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Hypertension. 1995;26:101-111

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*LOSARTAN POTASSIUM
*SPIRONOLACTONE

(Hypertension. 1995;26:101-111.)
© 1995 American Heart Association, Inc.

Articles

Left Ventricular Fibrosis in Renovascular Hypertensive Rats

Effect of Losartan and Spironolactone

Antonino Nicoletti; Didier Heudes; Nicole Hinglais; Marie-Dominique Appay; Monique Philippe; Caroline Sassy-Prigent; Jean Bariety; Jean-Baptiste Michel

From Unité 430 INSERM, Hôpital Broussais (A.N., D.H., N.H., M.-D.A., C.S.-P., J.B.) and Unité 367 INSERM (M.P., J.-B.M.), Paris, France.


*    Abstract
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Abstract Myocardial fibrosis resulting from arterial hypertension alters myocardial structure and function. Myocardial fibrosis is characterized by a pathological accumulation of types I and III collagens. We used an aldosterone antagonist (spironolactone) and an angiotensin II antagonist (losartan) to elucidate the respective role of these hormones and hypertension in the development of myocardial fibrosis in the Goldblatt model of two-kidney, one clip hypertension in the rat. Fibrosis was assessed by computer-assisted morphometry in the interstitial space, around coronary arteries, in microscar areas, and on left ventricular sections stained with Sirius red and by biochemical techniques. Morphometry was performed with both standard light and polarization microscopy; this latter method was used to quantify yellow-red and green collagen fibers. Concurrently, type I and type III collagen mRNAs were evaluated by a semiquantitative polymerase chain reaction method. The collagen content of the untreated two-kidney, one clip hypertensive rats increased mainly around the coronary arteries; the number and surface area of microscars also increased in chronic hypertension. Losartan treatment decreased systolic pressure and yellow-red collagen fiber content in all areas, whereas spironolactone treatment decreased green collagen fiber content without decreasing systolic pressure. mRNA levels for types I and III collagens showed profiles similar to those of yellow-red and green collagen fiber contents, respectively, suggesting that yellow-red collagen fibers are mainly type I collagen fibers and green collagen fibers are mainly type III collagen fibers. These results suggest that angiotensin II, possibly together with hypertension, and aldosterone, independently of hypertension, have a major influence on myocardial fibrosis, inducing type I and type III collagen deposits, respectively, mainly around coronary arteries.


Key Words: collagen • aldosterone • hypertension, renovascular rats • heart • angiotensin II • polymerase chain reaction


*    Introduction
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 *Introduction
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 down arrowResults
 down arrowDiscussion
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The experimental left ventricular overload induced by high arterial blood pressure is associated with quantitative1 and qualitative2 changes of cardiomyocyte phenotype. In hypertension, left ventricular hypertrophy (LVH) is also associated with fibrotic changes in the interstitial space and around coronary arteries.3 The main consequence of this interstitial activation is fibrosis,4 and interstitial fibrosis is one of the main causes of the cardiac disease resulting from hypertension.

The fibrosis of the left ventricle is usually described as interstitial, pericoronary, and replacement fibroses.4 The pathophysiology of the replacement fibrosis (myocyte death leading to fibrotic microscars), its dependence on myocardial stress, and its sensitivity to treatment, which decreases afterload, are now well accepted.5 In contrast, the pathophysiology, sensitivity to treatments, and distributions of the different forms of collagen are less well documented in interstitial and pericoronary fibroses. The left ventricular fibrosis results from an imbalance between the synthesis and degradation of extracellular matrix, composed mainly of type I and type III collagens. The cells that synthesize these collagens are the interstitial and pericoronary fibroblasts.3 6 The pathophysiological conditions causing fibroblasts to synthesize and accumulate collagen can be dissociated from the pathophysiological conditions activating cardiomyocyte hypertrophy. Fibrosis can be found in the hypertrophied left ventricle, in which work load is increased, as well as in the nonhypertrophied right ventricle with a normal work load,7 indicating that the regulation of fibrous tissue is distinct from myocyte growth. The synthetic activity of cardiac fibroblasts may be regulated by numerous interacting factors, including hemodynamic (ventricular overload, high coronary perfusion pressure) and humoral (angiotensin II [Ang II], aldosterone) factors. An experimental model in which Ang II and aldosterone are increased and reactive fibrosis is well developed is the two-kidney, one clip (2K1C) Goldblatt model of hypertension. In this model, the renin-angiotensin-aldosterone system is activated 1 month after clipping and progressively deactivated during the 2nd and 3rd months.8 It has been demonstrated that treatment with a specific aldosterone receptor antagonist prevented the increase in collagen density in rats in several experimental hypertension models, including the renovascular hypertension model, indicating that aldosterone is potentially a mediator of the collagen synthetic activity of fibroblasts.9 10 Otherwise, it has been postulated that Ang II could also be a potent regulator of fibroblast activities.9 11 12 13 14 15

