Modulation of Cardiac Mast Cell Mediated Extracellular Matrix ...

Articles in PresS. Am J Physiol Heart Circ Physiol (February 18, 2005). doi:10.1152/ajpheart.00765.2004

Modulation of Cardiac Mast Cell Mediated Extracellular Matrix Degradation by Estrogen

Amanda L. Chancey Jason D. Gardner David B. Murray Gregory L. Brower Joseph S. Janicki

Department of Anatomy, Physiology, and Pharmacology Auburn University, Auburn, AL 36849

Running Head: H-00765-2004.R1; Estrogen and Cardiac Mast Cells

Address Correspondence To:

Joseph S. Janicki, Ph.D. Dept. of Anatomy, Physiology, and Pharmacology 106 Greene Hall Auburn University Auburn, AL 36849-5517 (334) 844-3700 (office) (334) 844-3697 (fax) [email protected] (email)

Copyright © 2005 by the American Physiological Society.

Estrogen and Cardiac Mast Cells

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Abstract There are fundamental differences between males and females with regard to susceptibility to heart disease. Although numerous animal models of heart failure have demonstrated that premenopausal females are afforded cardioprotection and therefore fare better in the face of cardiac disease than their male counterparts, many questions as to how this occurs still exist. Recently, we have shown: 1) that increased mast cell density is associated with adverse ventricular remodeling; and 2) chemically induced mast cell degranulation using compound 48/80 resulted in remarkable changes in matrix metalloproteinase (MMP) activity, cardiac collagen structure, and cardiac diastolic function in normal male rats. With the known gender differences in cardiac disease in mind, we sought to examine the effects of chemically induced cardiac mast cell degranulation in isolated, blood-perfused hearts of intact females, ovariectomized females, and ovariectomized females treated with 17$-estradiol. In response to mast cell degranulation, no significant differences in cardiac function, MMP-2 activity, or collagen volume fraction (CVF) were observed between intact females or ovariectomized females treated with estrogen. Whereas, in the ovariectomized female group, a significant rightward shift in the left ventricular pressure-volume relationship was noted post 48/80, accompanied by a marked 133 % increase in active MMP-2 values over that seen in intact female hearts post 48/80 (p < 0.05) and a significant reduction in CVF below control (0.46 + 0.23 vs. 0.73 + 0.13, respectively; p < 0.05). These findings indicate that estrogen’s cardioprotective role can be partially mediated by its effects on cardiac mast cells, MMPs, and the extracellular matrix. Keywords: isolated heart, matrix metalloproteinase, gender, ventricular remodeling


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Introduction Despite the fact that gender differences in the prevalence and severity of cardiovascular disease have been identified in humans and in animal models of heart failure (24; 36), the mechanisms mediating cardioprotection in premenopausal females are poorly understood. We recently reported that male rats respond to chronic biventricular volume overload induced by an aortocaval (AV) fistula with a predictable pattern of significant left ventricular (LV) hypertrophy and dilatation and accompanying cardiac dysfunction (8). In contrast, female rats subjected to chronic volume overload did not develop LV dilatation and maintained compensated cardiac function with appropriate myocardial hypertrophy (14). Mast cell mediated activation of matrix metalloproteinases (MMPs) is one mechanism contributing to the development of ventricular dilatation in males. This finding was established by previous studies demonstrating an association between increased mast cell density, activation of MMPs, and the initiation of cardiac remodeling in volume overload induced by an AV fistula in male rats (4; 22) and by mitral regurgitation in dogs (38). We have also shown that mast cell degranulation induced by endothelin-1 or compound 48/80 in the isolated, blood perfused heart from male rats resulted in MMP activation, extensive collagen matrix degradation, and LV dilatation within 30 minutes (10; 31). 17$-estradiol, hereafter referred to as estrogen, has been shown to exert cardioprotective effects in animal studies (35; 37). It is interesting that a few studies have reported direct effects of estrogen on non-cardiac mast cells (16; 25), consistent with the observation that mast cells from the aorta of premenopausal women express estrogen receptors (33). Thus, the gender differences in the pattern of myocardial remodeling observed in response to an AV fistula may be related to hormonal modulation of mast cell


