Monocyte Infiltration and c-fms Expression in Hearts of Spontaneously Hypertensive Rats
Abstract To elucidate mechanisms of myocardial hypertrophy in spontaneously hypertensive rats (SHR), we examined by Northern blotting the expression of the proto-oncogenes c-myc, c-fos, c-sis, and c-fms in the hearts of 4- and 14-week-old SHR and normotensive Wistar-Kyoto (WKY) rats. No difference in c-myc or c-fos expression could be found between SHR and WKY rats. In SHR, c-sis gave a weak and c-fms a very strong signal at 14 weeks, whereas no signal for these oncogenes was found in either WKY rats or Sprague-Dawley controls. Since c-fms codes for the receptor of monocyte colony-stimulating factor, we next used in situ hybridization to localize the presence of c-fms in hearts of SHR at 14 weeks. We found strong signals for c-fms around small blood vessels and between cardiac myocytes in 14-week-old SHR but none in WKY rats. Immunohistochemical staining corroborated the presence of clusters of monocyte infiltration at these same perivascular sites in significantly greater numbers in SHR than in WKY rats. We conclude that c-fms expression and macrophage infiltration are increased in the perivascular space of hypertrophied hearts from SHR. We suggest that mononuclear cell recruitment and induction of c-fms may play a role in the development of hypertension-associated myocardial hypertrophy.
Epidemiological data indicate that left ventricular hypertrophy is an independent risk factor for cardiovascular morbidity and mortality in patients with arterial hypertension.1 Although the hypertrophic process is a compensatory response to maintain cardiac function in the face of altered hemodynamic demand, it is eventually associated with disorders of excitation, relaxation, and contraction. The structural remodeling of the myocardium that occurs in hypertension is associated with increased collagen matrix deposition, myocardial fibrosis, and increased myocardial stiffness.2 These pathological events may culminate in congestive heart failure.
Proto-oncogenes are normal cellular genes that encode critical regulatory proteins. Their potential role in cardiac hypertrophy has been reviewed by Simpson.3 In Wistar rats subjected to aortic banding, c-fos, c-myc, and c-Ha-ras levels were increased acutely in the myocardium compared with controls.4 Observations in normal Wistar rats from embryonal development to 200 days of age showed that c-myc was detected in fetal rats, whereas c-fos expression increased progressively during the life of the animals.4 We examined the expression of c-fos, c-myc, c-sis, and c-fms in the myocardium of spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) control rats. Because of our previous observations on the participation of monocytes in hypertension-induced renal injury,5 we were particularly interested in c-fms, which encodes for the colony-stimulating factor-1 (CSF-1) receptor on monocytes. We were able to localize c-fms expression to perivascular areas in which the presence of monocytes and macrophages was increased.
Experimental Animal Models
Male SHR and WKY rats were purchased (Ivanovas, Kisslegg, FRG) at 3 and 12 weeks of age and maintained in cages at 24±2°C. They were fed a standard rat diet (C-1000, Altromin) containing 0.2% sodium by weight and were allowed free access to tap water. At 4 and 14 weeks of age, the rats were killed instantly by decapitation, and the hearts were immediately removed.
RNA Isolation and Northern Gel Analysis
The freshly excised hearts were weighed, cut into small pieces, and homogenized by a polytron in the presence of RNAse inhibitor. Total RNA was then extracted using minor modifications of the guanidinium isothiocyanate/cesium chloride centrifugation method.6 Fifteen micrograms of total RNA was quantified by absorbance at 260 nm and run on a 1% agarose/1 mol/L formaldehyde gel in a buffer of (mmol/L) 3-[N-morpholino]propanesulfonic acid (MOPS) 20, sodium acetate 5, and EDTA 1, pH 7.0. The gels were electrophoresed at 100 V for 3 to 4 hours with constant circulation of the buffer. Gels were stained with ethidium bromide, and Northern blotting was performed by capillary transfer in 5× (SSC) (1× SSC equals 0.15 mol/L sodium chloride, 0.015 mol/L sodium citrate, pH 7.0). The blots were baked in a vacuum oven at 80°C for 2 hours and prehybridized at 42°C for 4 hours in a buffer containing 5× Denhardt’s solution, 15× SSC, 1% sodium dodecyl sulfate, and 200 μg/mL salmon sperm DNA. The blots were then hybridized in the same buffer at 42°C for 24 to 36 hours with 32P-labeled DNA. After hybridization, the membranes were washed for 5 minutes with 2× SSC at room temperature and then twice for 30 minutes with 1× SSC and 0.1× SSC at 45° and 56°C, respectively. Autoradiography was performed with Kodak X-Omat films with intensifying screens at −70°C. The developed films were then scanned with a densitometer to measure the relative signal intensity of the bands obtained. The size (in kilobases) of detected mRNAs was calculated on the basis of the 18S and 28S ribosomal RNA migration.