The present study addresses the ability of spironolactone and losartan to cause left ventricular fibrosis to regress rather than to prevent it in the 2K1C model of hypertension in rats. We have quantified yellow-red and green collagen fibers on Sirius red–stained sections by a computer-assisted morphometric method coupled to polarization microscopy. In fact, Sirius red–stained sections observed under polarized light show closely packed thick fibrils, giving an intense birefringence with a yellow-red color, and thin fibers composed of loosely packed thin fibrils giving a weak greenish birefringence. That the yellow-red collagen fibers could be mainly type I collagen and green collagen fibers could be mainly type III collagen is still debated.16 17 18 To test whether the profiles obtained with the types I and III collagen mRNAs could match with those of yellow-red and green collagen fiber contents, respectively, mRNAs for types I and III collagens were semiquantified from extracts of the same ventricles as those on which the quantification of yellow-red and green fibers was made. If verified as an accurate way of typing collagens, this morphometric technique coupled to polarization microscopy could be a useful tool for investigators working on the deregulation of extracellular matrix to quantify, on the same tissue sections, type I and type III collagens in situ.


*    Methods
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 *Methods
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Animals
A total of 63 Wistar rats underwent surgery under ether anesthesia. In 39 rats, the left renal artery was clipped (0.2 mm diameter; 2K1C Goldblatt model8 ) and the right renal artery left untouched. Twenty-four rats were sham operated. The rats were returned to their cages and fed a standard rat diet. Water was provided ad libitum.

The clipped rats were randomly divided into six groups 1 month after application of the clip. Rats in the first group (clip-1m, n=10) were killed by rapid decapitation, together with a control group of sham-operated rats (sham-1m, n=11). The study was continued for 18 weeks after application of the clip in the four other groups. The control group of sham-operated rats (sham-4m, n=13) received no treatment. The rats in the three other groups (clipped rats) were treated as follows from the beginning of the 5th week: Los-4m rats (n=12) were given 10 mg/kg losartan (DuP 753) by twice daily gavage; Spiro-4m rats (n=8) were given 20 mg/kg spironolactone by daily gavage; and the third group was given a placebo (clip-4m, n=9) by daily gavage.

Systolic pressure (tail-cuff method) and body weight were measured once a week at the same time of day from the 1st to the 18th week after application of the clip. Eighteen weeks after application of the clip, rats in the three groups of clipped rats and the sham-4m control group were killed by decapitation.

Animal care complied with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication 86-23, revised 1989, authorization 00577, 1989, Paris, France).

Humoral Parameters
Plasma Renin Activity
At the time of decapitation, the first 2 mL of blood from the unanesthetized rats was collected in heparinized tubes containing 20 µL of 15% EDTA (pH 6.5). Plasma renin activity (PRA) was measured by quantification of Ang I, after incubation of 250 µL plasma at 37°C for 1 hour. Then, Ang I was measured by radioimmunoassay with 125I-labeled Ang I as previously described.19

Urinary Aldosterone
Rats were housed in individual metabolic cages before death. Urine samples were collected for a 24-hour period. Aldosterone was extracted (Bond Elut column, Prolabo) and measured by radioimmunoassay with 3H-labeled aldosterone. Results were expressed as the total aldosterone extracted from the urine samples and represented an estimation of the daily aldosterone excretion.20

Collagen Quantification
Hearts were rapidly removed. Only left ventricles were analyzed in this study. Two equatorial slices of the left ventricles were taken. One slice was immediately frozen in liquid nitrogen for assessment of hydroxyproline content and for the polymerase chain reaction (PCR) study. The other was fixed in 10% formol, dehydrated at room temperature through ethanol series, and embedded in paraffin. Sections (5 µm thick) were cut and stained with Sirius red F3BA (0.5% in saturated aqueous picric acid).

Standardized transversal median ventricular sections stained with Sirius red were studied by a single investigator who was unaware of the nature of the experimental groups. Collagen was quantified by computer-assisted morphometry. The automated system included an image-analysis processor (Nachet 15000) based on mathematical morphology software21 connected to a Macintosh II computer (Apple) running customized algorithms written in C. Each field sent to the image analyzer was transmitted by a video camera connected to a macroscope or microscope and transformed into a digital binary image. Then, a sequence of mathematical and morphological operations allowed us to identify and quantify all structures of interest. Thus, parameters such as surface area, perimeter, and number of each histological structure were measured. The computer software guaranteed that the same analysis sequence was used in each field.

Macroscopic Study
The entire left ventricular surface area was included in one field (final magnification x15, giving a final resolution of 30 µm per pixel). This macroscopic field was used for quantification of the collagen area ("top hat" method) and the corresponding cardiac area under standard light microscopy. Only perivascular and microscar collagens were quantified at this magnification level.

Microscopic Study
Interstitial and perivascular fibroses. Sections were examined under a Nachet microscope fitted with cross-polarizing filters at a final magnification of x250 (final resolution, 0.48 µm per pixel), with or without band-pass filters. These filters were used to stop all wavelengths except a narrow-wavelength band defined according to Junqueira et al.22 Junqueira et al showed by spectral analysis of collagen color with polarization microscopy on Sirius red–stained sections that green collagen fibers (mainly type III collagen) present a characteristic peak at 700 nm, whereas yellow-red collagen fibers (mainly type I collagen) present a peak at 550 nm. We used appropriate band-pass filters (550±5 and 700±5 nm) to quantify each type of collagen fiber separately. The surface area of the total collagen was computed based on the result from the OR Boolean operator on the image obtained with the 550-nm filter and on the image obtained with the 700-nm filter.

