Type II diabetes promotes a myofibroblast phenotype in cardiac fibroblasts.

AIMS: Cardiovascular disease is the leading cause of death for individuals diagnosed with type II diabetes mellitus (DM). Changes in cardiac function, left ventricular wall thickness and fibrosis have all been described in patients and animal models of diabetes; however, the factors mediating increased matrix deposition remain unclear. The goal of this study was to evaluate whether cardiac fibroblast function is altered in a rat model of type II DM. MAIN METHODS: Cardiac fibroblasts were isolated from 14 week old Zucker diabetic and lean control (LC) adult male rat hearts. Fibroblasts were examined for their ability to remodel 3-dimensional collagen matrices, their adhesion, migration and proliferation on collagen and changes in gene expression associated with collagen remodeling. KEY FINDINGS: Cardiac fibroblasts from diabetic animals demonstrated significantly greater ability to contract 3-dimensional collagen matrices compared to cardiac fibroblasts from LC animals. The enhanced contractile behavior was associated with an increase in diabetic fibroblast proliferation and elevated expression of α-smooth muscle actin and type I collagen, suggesting the transformation of diabetic fibroblasts into a myofibroblast phenotype. SIGNIFICANCE: Cardiac fibrosis is a common complication in diabetic cardiomyopathy which may contribute to the observed cardiac dysfunction associated with this disease. Identifying and understanding the changes in fibroblast behavior which contribute to the increased deposition of collagen and other matrix proteins may provide novel therapeutic targets for reducing the devastating effects of diabetes on the heart.

Chelation: a fundamental mechanism of action of AGE inhibitors, AGE breakers, and other inhibitors of diabetes complications.

This article outlines evidence that advanced glycation end product (AGE) inhibitors and breakers act primarily as chelators, inhibiting metal-catalyzed oxidation reactions that catalyze AGE formation. We then present evidence that chelation is the most likely mechanism by which ACE inhibitors, angiotensin receptor blockers, and aldose reductase inhibitors inhibit AGE formation in diabetes. Finally, we note several recent studies demonstrating therapeutic benefits of chelators for diabetic cardiovascular and renal disease. We conclude that chronic, low-dose chelation therapy deserves serious consideration as a clinical tool for prevention and treatment of diabetes complications.

Alteration in myocardial prostaglandin D synthase expression in pressure overload-induced left ventricular remodeling in rats.

We hypothesized that acute pharmacological blockade of cyclooxygenase-2 (COX-2) using nimesulide (Nime) would prevent maladaptive changes in left ventricular (LV) structure and function secondary to abdominal aortic coarctation-induced pressure overload (PO). In vivo LV chamber dimension and function were assessed by pressure/volume admittance catheter at 14 days' postsurgery in three groups (n ≥ 6/group): sham-operated (Sham); untreated PO; and selective COX-2 inhibitor nimesulide-treated PO (PO + Nime; 25 mg/kg/d). Treatment was initiated 24 h prior to surgical induction of PO. Relative to Sham, there was a marked increase in LV mass index in the PO groups (2.2 ± 0.01 mg/g versus 2.9 ± 0.10 mg/g Sham versus PO, PO+Nime: 2.5 ± 0.03 mg/g). End diastolic volume, an indicator of chamber size, was significantly decreased in the PO animals compared with Sham (202 ± 17µL versus 143 ± 16 µL Sham versus PO, PO + Nime: 226 ± 9 µL). Collagen levels in PO rats assessed by hydroxyproline analysis were significantly elevated relative to Sham values. Nimesulide treatment attenuated: (1) the increase in LV mass index; (2) the reduction in end diastolic volume; and (3) the PO-induced increase in myocardial collagen. In summary, acute COX-2 inhibition with nimesulide attenuated the maladaptive changes in the LV after PO. Acknowledging the clinical failure of chronic COX-2 inhibitor use, we propose that acute treatment with COX-2 inhibition during the initial stages of cardiac remodeling can be beneficial in maintaining the normal cardiac structure and function during PO.

Tryptase activates isolated adult cardiac fibroblasts via protease activated receptor-2 (PAR-2).

