventricle)-derived tissue sections, strong anti-FGF-2 staining was observed mainly in association with CM periphery and with non-myocytes located in the vicinity of CMs (Figure 2B). These non-myocytes stained positive for vimentin, indicating that they were for the most part of fibroblastic nature.
Expression of FGF-2 by adult heart cells. (A) In vitro: representative western blot probed for FGF-2 and corresponding quantitative data for relative Hi-FGF-2 levels in adult heart-derived CNMs and CMs. **P < 0.01 (n = 3). (B) In vivo: predominant FGF-2 localization in adult rat heart non-myocytes. Adult rat heart ventricular sections were stained for FGF-2 (green), vimentin (red), and nuclei (blue), as indicated. Images a, c, and e show the same field stained for: a, FGF-2; b, vimentin as well as nuclei; and c, FGF-2 and vimentin. The region enclosed within the dotted-line square in a, c, and e is shown at higher magnification in, respectively, b, d, and f. White arrows point at fibroblastic cells (vimentin-positive) surrounding CMs. Sizing bars in a, c, and e or b, d, and f correspond to 50 or 20 µM, respectively.
3.2 Effect of Ang II and its receptors on Hi-FGF-2 accumulation
CNMs stimulated with increasing concentrations of Ang II and examined 1 day later showed a dose-dependent increase in relative Hi-FGF-2 levels; this increase was significant (compared with untreated cells) and maximal at 10−8–10−7 M Ang II (Figure 3A). Similar increases in Hi-FGF-2 were present at 36 and 48 h of incubation with Ang II (unpublished observations). Corresponding Lo-FGF-2 levels showed a trend towards increasing in response to Ang II, although values did not reach statistical significance. AT1 inhibition by losartan, but not AT2 inhibition by PD123319, significantly reduced the Ang II-induced Hi-FGF-2 up-regulation (Figure 3B). Adult heart-derived CNMs also responded to Ang II by a significant up-regulation of Hi-FGF-2, which was significantly reduced by losartan but not PD123319 (Figure 3C).
Ang II and Hi-FGF-2 accumulation in neonatal (A and B) or adult heart (C) CNMs. (A) Representative western blot images, probed for FGF-2, and corresponding cumulative data showing relative Hi- and Lo-FGF-2 levels in neonatal CNMs as a function of Ang II concentration, as indicated. (B and C) The effect of AT1and AT2 inhibitors (losartan and PD123319, respectively) on the Ang II (10−7 M)-induced Hi-FGF-2 up-regulation in, respectively, neonatal and adult heart CNMs, is illustrated in representative western blots probed for FGF-2 and corresponding quantitative data, as indicated. Blots were also probed for GAPDH, or β-tubulin, as loading controls. For all graphs, brackets point to comparisons between groups (n = 3). ***, **, and * correspond to P < 0.001, P < 0.01, and P < 0.05, respectively. NS, non-significant.
Additional pro-hypertrophic agents such as endothelin and isoproterenol (both tested at 10−7 M) significantly increased relative Hi-FGF-2 in CNMs, but not in CMs (Figure 4).
Effect of endothelin-1 (A and B) and isoproterenol (C and D) on Hi-FGF-2 accumulation by cardiac cells. (A and B) Representative western blots of CNM and CM lysates, respectively, probed for FGF-2, and corresponding quantitative data showing the effect of ET-1 (10−7 M) on Hi-FGF-2 (arbitrary optical density, OD, units) accumulation. (C and D) Representative western blots of CNM and CM lysates, respectively, probed for FGF-2, and corresponding quantitative data showing the effect of isoproterenol (10−7 M, Isp) on Hi-FGF-2 (arbitrary OD, units) accumulation. Staining for pan-actin is included as a loading control. In all panels, brackets point to groups (n = 3) being compared; ** and * correspond to P < 0.01 and P < 0.05, respectively. A value of P > 0.05 is indicated as non-significant (NS).
3.3 Hi-FGF-2 is exported by CNMs
We looked for Hi- and Lo-FGF-2 in conditioned medium from unstimulated and Ang II-stimulated CNMs, using western blotting of the heparin–sepharose-bound fraction of conditioned media. A clear anti-FGF-2 signal at 23 kDa corresponding to Hi-FGF-2 was detected only in conditioned medium from Ang II-treated cells (10−8 M). This experiment was repeated twice, with similar results.