We first studied the evolution in the mean and variance to determine the number of fields needed to obtain a convergent estimate of fibrosis density. We estimated collagen content in the interstitial space by analyzing 30 fields in the subendocardium for each rat, excluding fields in which there were microscars or coronary arteries. Interstitial collagen density was obtained from the ratio of the collagen area and the corresponding left ventricular area. Otherwise, all the fields in which there was a coronary artery were analyzed separately. The collagen in these fields was the collagen of the adventitial area and was considered to be perivascular if the mathematical morphology software found it to be proximal to the artery lumen. The criterion of proximity was computed in each field as a function of the artery diameter. The perivascular fibrosis measured in this way was normalized to the cross-sectional area of the vessel lumen. To avoid a cut effect variability, we used for the statistical analysis only the fields in which the arteries have a form factor


greater than 0.75 (the form factor of a perfect circle is 1). Thus, we used a mean of 23 fields per heart to compute perivascular fibrosis.

Microscar fibrosis. Given the size of the microscars, their surface areas were measured on the entire left ventricular section with cross-polarizing filters at a final magnification of x40. Band-pass filters were not used at this magnification level. The proportions of yellow-red and green collagen fibers in these microscars were estimated in a complementary study at a final magnification of x250 with cross-polarizing and band-pass filters. Fields in which there was a coronary artery were excluded.

Morphometric Estimation of Overall Collagen Content
Morphometric analyses provided an estimate of the collagen contents of the interstitial, perivascular, and microscar compartments. The sum of these estimations, weighed by the surface areas of each compartment, gave an estimate of the overall collagen content with the use of the following formula: Interstitial Collagen Densityx(Surface Area of LV-Surface Area of Perivascular Collagen-Surface Area of the Lumen of the Arteries-Surface Area of Microscars)+Surface Area of Microscars+Surface Area of Perivascular Collagen. The perivascular fields in which the form factor was more than 0.75 were not excluded for this computation. In a similar manner, the overall yellow-red and green collagen fiber contents were estimated for the entire left ventricle. The overall yellow-red and green collagen fibers were used for calculation of the absolute ratio of the yellow-red and green collagen fibers.

Polymerase Chain Reaction
Total RNA was isolated from 100 mg of frozen tissue of each left ventricle by the acid guanidium thiocyanate/phenol/chloroform extraction method.23 mRNAs (1 µg) were reverse transcribed into cDNA with oligo(dT) and MMLV reverse transcriptase (BRL) in a 20-µL final volume. Amplification of 2 µL of the reverse transcription products was carried out with primers for the mouse {alpha}1 (III) collagen (upper primer, 5'-TGC CCA CAG CCT TCT ACA CCT-3'; lower primer, 5'-CAG CCA TTC CTC CCA CTC CAG-3'; product length, 244 bp), for the mouse {alpha}2 (I) collagen (upper primer, 5'-TGT TCG TGG TTC TCA GGG TAG-3'; lower primer, 5'-TTG TCG TAG CAG GGT TCT TTC-3'; product length, 254 bp), and for GAPDH (upper primer, 5'-GTG AAG GTC GGA GTC AAC G-3'; lower primer, 5'-GGT GAA GAC GCC AGT GGA CTC-3'; product length, 299 bp). All PCRs were done in a 25-µL volume. The mixture contained 10 mmol/L dNTP, 1x PCR buffer (10 mmol/L Tris-HCl, pH 8.3, 50 mmol/L KCl, 40% dimethyl sulfoxide, 0.001% gelatin, 0.75 mmol/L MgCl2 for type III collagen primers, 1.5 mmol/L MgCl2 for type I collagen primers, and 1 mmol/L MgCl2 for GAPDH primers), and 2.5 U amplitaq polymerase (Perkin-Elmer Cetus). Each sample was incubated in a DNA thermal cycler (Perkin-Elmer Cetus) for 25 cycles, each cycle consisting of 2 minutes at 94°C; 2 minutes at 55°C for GAPDH, at 56°C for type I collagen, and at 58°C for type III collagen; and 3 minutes at 72°C. The PCR fragments were analyzed by 3% agarose gel electrophoresis and visualized by ethidium bromide staining. The band intensities of type I and type III collagens were normalized to the GAPDH band intensities. The contents of each RNA species were extrapolated to the entire left ventricle in each group.

Determination of Total Left Ventricular Hydroxyproline Content
A 50-mg sample of frozen tissue from each ventricle was dried and hydrolyzed for 24 hours at 110°C by addition of HCl to a final concentration of 6N. The hydroxyproline content was measured spectrophotometrically at 557 nm by its reaction with chloramine T, perchloric acid, and p-dimethylaminobenzaldehyde solution according to Woessner.24 Hydroxyproline values were determined directly from a standard curve; with the assumption that hydroxyproline makes up 12.7% of the total collagen, the collagen concentration was calculated. Collagen content was obtained by multiplication of collagen concentration by left ventricular weight.

Statistical Analysis
Results are expressed as mean±SEM. Statistical analysis was carried out with one- and two-factor ANOVA followed by Bonferroni/Dunn's test for comparison of multiple groups. Correlation coefficients were obtained by the least-squares method. Data were analyzed with STATVIEW 4.0 software (Abacus Concepts Inc).