Protease activated receptor-2 (PAR-2) derived cycloxygenase-2 (COX-2) was recently implicated in a cardiac mast cell and fibroblast cross-talk signaling cascade mediating myocardial remodeling secondary to mechanical stress. We designed this study to investigate in vitro assays of isolated adult cardiac fibroblasts to determine whether binding of tryptase to the PAR-2 receptor on cardiac fibroblasts will lead to increased expression of COX-2 and subsequent formation of the arachodonic acid metabolite 15-d-Prostaglandin J(2) (15-d-PGJ(2)). The effects of tryptase (100 mU) and co-incubation with PAR-2 inhibitor peptide sequence FSLLRY-NH(2) (10(-6)M) on proliferation, hydroxyproline concentration, 15-d-PGJ(2) formation and PAR-2/COX-2 expression were investigated in fibroblasts isolated from 9 week old SD rats. Tryptase induced a significant increase in fibroproliferation, hydroxyproline, 15-d-PGJ(2) formation and PAR-2 expression which were markedly attenuated by FSLLRY. Tryptase-induced changes in cardiac fibroblast function utilize a PAR-2 dependent mechanism.

The contribution of prostaglandins versus prostacyclin in ventricular remodeling during heart failure.

Although the role of Cox-2 in the heart's response to physiologic stress remains controversial (i.e. expression in myocytes versus other resident myocardial cells) the ever expanding role of prostanoids in multiple models of heart failure cannot be denied. Due to the fact that prostanoids are metabolized rather quickly (half life of seconds to minutes) it is believed these signaling mediators act in a paracrine fashion at the site of production. Evidence to date is quite convincing that these bioactive lipid derivatives are involved in physiologic homeostatic regulation as well as beneficial and maladaptive ventricular remodeling in heart failure. Thus, this review will assess the direct contribution of each PG on remodeling in the left ventricle (e.g. hypertrophy, functional effects, and fibrosis).

Inhibition of matrix metalloproteinase activity prevents increases in myocardial tumor necrosis factor-alpha.

TNF-alpha is known to cause adverse myocardial remodeling. While we have previously shown a role for cardiac mast cells in mediating increases in myocardial TNF-alpha, however, matrix metalloproteinase (MMP) activation of TNF-alpha may also be contributory. We sought to determine the relative roles of MMPs and cardiac mast cells in the activation of TNF-alpha in the hearts of rats subjected to chronic volume overload. Interventions with the broad spectrum MMP inhibitor, GM6001, or the mast cell stabilizer, nedocromil, were performed in the rat aortocaval fistula (ACF) model of volume overload. Myocardial TNF-alpha levels were significantly increased in the ACF. This increase was prevented by MMP inhibition with GM6001 (p< or =0.001 vs. ACF). Conversely, myocardial TNF-alpha levels were increased in the ACF+nedocromil treated fistula groups (p< or =0.001 vs. sham). The degradation of interstitial collagen volume fraction seen in the untreated ACF group was prevented in both the GM6001 and nedocromil treated hearts. Significant increases in LV myocardial ET-1 levels also occurred in the ACF group at 3days post-fistula. Whereas administration of GM6001 significantly attenuated this increase, mast cell stabilization with nedocromil markedly exacerbated the increase, producing ET-1 levels 6.5 fold and 2 fold greater than that in the sham-operated control and ACF group, respectively. The efficacy of the MMP inhibitor, GM6001, to prevent increased levels of myocardial TNF-alpha is indicative of MMP-mediated cleavage of latent extracellular membrane-bound TNF-alpha protein as the primary source of bioactive TNF-alpha in the myocardium of the volume overload heart.

Sympathetic nervous system modulation of inflammation and remodeling in the hypertensive heart.