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function. Accordingly, we sought to test the hypothesis that estrogen mediated effects on cardiac mast cells account for the lack of adverse remodeling in the female heart subjected to chronic volume overload. To this end, we sought to determine if differences in the response to acute, chemically-induced degranulation of cardiac mast cells exist among intact females, ovariectomized females, and ovariectomized females treated with estrogen. Our results indicate that the changes evoked in response to cardiac mast cell degranulation are strongly dependent on the presence of estrogen.


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Materials and Methods The surgical procedures and subsequent care of the animals conformed with the principles of the National Institutes of Health “Guide for the Care and Use of Laboratory Animals,” and the protocol was approved by Auburn University’s Institutional Animal Care and Use Committee. In all terminal procedures, the animals were deeply anesthetized using sodium pentobarbital (50 mg/kg, i.p.). In order to prevent possible effects of a soy based diet, all animals were maintained on a diet that contained minimal phytoestrogens (Harlan Teklad Diet 2014).

Surgical Preparation and Experimental Protocol Age-matched adult female Sprague Dawley rats were randomly divided into groups as follows: intact females (n=6), ovariectomized females (n=7), and ovariectomized females receiving 0.02 mg/kg/day estrogen (n=6). This dose of estrogen was chosen based on preliminary data from our laboratory indicating that ovariectomized females treated with 0.02 mg/kg/day estrogen are protected from adverse myocardial remodeling induced by an AV fistula (13). Therefore, females supplemented with estrogen were implanted subcutaneously with 0.25 mg time-release pellets (Innovative Research of America, Sarasota, FL) using a 10 gauge precision trochar at the time of ovariectomy to achieve a dosage of 0.02 mg/kg/day. The ovariectomy procedure was performed two weeks prior to obtaining isolated heart function. Briefly, with the rat under halothane inhalation anesthesia, the ovaries are approached via a ventral abdominal laparotomy, the ovarian pedicle ligated and the ovary


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excised. The abdominal musculature and skin were closed using standard techniques with absorbable sutures and autoclips, respectively.

Experimental Design The effects of cardiac mast cell degranulation on ventricular function was determined using an isolated, blood perfused heart preparation as previously described (8). Briefly, the ascending thoracic aorta in the anesthetized rat was cannulated for continuous retrograde perfusion of the heart via an apparatus consisting of a pressurized perfusion reservoir (100-105 mm Hg) and a venous collection reservoir connected in circuit with a female support rat. The perfusion apparatus was primed with blood obtained from female blood donors. In the case of the ovariectomized group, the support and blood donor rats were ovariectomized as well. Following positioning of a compliant balloon in the LV, pressure-volume relationships for each heart were obtained both prior to and 30 minutes after infusion of the mast cell secretagogue, compound 48/80 as previously described (10).

Administration of 1 ml of 0.9% sterile saline containing 7.2 mg of

compound 48/80 (Sigma, St. Louis, MO) to the isolated heart was accomplished by introducing the solution into the pressurized perfusion reservoir, allowing the solution to mix with the reservoir blood and perfuse through the beating heart. In a previous study, this dosage of compound 48/80 resulted in near 100% cardiac mast cell degranulation (10). The coronary venous blood containing 48/80 was not returned to the support rat. After completion of the functional studies, the atria and great vessels were removed and LV (plus septum) and right ventricle (RV) were separated and weighed. A complete transmural section of the LV at midventricle was placed in buffered formalin for fixation,


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and the remainder was minced into -1 mm cubes, snap-frozen in liquid nitrogen, and stored at !80° C. The formalin-fixed tissue was processed for routine histopathology and 5 micron thick paraffin-embedded sections were stained with either pinocyanol erythrocynate for visualization of mast cell morphology or picrosirius red for quantification of collagen volume fraction (CVF), presented as the percentage of collagen area relative to total myocardial area as previously described (10). Portions of the frozen LV tissue were used to determine the wet-to-dry weight ratio as a measure of myocardial water content and for western blot analysis of protein expression. These analyses were performed with the observer blinded as to the source of the tissue sections.