*    Results
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 up arrowMethods
 *Results
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Blood Pressure
Systolic pressure was elevated as early as the 2nd week after application of the clip, and hypertension continued until the 4th month in the untreated clipped group (Fig 1). Systolic pressure of the Spiro-4m group was significantly higher (F=34.6, P<.0001) than that of the age-matched sham controls (sham-4m) and did not differ from the values of the clip-4m placebo group. On the other hand, blood pressure decreased significantly in the Los-4m group compared with the clip-4m group (F=34.6, P<.0001) and the Spiro-4m group (F=34.6, P<.0001) but remained slightly although significantly higher than the blood pressure of the sham-4m group (F=34.6, P<.01).



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  		Figure 1. Line graph shows systolic blood pressure (mean±SEM). {blacksquare} indicates sham rats; {bullet}, clipped rats; {blacktriangleup}, losartan-treated rats (Los-4m); and *, spironolactone-treated rats (Spiro-4m).

Left Ventricular Hypertrophy
LVH, estimated by a significant increase in the ratio of left ventricular weight to body weight (LV/BW), was present from 1 month (clip-1m) in the clipped animals to 4 months (clip-4m, Table 1). LVH regressed in the losartan-treated rats and was not significantly different from the LV/BW ratio of the sham-4m group. Treatment with spironolactone did not completely regress LVH, because the LV/BW ratio was significantly higher in the Spiro-4m group than in the sham-4m group but significantly lower than in the clip-4m group.


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  		Table 1. Left Ventricular Hypertrophy

Plasma and Urinary Parameters
PRA was significantly higher in the clip-1m and clip-4m rats than in age-matched controls (Table 2). PRA was three times less elevated at 4 months after clipping than at 1 month, and this decrease was statistically significant (F=14.4, P<.0005). PRA of the Spiro-4m group was comparable to PRA of the sham-4m group. PRA of the Los-4m group was significantly higher than PRA of the clip-4m, sham-4m, and Spiro-4m groups and reached values close to those of the clip-1m group.


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  		Table 2. Renin and Aldosterone Parameters

The 24-hour urinary excretion of aldosterone was significantly elevated in the untreated clipped groups compared with the age-matched sham controls (Table 2). This parameter was twofold greater 1 month after the clip than 4 months after. Urinary aldosterone values of the Los-4m and Spiro-4m groups were similar and both significantly decreased. They were lower than in the clip-4m group but higher than in the sham-4m group. PRA and total excreted aldosterone (excluding the Los-4m group) were correlated (r=.94, P<.01).

Collagen Quantification
Hydroxyproline Content
The collagen content estimated from the hydroxyproline content was significantly greater in clipped untreated rats than in age-matched sham controls (Fig 2A). Rats treated with losartan had lower collagen contents than did the clip-4m group (not statistically different; F=1.81, P=.35) but higher than the sham-4m group (not statistically different; F=1.81, P=.33). The collagen contents of the rats treated with spironolactone were not significantly different from those of the clip-4m group.



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  		Figure 2. Graphs show collagen content estimated from hydroxyproline content (A) and by morphometry at a final magnification of x15 (B) (mean±SEM). C, Linear regression between biochemical and morphometric data (r=.66, F=49.6, P<.0001). {square} indicates 1-month sham rats (sham-1m); {circ}, 1-month clipped rats (clip-1m); {blacksquare}, 4-month sham rats (sham-4m); {bullet}, 4-month clipped rats (clip-4m); {blacktriangleup}, losartan-treated rats (Los-4m); and *, spironolactone-treated rats (Spiro-4m). Horizontal bars indicate significant differences, P<.05.

Macroscopic Study
The macroscopic study provided an estimate of the total collagen surface area (perivascular and microscar collagens). The collagen surface area per section was increased in the untreated clipped rats compared with sham-operated rats (Fig 2B). The collagen surface area in the Los-4m group was significantly lower than in the clip-4m group. The collagen surface area in the Los-4m group was also significantly lower than in the clip-1m group (F=5.0, P<.05), probably indicating that the treatment caused macroscopic fibrosis to regress. The collagen surface area in the rats treated with spironolactone did not differ from values in the clip-4m and clip-1m rats and was significantly higher than in the sham-4m group. The correlation between the collagen content estimated from the hydroxyproline content and the macroscopic collagen surface area was statistically significant (r=.66, F=49.6, P<.0001; Fig 2C).

Polarization Microscopic Study
The images obtained with cross-polarizing filters showed some areas around the coronary artery that appeared red under standard light (Fig 3, line 1) but were not birefringent (Fig 3, line 2). With the 550-nm band-pass filter (Fig 3, line 3), the bright areas corresponded to yellow-red collagen fibers, whereas with the 700-nm band-pass filter (Fig 3, line 4), the bright areas corresponded to green collagen fibers. These areas could contain both yellow-red and green collagen fibers. Therefore, the sum of the surface areas of the yellow-red and green collagen fibers was always greater than the total collagen surface area, because the total was computed from the resulting image of an "OR" Boolean operator between the image obtained with the 550-nm band-pass filter and the one obtained with the 700-nm band-pass filter. Fig 3 also shows a characteristic illustration of the morphometric quantifications: at 4 months after clip application, the perivascular collagen had increased in the untreated clipped rats, the yellow-red collagen fiber content had decreased in the Los-4m group, and the green collagen fiber content had decreased in the Spiro-4m group.