Chronic activation of the sympathetic nervous system is a key component of cardiac hypertrophy and fibrosis. However, previous studies have provided evidence that also implicate inflammatory cells, including mast cells (MCs), in the development of cardiac fibrosis. The current study investigated the potential interaction of cardiac MCs with the sympathetic nervous system. Eight-week-old male spontaneously hypertensive rats were sympathectomized to establish the effect of the sympathetic nervous system on cardiac MC density, myocardial remodeling, and cytokine production in the hypertensive heart. Age-matched Wistar Kyoto rats served as controls. Cardiac fibrosis and hypertension were significantly attenuated and left ventricular mass normalized, whereas cardiac MC density was markedly increased in sympathectomized spontaneously hypertensive rats. Sympathectomy normalized myocardial levels of interferon-gamma, interleukin 6, and interleukin 10, but had no effect on interleukin 4. The effects of norepinephrine and substance P on isolated cardiac MC activation were investigated as potential mechanisms of interaction between the two. Only substance P elicited MC degranulation. Substance P was also shown to induce the production of angiotensin II by a mixed population of isolated cardiac inflammatory cells, including MCs, lymphocytes, and macrophages. These results demonstrate the ability of neuropeptides to regulate inflammatory cell function, providing a potential mechanism by which the sympathetic nervous system and afferent nerves may interact with inflammatory cells in the hypertensive heart.

Estrogen attenuates chronic volume overload induced structural and functional remodeling in male rat hearts.

We have previously reported gender differences in ventricular remodeling and development of heart failure using the aortocaval fistula model of chronic volume overload in rats. In contrast to males, female rats exhibited no adverse ventricular remodeling and less mortality in response to volume overload. This gender-specific cardioprotection was lost following ovariectomy and was partially restored using estrogen replacement. However, it is not known if estrogen treatment would be as effective in males. The purpose of this study was to evaluate the structural and functional effects of estrogen in male rats subjected to chronic volume overload. Four groups of male rats were studied at 3 days and 8 wk postsurgery as follows: fistula and sham-operated controls, with and without estrogen treatment. Biochemical and histological studies were performed at 3 days postsurgery, with chronic structural and functional effects studied at 8 wk. Measurement of systolic and diastolic pressure-volume relationships was obtained using a blood-perfused isolated heart preparation. Both fistula groups developed significant ventricular hypertrophy after 8 wk of volume overload. Untreated rats with fistula exhibited extensive ventricular dilatation, which was coupled with a loss of systolic function. Estrogen attenuated left ventricular dilatation and maintained function in treated rats. Estrogen treatment was also associated with a reduction in oxidative stress and circulating endothelin-1 levels, as well as prevention of matrix metalloproteinase-2 and -9 activation and breakdown of ventricular collagen in the early stage of remodeling. These data demonstrate that estrogen attenuates ventricular remodeling and disease progression in male rats subjected to chronic volume overload.

ETA selective receptor antagonism prevents ventricular remodeling in volume-overloaded rats.

The objective of this study was to investigate the ability of selective endothelin receptor subtype A (ET(A)) endothelin receptor antagonism (ETA) to prevent the acute myocardial remodeling process secondary to volume overload. Left ventricular tissue from sham-operated (Sham) and untreated (Fist), and TBC-3214 (Fist + ETA, 25 mg.kg(-1).day(-1))-treated fistula animals was analyzed for mast cell density, matrix metalloproteinase (MMP) activity, and extracellular collagen volume fraction (CVF) 1 and 5 days following the initiation of volume overload. Compared with Fist, ETA treatment prevented the increase in left ventricular mast cell density at 1 day and 5 days. Additionally, at 1 day postfistula, a substantial decrease in MMP-2 activity below Sham levels was observed following endothelin receptor antagonism (1.7 +/- 0.7 vs. 0.3 +/- 0.3 vs. 0.9 +/- 0.2 arbitrary activity units, Fist vs. Fist + ETA vs. Sham, P < or = 0.05). This same effect was also seen at 5 days postfistula (1.9 +/- 0.3 vs. 0.5 +/- 0.1 arbitrary activity units, Fist vs. Fist + ETA, P < or = 0.05). The marked decrease in myocardial CVF seen in Fist hearts (0.7 +/- 0.1 vs. 1.6 +/- 0.1% myocardial area, Fist vs. Sham, P < or = 0.05) was prevented by ETA (1.7 +/- 0.1% Fist + ETA, P < 0.05 vs. Fist). This preservation of the collagen matrix was also present on day 5 in the TBC-treated group vs. the Fist group (1.0 +/- 0.1 vs. 1.4 +/- 0.1%, Fist vs. Fist + ETA, P < or = 0.01). Furthermore, an 8-wk preventative treatment with ETA significantly attenuated the increase in left ventricular end systolic and diastolic volumes compared with untreated fistula hearts. In conclusion, the novel findings of this study indicate that the activation of cardiac mast cells and subsequent MMP activation/collagen degradation during the acute stages of volume overload are prevented by blockade of the ET(A) receptor subtype. Furthermore, by preventing these events, ET-1 antagonism was efficacious in attenuating ventricular dilatation and limiting the development of structural and functional deficits.