Matrix Metalloproteinase Activity Matrix metalloproteinase activity in cardiac tissue extracts was performed by standard gelatin zymography procedures using a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) matrix containing gelatin (1 mg/ml) (9).

All of the

zymograms had two lytic bands corresponding to standards for the proenzyme (68 kd) and activated (62 kd) forms of gelatinase A (MMP-2) and the proenzyme (95 kd) and activated (82 kd) forms of gelatinase B (MMP-9; Chemicon International, Inc., Temecula, CA). Each gel was run in duplicate, and in order to compare results from different gels, extract from a single heart was used as a standard on all gels. The activity of the lytic bands in the other lanes of a gel were expressed as a percent of this standard’s activity.

Once

normalized in this fashion, the percent activities from hearts belonging to each group were averaged, with the intact female group set to 100%.


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Western Blot Analysis Expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-3 protein was determined by western blotting of immunoprecipitated samples. Protein concentration of LV homogenates was determined using BCA reagent (Pierce, Rockford, IL). Equal amounts of protein (20:g) were immunoprecipitated using the protein A agarose bead separation method (Sigma, St. Louis, Mo) and monoclonal rabbit anti-human TIMP-1 or TIMP-3 antibodies (Research Diagnostic Inc, Flanders, NJ). Appropriate negative controls were performed by substituting water in place of the primary antibody prior to the addition of agarose beads. Equal amounts of supernatant (4 ul) were separated by 10% SDSPAGE and transferred onto nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA). Membranes were probed with primary antibodies for TIMP-1 and TIMP-3 (1:1000) for 4 hrs at room temperature, washed (30 min) and incubated with anti-rabbit secondary antibody conjugated to horse radish peroxidase (Amersham Biosciences Corp, Piscataway, NJ). Immunoreactive bands were visualized using enhanced chemiluminescence reagents (ECL, Amerhsam Biosciences Corp) exposed to hyperfilm at the linear range of film density. The films were scanned and densitometric analysis was performed using Un Scan It Software (Silk Scientific Inc, Orem, Utah).

Statistical Analysis Statistical analyses were performed with Systat 9.0 software (SPSS Inc., Chicago, IL). All grouped data are expressed as means ± SD, unless otherwise noted. Grouped data comparisons were made by one-way analysis of variance with intergroup comparisons analyzed using Bonferroni post-hoc testing. Statistical significance was taken to be p #


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0.05. The pre- and post-48/80 pressure-volume curves for each heart were fit to a 3rd order non-linear regression (SigmaPlot, SPSS Science, Chicago, IL), and the volumes corresponding to pressure increments of 2.5 mm Hg were determined. The volumes were then averaged to obtain the final pressure-volume relationship for each group. Pressurevolume curves were analyzed using a two-factor repeated measures ANOVA.


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Results Histological examination demonstrated comparable, extensive 48/80 induced mast cell degranulation in all groups (i.e., > 95%).

An increase of approximately 3% in

myocardial water also occurred in the three groups, presumably from mast cell released histamine. However, in contrast to our findings in a previous study (10), mast cell degranulation did not result in increases in MMP activity in the hearts from intact and ovariectomized + estrogen females.

It was only in the ovariectomized females not

receiving supplemental estrogen that 48/80 caused a 133% elevation in MMP-2 activity above that of intact females and 112% above that of the ovariectomized + estrogen group (p < 0.007; Figure 1). Average density of the latent MMP-2 band present was not different among groups. While bands representative of latent and active MMP-9 were present, 48/80 had no effect on the density of these bands. Moreover, the intensity of the MMP-9 bands was markedly less than that of the MMP-2 bands.