The heparin–sepharose-bound fraction from non-conditioned medium (which contains 0.5% FBS) did not have detectable levels of FGF-2, unless it was supplemented with exogenous recombinant HisFGF-2 (Figure 5A). Thus, the heparin–sepharose approach is effective in ‘concentrating’ FGF-2 present in the medium, while the medium itself has undetectable levels of FGF-2.
Hi-FGF-2 is exported by CNMs. (A) Conditioned medium from Ang II-treated neonatal CNMs contains Hi-FGF-2: western blot, representative of three experiments, of the heparin–sepharose-bound proteins present in the conditioned medium of CNMs treated with 0, 10−9, and 10−8 M Ang II, as indicated, and probed for FGF-2. Arrow points to an immunoreactive 23 kDa band. Lanes labelled ‘Medium’ and ‘Medium + HisHi-FGF-2’ contain the heparin-binding fraction from non-conditioned medium without or with added HisHi-FGF-2 (1 ng), respectively. (B) A portion of cell-associated Hi-FGF-2 is removed by a 2 M NaCl wash. Representative western blots (for FGF-2 and GAPDH), and corresponding quantitative data, showing relative Hi-FGF-2 levels in lysates from CNMs (untreated or treated with Ang II, as indicated) and obtained ‘Before’ or ‘After’ elution of the cell surface/matrix-bound proteins by a 2 M NaCl wash, as indicated. Brackets point to the groups being compared; *significant (P < 0.05) difference between groups (n = 3). (C) LDH release in CNM culture medium is shown as a function of treatment with 10−7 M Ang II vs. untreated cells (Control). (D) LDH release by CNMs is shown as a function of either a PBS or a 2 M NaCl wash of the cell monolayer. In both (C) and (D), n = 3, P > 0.05 (non-significant, NS).
In addition to finding its way to the soluble phase (conditioned medium), exported FGF-2 may bind to the cell surface/matrix. To elute extracellular cell/matrix-bound FGF-2, we subjected CNMs to a brief, gentle wash with a 2 M NaCl solution; control cells were ‘washed’ with PBS. Lysates from 2 M NaCl- or PBS-washed cells were analysed for FGF-2. The high salt wash significantly reduced Hi-FGF-2 content of cell lysates, by 34% in non-Ang II-treated cultures and by 29% in Ang II-treated cultures (Figure 5B), consisted with the notion that Hi-FGF-2 is exported and can bind to the cell surface/matrix. The Ang II-treated cultures retained significantly (P < 0.05) more Hi-FGF-2 compared with untreated cells even after the salt wash.
Neither Ang II treatment nor the 2 M NaCl wash caused cell injury/permeabilization: relative lactic dehydrogenase (LDH) levels were similar in the conditioned medium from non-treated or Ang II-treated cells (Figure 5C). Relative LDH levels were also similar between the PBS and the 2 M NaCl cellular washes (Figure 5D).
3.4 Hi-FGF-2 export and caspase-1
We asked whether Hi-FGF-2 export by CNMs would be affected by caspase-1, as reported recently for several unconventionally secreted proteins including ‘FGF-2’ of undetermined isoform composition.26 We thus stimulated CNMs with Ang II in the absence or presence of the caspase-1 inhibitor YVAD and determined relative levels of exported Hi-FGF-2. As shown in Figure 6A, the Ang II-induced Hi-FGF-2 increase in conditioned medium was prevented in cells treated with YVAD. This experiment was done twice with similar results. We also looked for relative Hi-FGF-2 levels in the 2 M NaCl wash of the cell monolayer, containing FGF-2 from the cell surface/matrix. As inferred from the results in Figure 5B, Ang II up-regulated Hi-FGF-2 found in the salt wash. YVAD prevented this up-regulation (Figure 6B). This experiment was done twice with similar results. YVAD had no significant effect on the Ang II-induced up-regulation in total (intracellular and cell-bound) cell-extracted Hi-FGF-2 (Figure 6C).
