(Hypertension. 1995;25:809-813.)
© 1995 American Heart Association, Inc.
Articles |
Effect of Angiotensin II Blockade on the Fibroproliferative Response to Phenylephrine in the Rat Heart
From the Boston (Mass) University School of Medicine.
Correspondence to Peter Brecher, PhD, Boston University School of Medicine, 80 E Concord St, Boston, MA 02118.
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Abstract |
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Abstract In this study we infused phenylephrine into adult Wistar rats and used losartan to test for a possible role of angiotensin II in the phenylephrine-induced fibrosis. Phenylephrine, given by Alzet minipumps at a rate of 25 mg · kg-1 · d-1, produced a rapid and striking fibrotic response that was obvious after 1 day and progressed throughout a 3-day infusion period. Northern and Western blot analyses showed large increases in cardiac fibronectin expression and atrial natriuretic peptide mRNA, corresponding to fibroblast proliferation and myocyte hypertrophy, respectively. Cardiac fibrosis, fibronectin mRNA, and atrial natriuretic peptide mRNA were blocked by prazosin (7 mg · kg-1 · d-1). Administration of losartan (10 mg · kg-1 · d-1) resulted in a threefold decrease in interstitial and perivascular fibroblast proliferation, as measured by proliferating cell nuclear antigen immunoreactivity (P<.05), a marked reduction of fibronectin mRNA in the heart, and a moderate reduction of cardiac atrial natriuretic peptide mRNA. The data suggest that effects mediated by
1-adrenergic and angiotensin type 1 receptors may
promote cardiac fibrosis.
Key Words: fibrosis • angiotensin II • hypertension, experimental • extracellular matrix • receptors, adrenergic • losartan
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Introduction |
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Cardiac hypertrophy and fibrosis are complex responses involving changes in the size, phenotype, and number of several cell types, including myocytes, vascular cells, and interstitial fibroblasts.1 The cellular changes associated with such responses are thought to be initiated in part by trophic factors and influenced by the extracellular matrix.2 In a recent study, we described rapid increases in fibronectin expression associated with cardiac hypertrophy and fibrosis in rats treated with angiotensin II (Ang II) for only 3 days, and suggested that the cellular changes that occur were in response to direct affects of Ang II on cardiac cells.3 Ang II is known to stimulate fibroblast proliferation in vitro,4 5 and an important role for the angiotensin type 1 (AT1) receptor was documented in mediating diverse effects of Ang II on cardiac myocytes and fibroblasts.4 5 Furthermore, converting enzyme inhibitors have been used therapeutically for cardiac hypertrophy and fibrosis, both to regress and to prevent cardiac lesions in hypertensive animals and human beings.6 7
Although an important role for the renin-angiotensin system in
regulating cardiac structure and function is well established, there
clearly are pathophysiological situations in which other systems may
act, either alone or in concert with Ang II, to influence cardiac cell
phenotype. Previous in vivo studies have shown that cardiac hypertrophy
and fibrosis can occur after administration of
catecholamines,8 and in vitro studies have shown direct
effects of norepinephrine on myocyte hypertrophy9 and
cardiac fibroblasts.10 In the present study, cardiac
fibrosis was induced acutely in rats using the 1-
adrenergic agonist phenylephrine. The results indicate that the
cellular response was due, at least in part, to a cooperative
interaction between adrenergic agonists and Ang II.
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Methods |
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Drugs
Phenylephrine hydrochloride and prazosin hydrochloride were purchased from Sigma Chemical Co. Losartan was provided by Du Pont/Merck, and trandolapril was a gift from Knoll Pharmaceuticals.
Animals and Tissue Preparation
Male Wistar rats were purchased from Charles River Breeding
Laboratories at 10 weeks of age and acclimated to the facilities for 1
week. An Alzet osmotic minipump (Alza Corp) containing phenylephrine
hydrochloride was implanted subcutaneously with a delivery rate of
either 10 or 25 mg · kg-1 · d-1.