Cardiac mast cells mediate left ventricular fibrosis in the hypertensive rat heart.

Correlative data suggest that cardiac mast cells are a component of the inflammatory response that is important to hypertension-induced adverse myocardial remodeling. However, a causal relationship has not been established. We hypothesized that adverse myocardial remodeling would be inhibited by preventing the release of mast cell products that may interact with fibroblasts and other inflammatory cells. Eight-week-old male spontaneously hypertensive rats were treated for 12 weeks with the mast cell stabilizing compound nedocromil (30 mg/kg per day). Age-matched Wistar-Kyoto rats served as controls. Nedocromil prevented left ventricular fibrosis in the spontaneously hypertensive rat independent of hypertrophy and blood pressure, despite cardiac mast cell density being elevated. The mast cell protease tryptase was elevated in the spontaneously hypertensive rat myocardium and was normalized by nedocromil. Treatment of isolated adult spontaneously hypertensive rat cardiac fibroblasts with tryptase induced collagen synthesis and proliferation, suggesting this as a possible mechanism of mast cell-mediated fibrosis. In addition, nedocromil prevented macrophage infiltration into the ventricle. The inflammatory cytokines interferon-gamma and interleukin (IL)-4 were increased in the spontaneously hypertensive rat and normalized by nedocromil, whereas IL-6 and IL-10 were decreased in the spontaneously hypertensive rat, with nedocromil treatment normalizing IL-6 and increasing IL-10 above the control. These results demonstrate for the first time a causal relationship between mast cell activation and fibrosis in the hypertensive heart. Furthermore, these results identify several mechanisms, including tryptase, inflammatory cell recruitment, and cytokine regulation, by which mast cells may mediate hypertension-induced left ventricular fibrosis.

The metal chelators, trientine and citrate, inhibit the development of cardiac pathology in the Zucker diabetic rat.

PURPOSE: The objective of this study was to determine the efficacy of dietary supplementation with the metal chelators, trientine or citric acid, in preventing the development of cardiomyopathy in the Zucker diabetic rat. HYPOTHESIS: We hypothesized that dietary chelators would attenuate metal-catalyzed oxidative stress and damage in tissues and protect against pathological changes in ventricular structure and function in type II diabetes. METHODS: Animals (10 weeks old) included lean control (LC, fa/+), untreated Zucker diabetic fatty (ZDF, fa/fa), and ZDF rats treated with either trientine (triethylenetetramine) or citrate at 20 mg/d in drinking water, starting when rats were frankly diabetic. Cardiac functional assessment was determined using a Millar pressure/volume catheter placed in the left ventricle at 32 weeks of age. RESULTS: End diastolic volume for the ZDF animals increased by 36% indicating LV dilatation (P < .05) and was accompanied by a 30% increase in the end diastolic pressure (P

Cardiac and renal function are progressively impaired with aging in Zucker diabetic fatty type II diabetic rats.