Caspase-1 inhibition prevents Hi-FGF-2 export. (A) YVAD prevents Hi-FGF-2 release to the conditioned medium. Representative western blot of heparin–sepharose-bound fractions from (pooled; 60 mL per sample) conditioned media derived from unstimulated CNMs (Control); CNMs stimulated with 10−7 M Ang II in the absence or presence of YVAD (Ang II and Ang II + YVAD, respectively); YVAD alone (YVAD), and probed for FGF-2. Lane labelled ‘HisHi-FGF-2’ shows immunoreactivity of 0.5 ng recombinant HisHi-FGF-2 used as a positive control. Ponceau Red staining of the same blot (as indicated) shows a protein band present in all heparin–sepharose fractions in equivalent amounts. (B) YVAD down-regulates exported and cell/matrix-bound Hi-FGF-2. Representative western blot of heparin–sepharose-bound fractions from 2 M NaCl washes (pooled; 6 mL per sample) of the CNM monolayer following the same treatments as in (A), as indicated, and probed for FGF-2. Ponceau Red staining of the same blot (as indicated) shows a protein band present in all heparin–sepharose fractions in equivalent amounts. (C) YVAD has no effect on the Ang II-induced up-regulation of cell-associated Hi-FGF-2. Representative western blot and corresponding cumulative data (n = 3) of cell lysates from neonatal CNMs treated with Ang II, Ang II plus YVAD, YVAD, and probed for FGF-2 or β-tubulin. Brackets point to comparisons between groups; *, **, and NS denote P < 0.05, P < 0.01, and P > 0.05, respectively.
3.5 Secreted Hi-FGF-2 is pro-hypertrophic
Conditioned medium from Ang II-treated CNMs (*Medium), which contains secreted Hi-FGF-2 (Figures 5A and 6A), promoted a significant 20% increase in CM cell surface area, compared with CMs incubated with non-conditioned medium (Medium; Figure 7A and B). This increase was completely abolished in the presence of anti-FGF-2-neutralizing antibodies, indicating that Hi-FGF-2, secreted by CNMs, played a major role in the hypertrophic effect (Figure 7A and B). The pro-hypertrophic effect of *Medium was not due to residual Ang II. Direct stimulation of CMs with Ang II elicited, compared with unstimulated cells, a 7.5% increase in cell size which was significantly less potent than that of *Medium. Interestingly, anti-FGF-2-neutralizing antibodies did not make a substantial change on the direct effect of Ang II on CM size. Direct stimulation with recombinant Hi-FGF-2 (10 ng/mL) elicited a 27% increase in cell size compared with unstimulated cells, confirming its pro-hypertrophic effect.12
Secreted Hi-FGF-2 is pro-hypertrophic. (A) CM size (cell surface area) is shown as a function of incubation under several conditions for 2 days: a, ‘Medium’ (non-conditioned medium); b, ‘*Medium’ (conditioned medium from Ang II-treated neonatal CNMs); c, *Medium + anti-FGF Neu Abs’ (conditioned medium from Ang II-treated CNMs supplemented with 10 µg/mL anti-FGF-2-neutralizing antibodies); d, ‘Ang II’ (10−7 M Ang II); e, ‘Ang II + anti-FGF Neu Abs’ (10−7 M Ang II + 10 µg/mL anti-FGF-2-neutralizing antibodies); f, Hi-FGF-2 (10 ng/mL recombinant Hi-FGF-2). Culture media in a, b, d, and f included 10 µg/mL of mouse IgG. Brackets denote comparisons between groups, where *** is extremely significant (P < 0.001); n = 960 myocytes/group. (B) Representative CM images from the first three groups (a–c) shown in (A). Myocytes are stained for α-actinin (red), N-cadherin (green), and nuclei (DAPI; blue). Bar corresponds to 50 µM.
Our central, novel findings are that Hi-FGF-2, widely considered as a nuclear factor, is exported by CNMs and is capable of paracrine activity such as the induction of CM hypertrophy. In addition, Hi-FGF-2 export is stimulated by Ang II and requires caspase-1 activity, linking this isoform to maladaptive cardiac remodelling and the innate immune response, respectively.
Although fibroblasts are a known source for ‘FGF-2’ in the heart,4 we demonstrated that the bulk of CNM FGF-2 is composed of Hi-FGF-2. Compared with CNMs, CMs appear to be weaker Hi-FGF-2 producers on a per mass unit basis. Our in vitro studies were concordant with in vivo findings since, in adult cardiac sections, the intensity of anti-FGF-2 staining was pronounced in the fibroblastic cell populations.
The robust Ang II-induced up-regulation of Hi-FGF-2 protein is likely to result, at least in part, from increased gene expression.27,28 Ang II may have promoted preferential translation of Hi-FGF-2 or have different effects on Hi- versus Lo-FGF-2 turnover.
The biological effects of Ang II are exerted by binding and activating its G-protein-linked receptors (AT1 and AT2) at the cell surface of both CMs and CNMs.29,30 Because the Ang II-induced Hi-FGF-2 up-regulation was prevented by losartan but not PD123319, we conclude that the AT1 receptor mediates Hi-FGF-2 up-regulation. AT1 receptor blockers also prevent or attenuate the deleterious cardiac effects of Ang II such as fibrosis and hypertrophy,29,30 and therefore, AT1-dependent endogenous Hi-FGF-2 production may play a role in these pathologies.