Other drugs were administered in the drinking water 1 day before
implantation of the minipumps and given concurrently with phenylephrine
throughout the 3-day protocol. Drugs were given as follows: prazosin 7
mg · kg-1 · d-1, losartan 10
mg · kg-1 · d-1, and
trandolapril 1 mg · kg-1 · d-1.
Systolic pressure was determined in a controlled-temperature room by
tail-cuff plethysmography on unanesthetized rats at 26°C using a
photoelectric cell detector (IITC Inc–Life Science Instruments).
Measurements were made before drug treatment, 1 day after surgery, and
4 to 6 hours before the animals were killed, with multiple values
averaged for each time interval. Sodium pentobarbital (Abbott Labs) was
used as surgical anesthesia (50 mg/kg) and for overdosing (0.5 g/kg)
the rats. The procedures followed were in accordance with institutional
guidelines.
RNA and Protein Analysis
Total RNA from the left ventricle was extracted with a 10-fold
volume of guanidinium thiocyanate buffer for the initial
homogenization.11 Northern blot analysis was performed
as described previously.12 Complementary DNA (cDNA) probes
were generated by use of a random prime nucleotide synthesis kit
(Amersham International), and hybridization was performed at 65°C for
all cDNA probes. Laser densitometry was used to quantitate the relative
signal intensity of the bands obtained. The cDNA probes were those we
used in a previous study.12 Western blot analysis was
performed exactly as described previously.3
PCNA and Fibronectin Immunodetection
Horseradish peroxidase–conjugated monoclonal antibody (clone
PC10–mouse anti-human) to proliferating cell nuclear antigen (PCNA)
was obtained from DAKO Corp and a polyclonal rabbit anti-rat antibody
to fibronectin was obtained from Calbiochem. Tissues were fixed in
formalin for 24 hours, embedded in paraffin, and sectioned. PCNA
antibody was used prediluted as supplied by the manufacturer in the
Enhanced Polymer System (EPOS) kit, and the antibody to fibronectin was
diluted 1:2000. Diaminobenzidine staining was used for visualizing PCNA
and fibronectin. For morphometric analysis, all slides analyzed
were processed and stained in the same batch. PCNA was developed
without the need for a secondary antibody, whereas a biotinylated goat
anti-rabbit IgG was used for fibronectin in conjunction with the
Vectastain Elite Kit from Vector Laboratories. The biotinylated
secondary antibody was diluted 1:200. Rat intestinal epithelium, which
has a high rate of proliferation, was used as a positive control for
PCNA. Sections were counterstained with Gill hematoxylin. To calculate
a PCNA index, we counted the number of stained and unstained nonmyocyte
nuclei in 12 medium-power (x125) microscope fields of perivascular
regions from the four quadrants of the left ventricle, using a
400-point grid to assist in calculations. This value was expressed as a
ratio of nonmyocyte positive nuclei to nonmyocyte total nuclei. Vessel
lumen area was noted and found to be not significantly different among
groups. Statistics were assessed by ANOVA with the Bonferroni
t procedure or Scheffé's F test method for multiple
comparisons.
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Results |
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Fig 1A shows representative Northern blot analysis on heart RNA taken from rats given either a low (10 mg · kg-1 · d-1) or high (25 mg · kg-1 · d-1) dose of phenylephrine, which was administered for 3 days by an osmotic pump. Steady-state mRNA levels for fibronectin, which serves as a marker for cardiac fibroblasts, were clearly increased by the higher dose of drug, whereas the lower dose produced little change from control levels. The range of doses used is presumably selective for stimulation of the
1-adrenergic receptor, because at
extremely high doses of phenylephrine, nonspecific binding to
ß1-adrenergic receptors in the juxtaglomerular cells of
the kidney could stimulate the production of renin. Similar
dose-dependent responses were seen for atrial natriuretic peptide
(ANP), a useful marker for myocyte hypertrophy in the left ventricle,
whereas the gene for glyceraldehyde-3-phosphate dehydrogenase (GAPDH),
often used as a constitutive gene, did not change under these
conditions. Adminstration of prazosin, an 1-adrenergic
antagonist, in the drinking water of rats given the high dose of
phenylephrine markedly attenuated the cardiac response for each of the
marker genes. Densitometric analysis in the high-phenylephrine
group showed a significant sixfold increase in steady-state fibronectin
mRNA (P<.05, n=6), and after concurrent administration of
prazosin, mRNA levels were not significantly different from those of
controls. Experiments showing the temporal response and reversibility
of the cardiac changes are summarized in Fig 1B and 1C. Steady-state
mRNA levels for fibronectin increased after only 1 day of phenylephrine
infusion, although the increase was more pronounced by 3 days (Fig 1B).