This study investigated the temporal relationship between cardiomyopathy and renal pathology in the type II diabetic Zucker diabetic fatty (ZDF) rat. We hypothesized that changes in renal function will precede the development of cardiac dysfunction in the ZDF rat. Animals (10 weeks old) were divided into four experimental groups: Lean Control (fa/?) LC(n = 7), untreated ZDF rats (n = 7) sacrificed at 16 weeks of age, and LC (n = 7) untreated ZDF rats (n = 9) sacrificed at 36 weeks of age. LV structural/functional parameters were assessed via Millar conductance catheter. Renal function was evaluated via markers of proteinuria and evidence of hydronephrosis. LV mass was significantly less in the ZDF groups at both time points compared to age-matched LC. End diastolic volume was increased by 16% at 16 weeks and by 37% at 36 weeks of age (p < 0.05 vs. LC). End diastolic pressure and end systolic volume were significantly increased (42% and 27%respectively) at 36 weeks of age in the ZDF compared to LC. Kidney weights were significantly increased at both 16 and 36 week in ZDF animals (p < 0.05 vs. LC). Increased urinary albumin and decreased urinary creatinine were paralleled by a marked progression in the severity of hydronephrosis from 16 to 36 weeks of age in the ZDF group. In summary, there is evidence of progressive structural and functional changes in both the heart and kidney, starting as early as 16 weeks,without evidence that one pathology precedes or causes the other in the ZDF model of type II diabetes.

Effects of nonselective endothelin-1 receptor antagonism on cardiac mast cell-mediated ventricular remodeling in rats.

The objective of this study was to investigate the effect a nonselective endothelin-1 (ET-1) receptor antagonist (bosentan) had on the acute myocardial remodeling process including left ventricular (LV) mast cells and matrix metalloproteinase (MMP) activity secondary to volume overload. Additionally, we investigated the overall functional outcome of preventative endothelin receptor antagonism during 14 days of chronic volume overload. LV tissue from sham-operated (Sham), untreated-fistula (Fist), and bosentan (100 mg.kg(-1).day(-1))-treated animals (Fist + Bos) was analyzed for mast cell density, MMP activity, and myocardial collagen volume fraction at 1 and 5 days after the creation of an aortocaval fistula. When compared with untreated fistulas, bosentan treatment prevented the marked increase in LV mast cell density at 1 day postfistula (3.1 +/- 0.3 vs. 1.3 +/- 0.3 LV mast cells/mm2, Fist vs. Fist + Bos, P

Response of cardiac mast cells to atrial natriuretic peptide.

Previously, our laboratory demonstrated that cardiac mast cell degranulation induces adverse ventricular remodeling in response to chronic volume overload. The purpose of this study was to investigate whether atrial natriuretic peptide (ANP), which is known to be elevated in chronic volume overload, causes cardiac mast cell degranulation. Relative to control, ANP induced significant histamine release from peritoneal mast cells, whereas isolated cardiac mast cells were not responsive. Infusion of ANP (225 pg/ml) into blood-perfused isolated rat hearts produced minimal activation of cardiac mast cells, similar to that seen in the control group. ANP also did not increase matrix metalloproteinase-2 activity, reduce collagen volume fraction, or alter diastolic or systolic cardiac function compared with saline-treated controls. In a subsequent study to evaluate the effects of natriuretic peptide receptor antagonism on volume overload-induced ventricular remodeling, anantin was administered to rats with an aortocaval fistula. Comparable increases of myocardial MMP-2 activity in treated and untreated rats with an aortocaval fistula were associated with equivalent decreases in ventricular collagen (P < 0.05 vs. sham-operated controls). Cardiac functional parameters and left ventricular hypertrophy were unaffected by anantin. We conclude that ANP is not a cardiac mast cell secretagogue and is not responsible for the cardiac mast cell-mediated adverse ventricular remodeling in response to volume overload.

The relationship between myocardial extracellular matrix remodeling and ventricular function.

Elevations in myocardial stress initiate structural remodeling of the heart in an attempt to normalize the imposed stress. This remodeling consists of cardiomyocyte hypertrophy and changes in the amount of collagen, collagen phenotype and collagen cross-linking. Since fibrillar collagen is a relatively stiff material, a decrease in collagen can result in a more compliant ventricle while an increase in collagen or collagen cross-linking results in a stiffer ventricle. If continued elevations in wall stress exceed the ability of the heart to compensate, then the ventricular wall thickness is disproportionately reduced compared to chamber volume and diastolic and systolic dysfunction ensues. This review describes the structural organization of collagen within the myocardium, discusses its effect on ventricular function and considers whether therapy aimed at reducing fibrosis is efficacious in heart failure. The evidence indicates that chamber stiffness can clearly be affected by alterations in both collagen quantity and quality, with the effect of changes in collagen concentration being modified by the extent of collagen cross-linking. The limited evidence available regarding the effects of collagen on systolic function indicates that pharmacological attempts to reduce interstitial collagen have a negative impact. Accordingly, a shift in treatment strategies directed more specifically at affecting collagen cross-linking, rather than reducing the concentration of collagen, may be warranted in the prevention of the adverse impact of collagen alterations on myocardial remodeling.