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  		Figure 3. Micrograph shows Sirius red–stained sections of coronary arteries and reactive pericoronary fibrosis under standard light (line 1) and with polarized light without band-pass filter (line 2). With the 550-nm band-pass filter the highlighted regions are yellow-red collagen fibers (line 3), and with the 700-nm band-pass filter the highlighted regions are green collagen fibers (line 4). Magnification x100. Rat groups are as defined in Fig 2 legend and "Methods."

Interstitial fibrosis. The densities of interstitial total collagen (Fig 4A), interstitial yellow-red collagen fibers (Fig 4B), and interstitial green collagen fibers (Fig 4C) were not significantly different between clipped rats and sham controls. Treatment with spironolactone or losartan decreased slightly but not significantly the densities of interstitial total collagen and interstitial yellow-red collagen fibers compared with the clip-4m group. Treatment with spironolactone significantly decreased the density of interstitial green collagen fibers to below that of the clip-1m, clip-4m, and sham-4m groups, whereas treatment with losartan did not decrease this density compared with the clip-4m rats.



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  		Figure 4. Bar graphs show interstitial fibrosis in the endocardium estimated by morphometry at a final magnification of x250: total (A), yellow-red (B), and green (C) collagen fiber densities (mean±SEM). Horizontal bars indicate significant differences, P<.05. Rat groups are as defined in Fig 2 legend and "Methods."

Perivascular fibrosis. The perivascular total collagen and perivascular yellow-red collagen fiber contents in the clip-1m and sham-1m groups were similar (Fig 5A and 5B). However, the perivascular green collagen fiber content began to increase by 1 month after renal clipping in the untreated clipped rats (Fig 5C), and perivascular yellow-red and green collagen fiber contents (and thus total collagen) increased significantly between the 1st and 4th month after clipping. Losartan treatment significantly decreased perivascular total collagen and yellow-red collagen fiber contents, whereas it had no significant effect on perivascular green collagen fiber content compared with placebo treatment. Perivascular total collagen and perivascular yellow-red collagen fiber contents were not significantly decreased in the group of rats treated with spironolactone. However, spironolactone treatment significantly decreased green collagen fiber content in the perivascular area compared with placebo treatment to below that of the clip-1m and sham-1m groups.



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  		Figure 5. Bar graphs show pericoronary fibrosis estimated by morphometry at a final magnification of x250: total (A), yellow-red (B), and green (C) pericoronary collagen fibers (surface area of the perivascular collagen normalized to the cross-sectional area of the vessel lumen; mean±SEM). Horizontal bars indicate significant differences, P<.05. Rat groups are as defined in Fig 2 legend and "Methods."

Microscar fibrosis. The differences in the cumulative surface areas of microscars were never statistically significant from one group to another because of the large variation in this measurement.

The number of microscars increased significantly with age in untreated rats, and this increase was more pronounced in clipped rats than in sham controls (F=4.5, P<.05; Fig 6A). The cumulative surface areas of total and yellow-red collagen fibers in the microscars in untreated clipped rats were not different from the values in age-matched sham controls (Fig 6B and 6C). However, the cumulative surface area of green collagen fibers in the clip-4m rats increased to become greater than in the sham-4m group (P=NS, Fig 6D).



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  		Figure 6. Bar graphs show number and surface area of microscars: frequency of microscars per ventricle (A) and cumulative surface area of total (B), yellow-red (C), and green (D) collagen fibers (mean±SEM). Horizontal bars indicate significant differences, P<.05. Rat groups are as defined in Fig 2 legend and "Methods."

Losartan treatment did not decrease the number of microscars, but the microscar surface areas of the total and yellow-red collagen fibers in the Los-4m group were smaller than in the clip-1m, clip-4m, and sham-4m rats (P=NS). The microscar surface area of the green collagen fibers in the Los-4m group was smaller than in the clip-4m group but greater than in the sham-4m group (P=NS). The number of microscars in the Spiro-4m group was the highest of all groups. The cumulative surface areas of total and yellow-red collagen fibers in the Spiro-4m group were greater than in all other groups (P=NS), whereas the cumulative surface area of green collagen fibers was similar to that in the clip-4m group. Thus, losartan treatment did not decrease the number of microscars but did reduce their size, whereas spironolactone treatment increased the number of microscars. In the Spiro-4m group, the yellow-red collagen fiber content was increased in the microscars, whereas the green collagen fiber content was not affected by the treatment.

Overall yellow-red and green collagen fiber contents. The overall yellow-red (Fig 7B) and green (Fig 7D) collagen fiber contents in the clipped untreated rats were significantly increased 4 months after clipping compared with sham controls. These increases were significantly prevented by losartan treatment compared with the placebo group. Spironolactone did not decrease the overall yellow-red collagen fiber content but decreased significantly the overall green collagen fiber content compared with the clip-1m and clip-4m groups, even to a significantly lower level than did the losartan treatment.