When phenylephrine infusion was discontinued after 3 days and the
animals were allowed to recover for an additional 7 days, steady-state
mRNA levels for fibronectin returned to control levels. A reduction in
GAPDH mRNA was frequently observed in phenylephrine-treated animals,
which may reflect differential expression of GAPDH in cells induced by
phenylephrine. Western blot analysis (Fig 1C) showed that the
increases in fibronectin mRNA were reflected by increases in protein,
particularly after 3 days. After discontinuation of treatment for 1
week, protein levels decreased almost to control levels. Thus, the
experiments shown in Fig 1 characterize the response to phenylephrine
infusion, indicating a time- and dose-dependent change in gene
expression involving 1-adrenergic receptors.
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To determine whether the renin-angiotensin system had a role in mediating the effects of phenylephrine on the heart, experiments were performed in which either losartan or the converting enzyme inhibitor trandolapril was given concurrently with phenylephrine. Fig 2A shows a representative Northern blot analysis from such experiments, and Fig 2B summarizes densitometric analyses of Northern blot data for four to seven rats per treatment group. The data in Fig 2B are expressed relative to GAPDH mRNA to minimize loading differences. Although changes in GAPDH mRNA probably occur, such changes are not to the same extent as those in fibronectin or ANP. After a 3-day infusion with phenylephrine, large increases in steady-state mRNA levels for fibronectin and ANP were obvious. Treatment with losartan alone had no effect on cardiac gene expression; however, when losartan was given in conjunction with phenylephrine, a marked attenuation of fibronectin mRNA was obvious, whereas ANP mRNA decreased to a lesser degree. Interestingly, converting enzyme inhibition did not reduce fibronectin or ANP mRNA levels to the same extent as did losartan.
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A summary of blood pressure and heart weight data for animals subjected to the different treatments is shown in the Table. Measurements were difficult to obtain on phenylephrine-treated rats, probably because of constriction of the tail artery. Values averaged 141 mm Hg for rats given the high dose of phenylephrine for 3 days and were significantly higher than control levels of 118 mm Hg. Losartan given to phenylephrine-treated rats resulted in average pressures of 131 mm Hg. Heart weights were increased slightly but significantly after 3 days of phenylephrine treatment, indicating hypertrophy, and this increase was not reversed by losartan or trandolapril treatment.