For CNM-produced Hi-FGF-2 to have an auto- or paracrine function, it needs to be exported to the extracellular environment. We have found this to be the case, at least in culture, as Hi-FGF-2 was detected in both extracellular pools traditionally assigned to Lo-FGF-2. The latter include FGF-2 tightly bound to the cell surface/matrix due to its affinity for heparan sulfate proteoglycans,24 and in cell conditioned media (in culture) and body fluids (blood and pericardial fluid).31,32 Subjecting CNMs to a high salt wash, which breaks the ionic interaction between FGF-2 and heparin sulfate proteoglycans,33 decreased relative levels of cell-associated Hi-FGF-2, while increasing its levels in the wash, providing evidence that Hi-FGF-2 was present in the extracellular space and bound to the cell surface/matrix.
As Hi-FGF-2 became detectable in the conditioned medium only after Ang II treatment, it is possible that the strong overall Hi-FGF-2 up-regulation by Ang II allowed proportionally more Hi-FGF-2 to ‘escape’ to the soluble phase, possibly by saturating FGF-2-binding sites at the extracellular space. Alternatively, Ang II may have actively promoted ‘liberation’ of cell- or matrix-bound FGF-2 to the medium by matrix degradation. In support of the latter, Ang II/AT1 signalling increases matrix metalloproteinase (MMP) activity which in turn is responsible for shedding/liberating another heparin-bound factor, the heparin-bound epidermal growth factor to the medium.34 Thus, MMP action may also contribute to the release of cell-bound Hi-FGF-2, as has been demonstrated for ‘FGF-2’ in the retina.35
Because neither Ang II nor the high salt wash stimulated the release of a cytosolic enzyme, LDH, an indicator of cellular injury, we conclude that the increase in exported Hi-FGF2 was not a consequence of injury.36 Rather, we assume that the mechanism(s) mediating Hi-FGF-2 export by CNMs follows the non-conventional pathways of secretion operating for several proteins such as ‘FGF-2’ lacking a signal peptide for secretion. One of these pathways requires caspase-1 activity.26 Because caspase-1 inhibition, by YVAD, diminished the Ang II-induced increases in extracellular Hi-FGF-2 (both cell/matrix-bound and in the soluble phase), but not the Ang II increase in intracellular Hi-FGF-2, we conclude that indeed, caspase-1 activity targets the Hi-FGF-2 export mechanism and does not affect protein accumulation per se.
Caspase-1 is a protease activated by inflammasome(s), innate immune response protein complex(es) forming in response to stress signals.37 Activated caspase-1 cleaves and activates immunological cytokines such as interleukin 1β, stimulating their secretion; it is also required for the cellular export of several other unconventionally secreted cytokines.38 Hi-FGF-2 export by CNMs is thus linked to a generalized cellular response to stress stimuli mediated by caspase-1. It remains to be determined whether caspase-1 acts as a cargo protein, transporting Hi-FGF-2 to the cell exterior, or whether it affects another process linked to Hi-FGF-2 export. Regardless, it is of interest that failing human and murine hearts have elevated caspase-1 levels, and caspase-1 deletion is protective from myocardial infarction-induced heart failure.39 Caspase-1 deletion/inhibition would decrease externalized Hi-FGF-2 and thus reduce paracrine Hi-FGF-2 effects in vivo.
Our data suggest that Hi-FGF-2 up-regulation in CNMs is a common downstream event of several triggers of cardiac hypertrophy: in addition to Ang II, other bioactive molecules signalling via G-protein-linked receptors and associated with maladaptive hypertrophy such as endothelin-113 or isoproterenol29 also up-regulated Hi-FGF-2. It is noteworthy that isoproterenol (or endothelin) targeted CNMs selectively and had no discernible effect on CM Hi-FGF-2. We reported previously that isoproterenol stimulated FGF-2 promoter expression in the hearts of transgenic mice, but not isolated CMs.40 The present study suggests that the stimulatory effects of isoproterenol on FGF-2 promoter expression in the whole heart reflected an effect on CNMs rather than CMs. Thus, our data are in agreement with studies showing that isoproterenol exerted effects on CNMs (rather than CMs) in a chronic adrenergic stimulation model of myocardial hypertrophy.34
Our data indicated that Hi-FGF-2, secreted by Ang II-stimulated CNMs, is capable of inducing CM hypertrophy in a paracrine manner because: (i) conditioned medium from stimulated CNMs, containing Hi-FGF-2 but undetectable levels of Lo-FGF-2, increased CM size, in a manner similar to that of recombinant Hi-FGF-2 and (ii) this effect was reversed by antibodies capable of neutralizing Hi-FGF-2.21 In addition, Ang II-induced hypertrophy in vivo is likely a combination of both Hi-FGF-2-dependent (through CNMs) and -independent (directly on CMs) action. There is correlative evidence that Hi-FGF-2 plays a hypertrophic role in vivo: we recently reported a strong up-regulation of Hi-FGF-2 in a mouse model of exaggerated cardiac hypertrophy and fibrosis.41
Our model, neonatal CNM, is widely used for the study of cardiac fibroblast/myofibroblast properties, sharing many phenotypic characteristics with their adult counterparts.42 This was validated further by showing that key findings (expression and Ang II/AT1 regulation of Hi-FGF2) are similar between neonatal and adult heart CNMs.