Cardiac mast cell regulation of matrix metalloproteinase-related ventricular remodeling in chronic pressure or volume overload.

The chronic elevation in ventricular wall stress secondary to ventricular volume or pressure overload leads to structural remodeling of the muscular, vascular and extracellular matrix components of the myocardium. While initially a compensatory response, the progressive hypertrophy and ventricular dilatation induced by this condition ultimately have a detrimental effect on ventricular function, resulting in heart failure. Fibrillar collagen provides the skeletal framework which interconnects the cardiomyocytes, thereby maintaining ventricular shape and size and contributing to tissue stiffness. Accordingly, these myocardial collagen fibers must be disrupted for ventricular dilatation, sphericalization and wall thinning to occur. The presence of an abundant, latent matrix metalloproteinase (MMP) population which coexists with myocardial fibrillar collagen has been documented. Thus, the potential for collagen degradation to exceed synthesis exists should there be significant activation of this latent MMP system. Mast cells are known to store and release a variety of biologically active mediators including TNF-alpha and proteases such as tryptase and chymase, which can induce MMP activation. Increased cardiac mast cell density has been implicated in the pathophysiology of human end-stage cardiomyopathy and experimental myocardial infarction, hypertension and chronic volume overload secondary to mitral regurgitation and aorto-caval fistula. The potential role of cardiac mast cells in activating MMPs, which then results in fibrillar collagen degradation and adverse myocardial remodeling secondary to chronic volume and pressure overload will be the subject of this review.

Modulation of cardiac mast cell-mediated extracellular matrix degradation by estrogen.

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 showed that 1) 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 female rats, ovariectomized female rats, and ovariectomized female rats treated with 17beta-estradiol. In response to mast cell degranulation, no significant differences in cardiac function, MMP-2 activity, or collagen volume fraction were observed between intact female rats and ovariectomized female rats treated with estrogen. In the ovariectomized female group, a significant rightward shift in the left ventricular pressure-volume relation, accompanied by a marked 133% increase in active MMP-2 values over that in the intact female group, was noted after treatment with compound 48/80 (P < or = 0.05), along with a significant reduction in collagen volume fraction below control (0.46 +/- 0.23 vs. 0.73 +/- 0.13%, P < or = 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.

Endothelin-1 mediates cardiac mast cell degranulation, matrix metalloproteinase activation, and myocardial remodeling in rats.

The objective of this study was to determine whether elevated circulating levels of endothelin (ET)-1 are capable of mediating left ventricular (LV) mast cell degranulation and thereby induce matrix metalloproteinase (MMP) activation. After the administration of 20 pg/ml ET-1 to blood-perfused isolated rat hearts, LV tissue was analyzed for signs of mast cell degranulation and MMP activation. Relative to control, ET-1 produced extensive mast cell degranulation as well as a significant increase in myocardial water content (78.8 +/- 1.5% vs. 74.2 +/- 2.2%, P <0.01), a marked 107% increase in MMP-2 activity (P <0.05), and a substantial decrease in collagen volume fraction (0.69 +/- 0.09% vs. 0.99 +/- 0.04%, P <0.001). Although the myocardial edema would be expected to increase ventricular stiffness, compliance was not altered, and moderate ventricular dilatation was observed (end-diastolic volume at end-diastolic pressure of 0 mmHg of 330.2 +/- 22.1 vs. 298.9 +/- 17.4 microl in ET-1 treated vs. control, respectively, P=0.07). Additionally, pretreatment with the mast cell stabilizer nedocromil prevented ET-1-induced changes in MMP-2 activity, myocardial water content, collagen volume fraction, and end-diastolic volume. These findings demonstrate that ET-1 is a potent cardiac mast cell secretogogue and further indicate that ET-1-mediated mast cell degranulation is a potential mechanism responsible for myocardial remodeling.