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Morphological changes induced by phenylephrine and losartan treatment are summarized in Fig 3. Fig 3A is a hematoxylin and eosin–stained papillary muscle of the left ventricle showing interstitial fibrosis in the left ventricle of a phenylephrine-treated rat. Both perivascular and interstitial fibrosis were invariably present in left and right ventricles of these treated rats. Fibronectin, detected by immunohistochemical staining, was present in regions of fibrosis (Fig 3B). PCNA, a nuclear antigen expressed during the replicative stages of the cell cycle, was used as a marker for fibroblast proliferation. In untreated animals, there was almost no evidence of fibrosis, and only occasionally was an interstitial fibroblast positive for PCNA. After 3 days of phenylephrine infusion, the focal regions of fibrosis that were obvious throughout the left and right ventricles were associated with PCNA-positive fibroblasts (Fig 3C). However, virtually no PCNA-positive myocytes or endothelial cells were detected, although inflammatory cells and vascular smooth muscle cells in perivascular lesions did show a positive signal for PCNA (data not shown). Morphometric analysis of PCNA-positive nonmyocytes (mostly fibroblasts) was performed in ventricular tissue from control rats and those treated with phenylephrine or both phenylephrine and losartan. The data are summarized in Fig 3D and indicate that treatment with losartan markedly diminished the number of PCNA-positive nonmyocytes. Histological observations were consistent with the near absence of fibrosis in the losartan-treated animals. Prazosin-treated rats also were examined, and their cardiac tissue appeared normal on histological analysis.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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These studies characterize an acute in vivo model for induction of both cardiac hypertrophy and fibrosis by use of phenylephrine infusion that is at least partially reversible and dependent on both adrenergic and AT1 receptor–mediated responses. ANP and fibronectin mRNA expression were used as biochemical markers for the induction of myocyte hypertrophy and fibroblast proliferation, respectively. Both morphological and chemical assessments of cardiac tissue were consistent with association of increased fibronectin expression with perivascular and interstitial fibrosis, in agreement with the findings of a previous study of an in vivo model in which Ang II was administered by osmotic minipump.3 In that study, increased fibronectin expression was induced by Ang II infusion by mechanisms independent of blood pressure. Fibronectin is thought to have an important role in diverse cellular processes, including wound healing and fibrosis.13 We have previously characterized fibronectin expression in several experimental models of cardiac hypertrophy and found increases in its mRNA and protein.3 12 Changes in fibronectin mRNA preceded changes in collagen gene transcripts, and the data indicated that fibronectin was a useful early indicator of fibrosis in the left ventricle.
Of particular interest was the attenuating effect of losartan on the
development of phenylephrine-induced fibrosis. Our data suggest direct
and perhaps selective effects of phenylephrine and Ang II on cardiac
fibroblasts to induce proliferation and subsequent fibrosis.
Relationships between Ang II and 1-adrenergic agonists
have been well documented in many pathophysiological conditions
relating to blood pressure regulation. Low doses of Ang II, either
given exogenously14 or circulating
endogenously,15 are known to potentiate the blood pressure
response to phenylephrine, and recently it was shown that losartan
reduced the mean arterial pressure response to
phenylephrine.16 Although direct effects of
1 agonists9 17 and Ang II4 18
on myocytes or the induction of cardiac hypertrophy have been described
extensively, there is less information available on the effects of
these substances on cardiac fibroblasts.10 19 The
converting enzyme inhibitor trandolapril did not reduce fibronectin to
the same degree as did losartan. Perhaps a locally generated
renin-angiotensin system exists that is relatively insensitive to
trandolapril.20 Alternatively, a higher dose of
trandolapril may have reduced the phenylephrine-induced changes in
the heart. Interestingly, Sadoshima et al21 have recently
reported that stretch of cardiac myocytes may release Ang II into the
culture medium. In that study, pretreatment with losartan, but not with
captopril, was shown to block the autocrine and paracrine effects
of stretch-released Ang II.
The expression of PCNA is maximal during the S phase of the cell cycle22 and has been used as a histological index of proliferation.23 Perivascular and interstitial fibroblasts, as well as vascular smooth muscle cells, were immunoreactive for PCNA, whereas the cardiac myocytes were completely negative. This implies that proliferation of nonmyocytes (predominantly fibroblasts) is important in phenylephrine-induced cardiac injury. Losartan, an AT1 receptor antagonist, completely blocked the PCNA reactivity in both fibroblasts and smooth muscle cells, although it was less effective at reducing ANP mRNA. This suggests that the effect of Ang II can act differentially with respect to cell type, and these effects may be influenced by metabolic and hemodynamic factors.
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Acknowledgments |
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This work was supported by grant HL-47124 from the National Institutes of Health, Bethesda, Md.
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