Similarly there were no

differences in TIMP-1 and TIMP-3 expression amongst the three groups (Figure 2). The significant increase in MMP-2 activity in the hearts from ovariectomized females post 48/80 was associated with a significant reduction in collagen volume fraction (CVF) below that of intact females (0.46 + 0.23 vs. 0.73 + 0.13, p < 0.05). Conversely, estrogen replacement in ovariectomized females prevented this significant 48/80 induced decrease in CVF (0.60 + 0.09, p < 0.05 vs. ovariectomized female group; Figure 3). Consistent with our previous findings of cardioprotection in intact females, hearts from normal intact females exposed to 48/80 did not develop an alteration in the LV pressure-volume curve following mast cell degranulation (Table 1). However, hearts from ovariectomized females developed a significant parallel rightward shift in the pressure-


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volume relationship following exposure to 48/80. Estrogen replacement in ovariectomized females effectively prevented a 48/80 induced rightward shift in the pressure-volume relationship, reproducing the findings in intact females. Concurrent with a significant rightward shift in the pressure-volume curve post 48/80 in ovariectomized female hearts, end diastolic volume at a pressure of 0 mm Hg (V0) increased significantly (Figure 4). However, V0 was not significantly increased in the intact female and ovariectomized + estrogen female groups. Myocardial compliance, as assessed by the volume required to increase end diastolic pressure from 0 to 25 mm Hg ()V25), was not different in any of the groups post 48/80 (Table 1).


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Discussion Gender differences in the susceptibility to cardiovascular disease have been established in both humans and animal models (6; 11; 14; 20; 28; 36), but the mechanisms affording this cardioprotection in females are not understood. An increased incidence of cardiovascular disease in postmenopausal women suggests that ovarian hormones may play a role in this cardioprotection (24).

Estrogen acts through receptor-mediated

pathways, and among other things has been shown to inhibit the development of atherosclerosis (2; 19), and increase endothelial and inducible nitric oxide synthase (eNOS and iNOS) activity (1; 3; 21; 34). However, reduced formation of atherosclerosis and vasodilation cannot fully account for the cardioprotective actions of estrogen. Thus, while numerous possible actions of estrogen have been examined, this is the first study to evaluate the effect of estrogen on cardiac mast cell mediated myocardial remodeling. Given our previous studies demonstrating that cardiac mast cell degranulation mediates MMP activation and extracellular matrix degradation (10; 23; 31), our hypothesis was that cardioprotection in intact females would be due to estrogen mediated prevention of mast cell degranulation. Accordingly, the purpose of this study was to determine if gender differences exist in the susceptibility to mast cell degranulation or in the initiation of myocardial remodeling following chemically-induced mast cell degranulation. However, since extensive mast cell degranulation was noted in all of the female groups stimulated with compound 48/80, mast cell stabilization does not appear to be the mechanism responsible for estrogen mediated cardioprotection. Despite our initial hypothesis being incorrect, this is the first study to demonstrate that estrogen prevents the mast cell mediated MMP activation and extracellular matrix degradation previously demonstrated in


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male rats (10; 31). While this study did not address whether the estrogen cardioprotective effect was receptor mediated, preliminary data from our laboratory indicate the presence of estrogen receptors on mast cells isolated from male and female rat hearts. Therefore, greater insight into mechanisms of action will require studies using estrogen receptor antagonists. MMP-2 activity in this study was assessed using gelatinase zymography. While this technique does not directly examine collagenase activity, one would expect the activities of the various MMPs in the collagen degradation cascade to be tightly coupled (32). This was demonstrated by Gunja-Smith et al. (17), who found zymographic gelatinase activity to be closely correlated with collagenase activity determined by tritium-labeled telopeptidefree collagen degradation. Direct evidence demonstrating the ability of cardiac mast cells to activate MMPs in normal male hearts was established by recent studies from our laboratory (10). This study examined the response to chemically induced mast cell degranulation using compound 48/80, and found that cardiac mast cell degranulation resulted in a significant increase in myocardial MMP-2 activity, degradation of the myocardial extracellular matrix, and ventricular dilatation. The findings reported herein are consistent with this study in males, in that 48/80 administered to hearts from the ovariectomized female group significantly increased MMP-2 activity relative to intact females and ovariectomized + estrogen treated females. Since latent MMP-2 levels were similar for all groups, this difference in MMP-2 activity does not appear to be due to estrogen mediated suppression of MMP expression. Neither can these differences in MMP-2 activity be attributed to estrogen mediated alterations in TIMP-1 and TIMP-3 expression. Rather these findings indicate that decreased release of the mast cell