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  		Figure 7. Bar graphs show overall yellow-red (B) and green (D) collagen fiber contents (mean±SEM) and mean relative content (sham-1m=1) of type I collagen (A) and type III collagen (C) mRNAs. Horizontal bars indicate significant differences, P<.05. Rat groups are as defined in Fig 2 legend and "Methods."

The ratio of yellow-red to green collagen fiber ranged from 1.22±0.16 in the Los-4m group to 4.17±0.52 in the Spiro-4m group (sham-1m, 2.06±0.23; clip-1m, 2.69±1.11; sham-4m, 2.06±0.27; clip-4m, 1.92±0.19). This ratio for the Spiro-4m rats was significantly higher than for all the other age-matched groups.

Overall total collagen content. The estimated overall total collagen content increased uniformly in all fibrotic compartments in the clip-1m and clip-4m groups compared with age-matched sham groups; this increase was more pronounced at 4 months than at 1 month after clipping (Table 3). The increase in the number of microscars and in the pericoronary and interstitial fibrosis in the Spiro-4m group resulted in the dramatically greater overall total collagen content compared with the sham-4m group. The overall total collagen content in the Los-4m group was slightly lower than in the clip-1m group and close to that of the sham-1m group, as it was shown in the macroscopic study.


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  		Table 3. Total Collagen Content in Each Fibrotic Compartment

Analysis of the PCR Products
We analyzed the agarose gel electrophoresis visualized by ethidium bromide staining (Fig 8) by videodensitometry to semiquantify the PCR products and by extrapolation the mRNA contents. The mRNA contents for the type I (Fig 7A) and type III (Fig 7C) collagens were similar to the overall yellow-red (Fig 7B) and green (Fig 7D) collagen fiber contents, respectively, except in the losartan group, in which the decrease of type III collagen mRNA content was more pronounced than the decrease of green collagen fiber content.



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  		Figure 8. Agarose gel electrophoresis of polymerase chain reaction products visualized by ethidium bromide staining. MW indicates 100-bp DNA molecular weight markers. Data are from one representative experiment. Rat groups are as defined in Fig 2 legend and "Methods."


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
Left ventricular overload associated with arterial hypertension leads to LVH and myocardial fibrosis. Alterations in myocyte size and shape are the major determinants in ventricular dimensions (for review, see Reference 2525 ). Cardiac fibroblasts are believed to be solely responsible for producing and depositing fibrillar collagen, mainly types I and III collagens, within the cardiac extracellular matrix.6 26 Factors regulating collagen synthesis in fibroblasts are still debated. It has been postulated that hormonal (aldosterone, Ang II) and/or hemodynamic (ventricular overload, high coronary perfusion pressure) factors could mediate directly or indirectly fibroblast "activation." The matrix accumulation seems to begin in the adventitia of coronary arteries (pericoronary fibrosis) and extend into the adjacent intermuscular interstitial network (interstitial fibrosis). Pericoronary and interstitial fibroses have been associated with abnormalities in myocardial stiffness.27 28 In parallel with interstitial and pericoronary fibroses, replacement fibrosis begins after myocyte necrosis, inducing diastolic and systolic dysfunctions in chronic hypertension.29 In the present study we have followed, in the renovascular hypertensive rat model, changes in the content and nature of the collagen in left ventricular fibrotic compartments. To assess the respective roles of hormonal and hemodynamic factors, we used specific inhibitors of Ang II (losartan) and aldosterone (spironolactone).

Left ventricular collagen was analyzed and quantified with polarization microscopy. Pickering and Boughner30 showed that this technique enhanced the contrast between collagen and cardiac tissue, thus allowing a reliable computer-assisted morphometric evaluation of fibrosis. They validated this approach by correlating morphometric quantifications with biochemical measurements. The present study confirms and extends earlier data for collagen estimates ranging from only 4% to 6% (Fig 2). Using polarization microscopy, we have found that collagen densities in the interstitial fibrotic compartment are lower than in studies in which collagen is estimated by Sirius red staining under standard light. This is probably due to the choice of exclusion criteria; the interstitial fields contained no vessels or microscars. Furthermore, some of the areas appearing red under standard light were not birefringent under polarized light (Fig 3). The Sirius red enhanced the natural birefringence of the collagen but is not birefringent by itself. This demonstrates that the polarized light and standard light approaches cannot provide comparable absolute collagen values.