Oligonucleotides for c-fos and c-myc were purchased from Dianova. DNA probes for c-fms were obtained from Clontech and for c-sis from Dianova.
In Situ Hybridization
Hearts were rapidly removed, snap-frozen in liquid nitrogen, and stored in −80°C deep freezers. Transverse sections, 5 μm thick, were cut on a cryostat at −20°C and mounted onto RNAse-free clean glass slides that had been coated with aminopropyltriethoxysilane. The slides were first dried on a hot plate (1 to 3 minutes) and were further air dried for an additional 1 to 2 hours. The sections were then fixed for 20 minutes in a fresh 4% paraformaldehyde solution in 0.1 mol/L phosphate-buffered saline (PBS) (pH 7.4) and were dehydrated in graded ethanol (30% to 100%), after which they were air dried for 30 to 60 minutes. For hybridization, the sections were immersed for 20 minutes in 0.2N HCl and incubated for 10 minutes in 0.125 mg/mL pronase (Boehringer Mannheim) in 0.1 mol/L PBS (pH 7.4) at room temperature and quickly dipped in 0.1 mol/L glycine (30 seconds). The sections were once more fixed for 20 minutes in 4% paraformaldehyde solution in 0.1 mol/L PBS (pH 7.4) and treated with 0.25% acetic acid anhydride in 0.1 mol/L triethanolamine (pH 8.0) for 10 minutes to prevent nonspecific binding of the probe to glass. Afterwards, sections were washed in 0.1 mol/L PBS, dehydrated in graded ethanol, and arranged in a hybridization chamber. The hybridization probe we used was a human c-fms cDNA probe (Clontech) labeled with uridine 5′-(α-thio) triphosphate (35S-UTP) (specific activity, 1.306 Ci/mmol; NEN–Du Pont) by in vitro transcription. The synthesis of antisense and sense riboprobes was carried out using SP6, T7, or T3 RNA polymerases (Boehringer Mannheim). Afterwards, cDNA templates were digested with RNase-free DNase (Boehringer Mannheim), and samples were extracted with phenol/chloroform and precipitated with ethanol. The sections were hybridized with 25 μL (for each section) of a solution containing 50% deionized formamide, 0.3 mol/L NaCl, 10 mmol/L Tris-HCl (pH 7.5), 10 mmol/L Na2HPO4 (pH 6.8), 1× Denhardt’s solution, 250 μg/mL yeast tRNA, 5 mmol/L EDTA, 10 mmol/L dithiothreitol, 10% dextran sulfate, and 2×105 cpm/mg 35S-UTP probe (sense and antisense, respectively), which was denatured previously at 80°C for 2 minutes, under a silicone-treated coverslip for 18 hours at 50°C in a sealed hybridization chamber. The chamber had been moistened with 50% formamide and 10% 10× salts in distilled water. After hybridization, the slides were washed at 56°C with gentle agitation successively in a washing solution (50% deionized formamide, PBS, 1.5 mmol/L dithiothreitol and distilled water) for 60 minutes. The washing was repeated three times (each 60 minutes), after which the slides were immersed in N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid (TES) solution at 37°C for 15 minutes, for 30 minutes in TES/RNase A (20 mg/mL) solution, and once more for 15 minutes in TES solution in a shaking bath. Thereafter, the slides were washed sequentially for 15 minutes each at room temperature in descending concentrations of SSC solution (2×, 1×, 0.1×, 0.05×) and were dehydrated in graded ethanols with 0.3 mol/L NH4Ac and air dried. The dried slides were dipped in photoemulsion (Ilford K5) for autoradiography, were air dried for 60 minutes, and were placed in a light-proof box at 4°C for 1 to 3 weeks. The slides were then developed in Kodak D 19, fixed, rinsed in distilled water, and counterstained with hematoxylin and eosin.