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enzymes responsible for MMP activation prevented the increase in MMP activity in the intact female and ovariectomized + estrogen treated groups. In support of this conclusion is the finding of Harnish et al. (18) that non-cardiac mast cell proteases were downregulated by estrogen. The development of extracellular matrix degradation and ventricular dilatation in the ovariectomized female hearts is consistent with the observed changes in MMP-2 activity. These changes were also comparable to our previous findings demonstrating a parallel rightward shift in the pressure-volume relationship in normal male hearts post 48/80 (10). In contrast to the significant LV dilatation and degradation of interstitial collagen (i.e., CVF) in the ovariectomized group, the pressure-volume relationships prior to and following administration of 48/80 were unchanged in the hearts from the intact female and ovariectomized + estrogen rats. Estrogen replacement also effectively prevented the significant decrease in the collagen volume fraction observed in the untreated ovariectomized females. Mast cell derived histamine is known to increase vascular permeability. Accordingly, it was not surprising that myocardial edema was seen secondary to mast cell degranulation. In all likelihood this edema was responsible for the non-significant trend of increased ventricular stiffness (i.e., reduction in )V25, Table 1) post-48/80. However, the fact that the extent of myocardial edema was comparable in all three groups indicates that histamine did not contribute to the rapidly induced myocardial remodeling observed. Comparable ventricular dilatation and extracellular matrix degradation independent of histamine were reported to occur within a similar 30 minute period by MacKenna et al. (29), utilizing rat hearts perfused with bacterial collagenase.


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These differences in MMP-2 activity and extracellular matrix degradation mediated by estrogen suggest an explanation for the different patterns of myocardial remodeling produced in male and female rats in the AV fistula model of heart failure (6; 8; 14). Males develop eccentric hypertrophy, marked ventricular dilatation and heart failure within 20 wks post-fistula (8), whereas intact females develop concentric hypertrophy without ventricular dilatation and ventricular function is maintained (14). However in a similar long-term study, ovariectomy eliminated this cardioprotective effect, with an AV fistula producing eccentric hypertrophy and ventricular dilatation comparable to that seen in males (6).

The

prevention of ventricular dilatation by estrogen is significant in light of the fact that ventricular dilatation is an independent risk factor in the progression to decompensated heart failure (26). Although estrogen has been shown to significantly affect MMP activity in the female reproductive system (27; 30), our understanding of the effect of estrogen on cardiac MMPs is limited (41). The study by Xu et al. (41) found that ovariectomy in otherwise normal aged rats resulted in a significant reduction in myocardial MMP-2 protein expression, with this decrease in MMP-2 expression being reversed by estrogen replacement. Conversely, while we saw no effect of ovariectomy on latent MMP-2 expression, we found a significant increase in mast cell mediated MMP-2 activation in hearts from ovariectomized rats. The reason for these disparate findings is not clear, but may be related to differences in age and duration of ovariectomy induced menopause between the studies. Estrogen has been shown to have anti-inflammatory effects (18; 25; 39; 40). Thus, alterations in mast cell content due to estrogen presents another possible mechanism. Harnish et al. (18) found that estrogen treatment was associated with a reduction in