In conclusion, we propose that CNM-derived Hi-FGF-2 may exert paracrine and/or autocrine activities in the heart. This is significant in view of the distinct (compared with Lo-FGF-2) biological activities of extracellular acting Hi-FGF-2: CNM-produced Hi-FGF-2 could directly promote CM hypertrophy.12 It could, in an autocrine manner, stimulate the release of cardiotrophin-112 which is also hypertrophic.43 Hi-FGF-2 would prevent endothelial cell migration and angiogenesis,44 subjecting hypertrophying CMs to an ischaemic environment and compound their hypertrophic response to Hi-FGF-2 and cardiotrophin-1.12 Targeting endogenous Hi-FGF-2 accumulation (translation, secretion, and/or degradation) could provide a strategy to prevent maladaptive cardiac remodelling after myocardial infarction.
This study was funded by the Canadian Institutes for Health Research (E.K.) and the St Boniface General Hospital Research Foundation (E.K.). J.-J.S. and X.M. have studentship awards from the National Sciences and Engineering Research Council, and the Manitoba Health Research Council, respectively.
. Thrombin cleaves the high molecular weight forms of basic fibroblast growth factor (FGF-2): a novel mechanism for the control of FGF-2 and thrombin activity. Oncogene 2008;27:2594-2601. doi:10.1038/sj.onc.1210899.
. High and low molecular weight fibroblast growth factor-2 increase proliferation of neonatal rat cardiac myocytes but have differential effects on binucleation and nuclear morphology. Evidence for both paracrine and intracrine actions of fibroblast growth factor-2. Circ Res 1996;78:126-136.pmid:8603495
. Distinctive patterns of basic fibroblast growth factor (bFGF) distribution in degenerating and regenerating areas of dystrophic (mdx) striated muscles. Dev Biol 1991;147:96-109. doi:10.1016/S0012-1606(05)80010-7.
. Characterization of two preparations of antibodies to basic fibroblast growth factor which exhibit distinct patterns of immunolocalization. Growth Factors 1990;4:69-80. doi:10.3109/08977199009011012.
. Proliferation of pulmonary interstitial fibroblasts is mediated by transforming growth factor-beta1-induced release of extracellular fibroblast growth factor-2 and phosphorylation of p38 MAPK and JNK. J Biol Chem 2005;280:43000-43009. doi:10.1074/jbc.M510441200.
. Involvement of the serotonin 5-HT2B receptor in cardiac hypertrophy linked to sympathetic stimulation: control of interleukin-6, interleukin-1beta, and tumor necrosis factor-alpha cytokine production by ventricular fibroblasts. Circulation 2004;110:969-974. doi:10.1161/01.CIR.0000139856.20505.57.
. Quantification of vascular endothelial growth factor, interleukin-8, and basic fibroblast growth factor in plasma of cancer patients and healthy volunteers—comparison of ELISA and microsphere-based multiplexed immunoassay. Clin Chem Lab Med 2008;46:1256-1264. doi:10.1515/CCLM.2008.249.
. Cardiac fibroblast to myofibroblast differentiation in vivo and in vitro: expression of focal adhesion components in neonatal and adult rat ventricular myofibroblasts. Dev Dyn 2010;239:1573-1584. doi:10.1002/dvdy.22280.
Jon-JonSantiago, XinMa, Leslie J.McNaughton, Barbara E.Nickel, Brian P.Bestvater, LipingYu, Robert R.Fandrich, ThomasNetticadan, ElissavetKardamiCardiovasc Res(2011)89 (1):
139-147DOI: http://dx.doi.org/10.1093/cvr/cvq261First published online: 9 August 2010 (9 pages)