Polarization microscopy and Sirius red staining were first used for assessment of collagen phenotype by Junqueira et al.22 They postulated that this method can distinguish between type I and type III collagens and have shown by spectral analysis of collagen color with polarization microscopy that type III collagen presents a characteristic peak at 700 nm (green fibers), whereas type I collagen presents a peak at 550 nm (orange-colored fibers). Various studies have confirmed the capacity to distinguish between the types of collagens using both electronic and polarization microscopy.17 31 32 33 34 Later, Junqueira et al16 showed that the colors and intensities of birefringence displayed by types I or III collagens in routine histological slides (5 to 7 µm) are due to differences in their patterns of physical aggregation. More recently, Ogbuihi et al35 have also used quantitative polarizing microscopy for the evaluation of types I and III collagens on lung sections. However, collagen typing by means of fibril diameter morphometry remains a controversial issue since Keene et al36 have postulated that type III collagen fibers would not form distinct fibrils but would be incorporated throughout the resulting banded collagen fibrils, together with type I collagen fibrils, regardless of fibril diameter. In vitro, Dayan et al18 studied the polarization colors of various purified collagens in fibers of similar thickness and showed that thin fibers of collagens and procollagens gave green to yellowish-green polarization colors, whereas thick fibers showed yellowish-orange to red polarization colors, except for thick fibers of procollagens. Thus, procollagens and thin fibers would appear green, and thick fibers would appear orange-colored, whatever the collagen type. However, that study18 was made on purified collagens, and the colors were estimated by one investigator who was not computer assisted. In vivo, the type III collagen triple helix is absent from the surface of large-diameter fibrils,37 38 indicating that large-diameter fibrils are not type III collagen fibrils. Last, studies in the heart strongly suggest that thin fibers represent mainly type III collagen fibers and thick fibers mainly type I collagen. A study by Pick et al39 showed that collagen accumulated in the evolutionary phase of hypertrophy in the nonhuman primate myocardium with pressure overload, including a disproportionate amount of newly formed thin perimysial fibers. They used pepsin extraction and electrophoresis to show that the amount of type III rose significantly from its normal value of 11% to 16%, suggesting that the increase in thin perimysial fibers was mainly composed of type III collagen. Moreover, the ratio of type I to type III collagens in the heart ranges from 1.4 to 1.940 and is thus comparable to the ratio of yellow-red to green collagen fiber contents we have obtained. Otherwise, a semiquantitative analysis of the mRNA for type I and type III collagens was done. The profiles obtained for the mRNAs matched with the profiles of the yellow-red and green collagen fibers assessed by polarization microscopy except in the Los-4m group. In this group, green collagen fiber content is slightly decreased, whereas the type III collagen mRNA level is dramatically decreased compared with the clip-4m and Spiro-4m groups. However, the mRNA content quantified 4 months after clipping is the result of the production at this time and thus possibly cannot match with the protein content quantified at the same time. Nonetheless, these data suggest that the yellow-red collagen fiber composed of closely packed thick fibrils giving an intense birefringence with a yellow-red color could be mainly type I collagen fibers, whereas the green collagen fibers forming thin fibers composed of loosely packed thin fibrils giving a weak greenish birefringence could be mainly type III collagen fibers. Coupling polarization microscopy to computerized morphometry provides a simple way to quantify simultaneously these two types of collagens in situ, a task difficult to achieve with antibodies.

In the present study, pericoronary collagen fiber content was increased 4 months after clipping. The density of total interstitial collagen fibers decreased 1 month after clipping because left ventricular weight was greatly increased, whereas the interstitial collagen content increased to a lesser extent (except for green collagen fiber content). That the interstitial collagen density was not increased while the pericoronary collagen was increased provides further support for the hypothesis that interstitial fibrosis begins in the adventitia of coronary arteries and then extends in the neighboring interstitial space. As shown by Ratajska et al41 in response to long-term elevations in plasma Ang II in rats, and by Reddy et al42 after injection of Ang II in dogs, coronary arteries are the site of permeability changes that could result in perivascular fibrosis. An increase in coronary perfusion pressure could also be the direct or indirect activation signal for the adventitial fibroblasts. Indeed, collagen production can be mechanically stimulated in vitro in cardiac fibroblasts,43 and there is no pericoronary fibrosis in models in which the coronary perfusion pressure is normal. For instance, the experimental infarct induced by coronary ligation is a model in which there is no pericoronary fibrosis despite replacement subendocardial fibrosis (unpublished observations). Similarly, there is no pericoronary fibrosis in cardiac eccentric hypertrophy due to volume overload (aorto-caval fistula model).44 Nevertheless, the study of Brilla et al7 indicates that perivascular fibrosis would not be related to arterial pressure because in the infrarenal aortic banding model, no perivascular fibrosis was found. These contradictory results reveal that perivascular fibrosis probably develops when several factors (Ang II, aldosterone, blood pressure, peptides, growth factors, etc) act together.

Ang II not only induces hypertrophy of cardiomyocytes in vitro but also a mitogenic response through type 1 (AT1) receptors in neonatal cardiac fibroblasts.11 14 45 Despite the presence of AT1 receptors at the surface of adult cardiac fibroblasts, Villarreal et al13 have shown that Ang II failed to induce a proliferative response, whereas Crabos et al12 have shown that Ang II induced DNA synthesis. There is also evidence that Ang II causes upregulation of transforming growth factor-ß1 in cardiomyocytes.45 This growth factor can stimulate type I collagen synthesis.46 47 In vitro, it has been shown that Ang II stimulation of AT1 receptors triggers an increased gene expression of extracellular matrix proteins in cardiac fibroblasts.12 13 In vivo, Crawford et al15 have shown that Ang II stimulates fibronectin and type I and type IV collagen expression within nonmyocytic cells via AT1 receptors. Moreover, the overexpression of cardiac angiotensin-converting enzyme associated with high blood pressure–induced LVH48 could locally modulate Ang II production. In our experiment, rats treated with losartan had lower total, yellow-red (type I), and green (type III) collagen fiber contents and lower mRNA levels, further indicating a possible action of Ang II on cardiac fibroblasts. However, given that blood pressure was decreased in the losartan group, the results observed in this group cannot be definitively attributed to Ang II or to hemodynamic factors, although the blood pressure in the Los-4m group was slightly higher than in sham controls. Several studies have shown that a range of treatments could prevent the development of fibrosis (for review, see Reference 44 ), but in our study fibrosis was established 1 month after clipping, before treatment began, and the morphometrically estimated collagen content decreased in the rat group treated with losartan compared with 1-month clipped rats, indicating that losartan causes fibrosis to regress.