Semiquantitative data on c-fms mRNA levels were obtained by measurement of the gray values of the photographs using an IBAS image-analyzing system (Koutron). The photomicrographs were digitalized directly via a TV camera on a screen, allowing the measurement of several cross-sectional perivascular areas at once. The areas of interest were selected by means of a light pen, and the mean gray tone value was measured (mean gray tone value of the total labeling). The specific mean gray values were defined as those obtained by subtracting the nonspecific values (sections incubated with labeled sense RNA probe) from the total values (sections incubated with the labeled antisense RNA probe) after correcting for the background.
Cardiac tissue was rapidly removed (see above) and frozen in liquid nitrogen. Transverse cryostat sections (5 mm thick) were prepared and air dried. The sections were fixed in cold acetone, air dried, and immersed in Tris-buffered saline (pH 7.4). For immunohistochemical staining of mononuclear cell infiltration, the sections were incubated with the rat macrophage–specific monoclonal antibody Ki-M 2R or HIS 36 (both from Dianova) diluted in 1% bovine serum albumin/RPMI (Seromed) for 30 minutes at room temperature in a humid chamber. After the sections were washed with Tris-buffered saline, they were incubated with an anti-mouse bridging antibody. Immunoreactivity was visualized with an alkaline phosphatase/anti-alkaline phosphatase (APAAP) method (Dualsystem, Dianova) using the neufuchsin substrate for detection (E Merck). The endogenous alkaline phosphatase is blocked by an addition to the substrate solution of levamisole (Sigma Chemical Co) at an end concentration of 10 mmol/L. The sections were light counterstained in hematoxylin and mounted with glycerin gelatin (E Merck). Control sections, which were incubated without the primary antibody, showed no nonspecific staining.
The degree of macrophage infiltration was determined by visually counting the stained macrophages in the perivascular areas across whole heart sections. To quantify the results, we used a grading scale in which 0 indicated that no macrophages were present, 1 indicated that two to three macrophages were identified, and 2 indicated infiltrates of more than five macrophages. Hearts from six SHR and WKY rats were used for this purpose. Multiple sections were examined so that at least 50 separate vessels were evaluated from each of the two rat strains.
Statistical analysis was conducted (Macintosh computer) with t tests and two-by-two contingency tables. A value of P<.05 was considered significant.
At 4 weeks, SHR and WKY rats had similar body weights (120±2 versus 127±3 g, n=10) and similar heart weights (787±67 versus 698±75 mg, n=10). At 14 weeks, SHR weighed less (P<.05) than WKY rats (381±2 versus 397±2 g, n=10), had larger (P<.05) hearts than WKY rats (1488±98 versus 1300±134 mg, n=10), and higher (P<.05) systolic (tail-cuff) blood pressures than WKY rats (148±3 versus 100±3 mm Hg, n=10).
Fig 1⇓ shows c-fos (top) and c-myc (bottom) expression, as well as that of β-actin as control marker, at 14 weeks in SHR and WKY rats by Northern blot. Representative blots from four to six experiments are shown. No difference in the expression of these oncogenes was found. Fig 2⇓ (top) shows the expression of c-sis at 4 and 14 weeks in SHR and WKY rats by Northern blot. At 14 weeks, a weak but discernible signal was observed in SHR but not in WKY rats. At 4 weeks, c-sis could not be detected in either strain. Fig 2⇓ (bottom) shows c-fms expression. In contrast to c-sis, the c-fms signal was remarkably strong at 14 weeks in SHR. At 4 weeks, c-fms was undetectable in SHR. In WKY rats, c-fms was never detected. To minimize the possibility that non–hypertension-associated genetic differences could be responsible for the observed difference in c-fms expression, we also measured c-fms expression in myocardial tissue from normotensive Sprague-Dawley rats. No c-fms expression was observed in the hearts of these rats (data not shown).
We used in situ hybridization to localize c-fms. Fig 3⇓ shows a hematoxylin and eosin–stained light photomicrograph with superimposed, radiolabeled antisense probe for c-fms (representative of four independent experiments). The WKY control tissue (top) shows only the nonspecific background signal. In the SHR myocardium (bottom), increased signals were observed between myocytes and particularly in the perivascular space and within the walls of small blood vessels. We then tried to measure mRNA levels of the in situ hybridization experiments. Using an image-analysis system, we assessed the total sectional area as well as the perivascular area for c-fms expression at the mRNA level. Fig 4⇓ shows the semiquantitative analysis of the in situ hybridization experiments. Expression of c-fms was significantly increased in the total sections of the myocardial tissue from SHR and in the perivascular areas (n=10, P<.05) compared with WKY tissues. Fig 5⇓ shows the immunohistochemical staining of monocytes and macrophages using the rat macrophage–specific monoclonal antibody Ki-M 2R. In the WKY control tissue (top), only a few cells can be identified. On the other hand, in SHR myocardium (bottom), numerous cells show positive staining, again with perivascular prominence.