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expression of mast cell proteases in colons of diseased animals, and improved intestinal health in a rat model of inflammatory bowel disease. Further, this study demonstrated that estrogen pretreatment of bone marrow-derived mast cells repressed production of several cytokines including tumor necrosis factor-" (TNF-"), interluekin-6, and interleukin-13. While all cardiac cell types have the ability to synthesize TNF-", emerging evidence indicates that constitutive expression of TNF-" is localized in cardiac mast cells (5; 12; 15). In addition, preliminary findings indicate suppression of TNF-" synthesis and/or release by ovarian hormones as the mechanism responsible for the gender specific cardioprotection from adverse myocardial remodeling secondary to chronic volume overload (7). However, the relationship between TNF-" and MMP activation in modulating myocardial remodeling remains to be elucidated. In summary, gender differences exist in the response of the isolated heart to 48/80induced mast cell degranulation. Hearts from normal males and ovariectomized females respond to mast cell degranulation with significant increases in active MMP-2 levels, significant reductions in CVF, and a rightward shift in the pressure-volume relationship. In contrast, hearts of intact females and ovariectomized females receiving estrogen supplementation do not undergo any of these changes in response to 48/80 administration. Although current knowledge of the effects of estrogen on mast cells is limited, the results of this and other studies clearly indicate that estrogen can affect the composition and/or release of mast cell contents; which in turn affect MMP activation, extracellular matrix degradation and cardiac remodeling.


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Acknowledgments This work was supported in part by NIH Grants R01-HL-62228 and R01-HL-73390 (JSJ), American Heart Association Scientist Development Grant 0435298N (JDG), American Heart Association Southern Research Consortium Postdoctoral Fellowship 0325228B (DBM) and American Heart Association Southern Research Consortium Predoctoral Fellowship 0215060B (ALC). The authors would like to thank Dr. James A. Stewart, Jr. and Mr. Steven Trott for their technical assistance.


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Figure Legends

Figure 1.

Matrix metalloproteinase (MMP-2) activity for the three experimental groups following administration of the mast cell secretagogue compound 48/80. Data presented as mean + SEM. * p < 0.05 vs. intact female; # p < 0.05 vs. ovariectomized female treated with estrogen. OX = ovariectomized female, OX + Est = ovariectomized female treated with 0.02 mg/kg/day of estrogen.

Figure 2.

Expression of tissue inhibitor of metalloproteinase-1 and -3 (TIMP-1 and TIMP-3, respectively) protein as determined by western blotting of immunoprecipitated samples. Data presented as mean + SD.

Figure 3.

Myocardial collagen volume fraction following administration of the mast cell secretagogue compound 48/80. Data presented as mean + SD. * p < 0.05 vs. intact female; # p < 0.05 vs. ovariectomized female treated with estrogen.

Figure 4.

Percent increase in left ventricular volume at an end diastolic pressure of 0 mm Hg (V0) following administration of the mast cell secretagogue compound 48/80. Data presented as mean + SEM. * p < 0.05 vs. intact female; # p < 0.05 vs. ovariectomized female treated with estrogen.

Estrogen and Cardiac Mast Cells Table 1.

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Change in V0 and )V25 in response to 48/80-induced mast cell degranulation. V0 (:l)

)V25 (:l)

pre 48/80

292.0 + 33.6

91.2 + 34.5

post 48/80

303.3 + 30.3

77.0 + 27.9

pre 48/80

312.8 + 20.3

46.1 + 16.5

post 48/80

360.2 + 21.1

36.7 + 12.3

pre 48/80

321.0 + 14.8

67.5 + 18.2

post 48/80

327.1 + 11.0

53.0 + 12.5

Group: Intact Female

OX Female

OX + Estrogen Female

Mean ± SEM.

Abbreviations:

OX = ovariectomized; V0 = left ventricular volume at an end diastolic pressure (EDP) of 0 mm Hg; )V25 = the change in LV volume between EDP 0 - 25 mm Hg.

Estrogen and Cardiac Mast Cells Figure 1.

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