Aldosterone type I receptors are present in the cardiac myocytes and fibroblasts of rabbit hearts.49 Glucocorticoid and mineralocorticoid hormones have cross-affinities for their respective type I and type II receptors. Since the plasma glucocorticoid hormone concentration is 100- to 1000-fold greater than that of aldosterone, the mineralocorticoid response can occur only in the presence of 11ß-hydroxysteroid dehydrogenase.50 This enzyme transforms glucocorticoid into a metabolite that has a low affinity for type I and type II receptors, leaving them free for mineralocorticoid binding. Its presence in the heart is still debated.50 51 Irrespective of the mode of action of aldosterone, Brilla and Weber10 showed that spironolactone (at the same dose as in our protocol, 20 mg/kg per day) prevented the increase in total and interstitial collagen densities in Sprague-Dawley rats as well as in the surface area of perivascular and microscar collagens in several experimental hypertension models, including the renovascular hypertension model. Although the study of Brilla et al has similarities to the present one, there are significant differences: Brilla et al pretreated their rats subcutaneously with spironolactone for 1 week before clipping and thereafter during 8 weeks, and they estimated the fibrosis on sections stained with Sirius red under standard light. In our study, treatment began 1 month after clipping when hypertension was established and fibrosis was already increased. This crucial point distinguishes our study from that of Brilla et al: they tested the effect of spironolactone on the establishment of the fibrotic process (prevention), whereas we tested the capacity of spironolactone to stop and regress this process. In addition, our treatment was administered orally, and the bioavailability of spironolactone could be different after subcutaneous injection; thus the concentrations used may not be equipotent. Above all, the average systolic pressure of the rats treated with spironolactone was 214±28 mm Hg, and it was 225±16 mm Hg in the placebo group in our study. In the study of Brilla et al, the clipped rats preventively treated with spironolactone had a systolic pressure of 156±10 mm Hg, and that of the placebo group was 202±12 mm Hg. This difference of 60 mm Hg between the spironolactone-treated groups could result in differences in the potential action of spironolactone. In our study, spironolactone not only prevents the increase in green (type III) collagen fiber content in all fibrotic compartments, it even causes the green (type III) collagen fiber content to become lower than in the age-matched sham rats and in the clip-1m rats (except in the microscars). The effect of spironolactone on green collagen fiber content is related to a reduction of type III collagen mRNA level. Conversely, spironolactone had a significant effect neither on the yellow-red (type I) collagen fiber content nor on the type I collagen mRNA level. This suggests that aldosterone specifically stimulates the deposition of type III collagen, whereas it would have no action on type I collagen deposition. That the content in green collagen fibers (type III) in the spironolactone group is below that of control groups could mean that aldosterone could be a physiological mediator of type III collagen synthesis, its blockade leading to a decrease in type III collagen content below that of controls in which a nonantagonized basal level of aldosterone would ensure a basal synthesis of type III collagen. The decrease in the overall green (type III) collagen fiber content in the Los-4m group is probably due to a decrease of the aldosterone in this group (Table 2).

There is evidence that an elevated circulating Ang II concentration is involved in the generation of cardiac myocyte necrosis,52 possibly dependent on vasoconstriction and the resultant ischemia. This would suggest that the number of microscars should decrease after losartan treatment, which was not observed (Fig 6). This may have been because the microscar process was probably initiated before the treatment began 1 month after clipping (Fig 6A). Several studies in which rats receiving Ang II presented microscopic scars after only 2 weeks of treatment confirm this hypothesis.52 53 54 However, the collagen fraction of the microscars in the Los-4m group was normalized compared with the sham-4m group (Fig 6B), whereas the number and collagen fraction of the microscars in the rats treated with spironolactone were much higher than those of the clipped placebo group. We have no explanation for the latter observation, but we propose the hypothesis that long-term administration of a mineralocorticoid antagonist could imbalance the ionic pools within the heart and thus could influence cardiac function and structure.

In conclusion, we have obtained evidence that aldosterone stimulates green (type III) collagen deposition independently of hypertension but does not alter yellow-red (type I) collagen accumulation. On the contrary, Ang II seems to enhance yellow-red (type I) collagen deposition and LVH in association with its hypertensive activity.


*    Acknowledgments
 
This work was supported by grants from Searle Laboratories and the Ministry of Research (France) (CIFRE No. 156/92). We thank Irene Laboulandine for technical assistance.


*    Footnotes
 
Reprint requests to A. Nicoletti, Hôpital Broussais, INSERM U430, 96, rue Didot, 75014 Paris, France. E-mail nicolett@citi2.fr.

Received December 8, 1994; first decision January 3, 1995; accepted February 8, 1995.


*    References
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