The number of blood vessels (from a total of 50 counted from six individual animals) involved with monocytic infiltration was compared in SHR and WKY rats (Fig 6⇓). Monocytic infiltration was found in only 13 vessels in WKY rats compared with 43 in SHR (P<.05). We also quantified the numbers of monocytes. In the right panel of Fig 6⇓, vessels with more than five monocytes per section were compared in SHR and WKY rats. In WKY rats, only five such vessels were found compared with 36 in SHR (P<.05).
We found no difference in c-fos and c-myc expression at the time points tested in SHR and WKY rats. Instead, we showed a barely detectable expression of c-sis in SHR at 14 weeks but not in WKY rats. A very intense signal for c-fms was observed in SHR at 14 weeks, which was also not present in WKY rats. In situ hybridization localized the c-fms signal between the cardiac myocytes and to perivascular areas. A prominent infiltration of these areas with monocytes was displayed with immunohistochemical staining.
The proto-oncogenes c-fos and c-myc encode for nuclear transcription factors.3 Komuro et al4 found that c-fos and c-myc expression increased in minutes to hours after aortic banding and that the signals decreased to control values by 48 hours. The fos genes show a complex pattern of tissue, cell type, and stage-specific expression, suggesting a role for these genes in cellular differentiation and proliferation.7 8 The gradual increase in c-fos expression throughout the life of Wistar rats suggests that fos may play a role in hypertrophy and aging. The proto-oncogene c-myc may also play a role in cell proliferation.9 10 11 Recently, the expression of c-myc was reported to be increased in cultured cardiac myocytes and in induced hypertrophy by α-adrenergic agents.12 13 Komuro et al4 found c-myc expression to be acutely increased in pressure-overloaded hearts and also showed that c-myc expression was prominent in the embryonal period, when cell proliferation is rapid. We found no difference in either c-fos or c-myc expression between SHR and WKY rats. We examined expression at 4 and 14 weeks, which we reasoned were times when hypertrophy was actively occurring and after hypertrophy was established. Our findings are in accord with those of Green et al,14 who also failed to detect an increased expression of the early-response genes in hypertrophied myocardium of genetically hypertensive animals. Conceivably, an earlier time point might have shown a difference in the expression of these proto-oncogenes; therefore, we cannot exclude a role for either proto-oncogene in the process of hypertrophy.
The proto-oncogene c-sis encodes for the β-chain of platelet-derived growth factor (PDGF).15 We found a slight increase in c-sis expression in SHR at 14 weeks compared with WKY rats, supporting a role for PDGF in cardiac hypertrophy. However, we have not examined PDGF synthesis per se and thus can only speculate on the meaning of these observations. Komuro et al4 could show no increase in c-sis expression in pressure-overloaded hearts.
We observed a marked expression of the proto-oncogene c-fms in hypertrophied myocardial tissue. The proto-oncogene c-fms encodes for the CSF-1 receptor, which is found on the surface of monocytes and macrophages. Claycomb and Lanson16 have previously shown that isolated cardiac myocytes express c-fms and that c-fms expression in cardiac tissue is caused by infiltrating cells. We reasoned that the appearance of c-fms might be associated with an increase in monocytes. With in situ hybridization, we were able to show that the c-fms expression did not emanate from the cardiac myocytes themselves but rather from perivascular areas and sites between the cardiac myocytes.
Histochemical staining with a specific monocyte marker antibody corroborated the presence of increased monocytes at perivascular sites in the hypertrophied myocardium. Infiltrating monocytes and macrophages are capable of elaborating cytokines including PDGF,16 which could conceivably explain the increased expression of c-sis that we observed in SHR. It is likely that a variety of growth factors are involved in the fibrotic response. We have not yet shown for certain that CSF-1 receptor protein synthesis is increased; however, we suggest that an increase in this product could result in monocyte proliferation within the perivascular areas of the myocardium. The monocytes may be integrally involved in the scarring process.
In addition to hypertrophy of cardiac myocytes, the pathological cardiac hypertrophy associated with hypertension includes an increased deposition of matrix collagen proteins and fibrosis.2 In hypertensive rat models, perivascular and interstitial fibrosis, with substantial increases in collagen types I, III, and IV, is observed after 4 to 8 weeks of sustained hypertension.17 The perivascular and interstitial myocardial fibrosis is accompanied by progressive cardiac myocyte loss and an increase in ventricular stiffness.18 The mechanisms responsible for these dramatic changes are not known; however, humoral mediators such as components of the renin-angiotensin-aldosterone axis, altered collagen metabolism, hemodynamic factors, and metabolic factors have all been implicated.19 20 One speculation from our results is that monocyte infiltration in the perivascular space plays a role in the fibrotic process.
We recently identified early interstitial changes in the kidneys of rats with two-kidney, one clip renovascular hypertension5 and were struck by a resemblance of the changes reported here to those observed within the kidney. We observed increased interstitial deposition of the same collagens associated with cardiac fibrosis.17 Furthermore, a progressive infiltration of monocytes and macrophages was identified in the walls of blood vessels and within the glomeruli. T lymphocytes were found to infiltrate the interstitium, which made the entire process resemble a chronic inflammatory response.
We have not elucidated the initial signal for monocyte infiltration nor the subsequent behavior of monocytes. The signaling may involve the renin-angiotensin-aldosterone system, some components of which may be produced locally.22 This system has been implicated in the progression of cardiac hypertrophy. Angiotensin-converting enzyme arising from endothelial cells is widely distributed among various tissues.23 Sun et al24 recently showed that angiotensin-converting enzyme was increased in the myocardium of rats exposed to either chronic angiotensin II or aldosterone. Interestingly, chronic granulomatous illnesses, which involve monocyte proliferation, also are associated with increased angiotensin-converting enzyme activity.25 We believe that increased c-fms expression is merely a step in a complicated process involving multiple signals and pathways. Nevertheless, c-fms expression may be a particularly important clue because it indicates the participation of inflammatory cells in a process that is not usually associated with inflammation.
Expression of c-fms is a marker of monocyte differentiation.26 Our findings suggest that increased c-fms expression not only demonstrates the presence of monocytes but also implies that monocytes display a differentiated phenotype with increased cytokine receptor expression. The mechanisms for the c-fms signaling we observed remain to be elucidated. We believe that their clarification will shed important light on how increased pressure mediates scarring in affected end organs.
This work was supported by a grant-in-aid to H.H. from the Deutsche Forschungsgemeinschaft.
- Received July 20, 1994.
- Revision received August 31, 1994.
- Accepted August 31, 1994.
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Dolittle RF, Hunkapiller MW, Hood LE. Simian sarcoma virus oncogene, v-sis, is derived from the gene (orgenes) encoding a platelet-derived factor. Science. 1983;221:275-277.
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Weber KT, Janicki JS, Shroff SG, Pick R, Chen RM, Bashey RI. Collagen remodeling of the pressure-overloaded hypertrophied nonhuman primate myocardium. Circ Res. 1988;62:757-765.
Jalil JE, Janicki JS, Pick R, Abrahams C, Weber KT. Fibrosis-induced reduction of endomyocardium in the rat after isoproterenol treatment. Circ Res. 1989;65:258-264.
Dzau VJ. Significance of the vascular renin-angiotensin pathway. Hypertension. 1986;8:553-559.
Brilla CG, Janicki JS, Weber KT. Cardioreparative effects of lisinopril in rats with genetic hypertension and left ventricular hypertrophy. Circulation. 1991;83:1771-1779.
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Lindpaintner K, Ganten D. The cardiac renin-angiotensin system: an appraisal of present and experimental evidence. Circ Res. 1991;68:905-921.
Campbell DJ. Circulating and tissue angiotensin systems. J Clin Invest. 1987;79:1-6.
Chambers SK, Gilmore-Hebert M, Wang Y, Rodov S, Benz E Jr, Kacinski BM. Posttranscriptional regulation of colony-stimulating factor-1 (CSF-1) and CSF-1 receptor gene expression during inhibition of phorbol-ester-induced monocytic differentiation by dexamethasone and cyclosporin A: potential involvement of a destabilizing protein. Exp Hematol. 1993;21:1328-1334.