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Am J Physiol Heart Circ Physiol 288: H2770-H2777, 2005. First published January 28, 2005; doi:10.1152/ajpheart.01146.2004
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Protease-activated receptor and endothelial-myocyte uncoupling in chronic heart failure

Karni S. Moshal, Neetu Tyagi, Brooke Henderson, Alexander V. Ovechkin, and Suresh C. Tyagi

Department of Physiology and Biophysics, University of Louisville School of Medicine, Louisville, Kentucky

Submitted 15 November 2004 ; accepted in final form 26 January 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We examined the hypothesis that oxidants generated nitroso derivatives, activated latent matrix metalloproteinase (MMP), and induced proteinase-activated receptor 1 (PAR-1), leading to disconnection between the endothelium and myocytes. Administration of cardiospecific tissue inhibitor of metalloproteinase-4 (TIMP-4/CIMP) ameliorated the oxidative-proteolytic stress and endothelial-myocyte uncoupling in chronic heart failure (CHF) in mice. Aortic-vena cava fistula (AVF) was created in 30 male mice (C57BL/6J) and studied at 0-, 2-, and 8-wk AVF. To reverse cardiac remodeling, as measured by MMP activation, purified CIMP was administered by an osmotic minipump subcutaneously after 8-wk AVF, and groups of mice (n = 6 mice/group) were examined after 12 and 16 wk. Levels of PAR-1 in the left ventricle (LV) were increased at 2 and 8 wk (compared with 0 wk of no CIMP treatment) but were normal at 12 and 16 wk after CIMP treatment, as measured by Western blot analysis. Similar results were obtained for LV levels of nitrotyrosine, MMP-2 and -9 activities, and TIMP-1 and -3. However, the levels of TIMP-4, endothelial cell density, and responses of cardiac rings to acetylcholine and bradykinin were attenuated at 2 and 8 wk and normalized after CIMP administration in AVF mice. CIMP induced nitric oxide in microvascular endocardial endothelial cells. The results suggest that nitro generation activated MMP and PAR-1, leading to endothelial-myocyte uncoupling. CIMP treatment normalized PAR-1 expression and ameliorated endothelial-myocyte uncoupling by decreasing oxidant-mediated proteolytic stress in CHF.

proteomics; shedding; nitric oxide; NADPH oxidase; cardiac ring; contraction; relaxation; dilatation; matrix metalloproteinase; nitrotyrosine; tissue inhibitors of metalloproteinase; coupling

NORMAL MYOCARDIUM contains latent matrix metalloproteinases (MMPs) (30). Studies from our laboratory demonstrated that the latency of these MMPs is, in part, due to their coupling with nitric oxide (NO) (10). An increase in oxidative stress and a decrease in NO levels lead to activation of latent MMPs and is an attempt to reduce wall stress during ventricular load (18). The chronic increase in oxidative and proteolytic insults disconnects the endothelium from the myocyte and causes impaired diastolic relaxation. This response contributes to decreased levels of cardiac tissue inhibitor of metalloproteinase-4 (TIMP-4/CIMP) and inactivation of CIMP by oxidative modification in chronic heart failure (CHF) (4, 5, 10, 27).

Normally, a critical balance between neutral serine proteinase inhibitor (serpin) and TIMP is maintained (26). In addition to its inhibitory role, serpin activates cell surface receptors, cell migration, and signal transduction (12, 15). Several lines of evidence also suggest that TIMP-1 induces antimitogenic activity (9). We showed that TIMP-1 caused proliferation of endothelial cells (31). In addition, TIMP-1 is associated with cardiac fibrosis (13) and is inactivated by oxyradicals (7). TIMP-2 had been shown to be a growth stimulatory protein for transformed fibroblasts (19). Baker et al. (1) demonstrated that TIMP-3 induced apoptosis in vascular smooth muscle cells and regressed neointimal growth. Results from our laboratory have shown that CIMP (TIMP-4) induced apoptosis in transformed cardiac fibroblasts but had no effect in normal fibroblast cells (25).

Serpin and TIMPs are sensitive to oxidative inactivation (28), and, during inflammatory pathogenesis, oxidants disrupt the balance between proteinase and antiproteinase (23, 24). In vitro, serine proteinases activate latent resident myocardial MMP (32). Serine proteinase degrades inhibitors of metalloproteinase, and metalloproteinase degrades inhibitors of serine proteinase (32). Therefore, a vicious cascade of activation/inactivation of proteinase antiproteinase is initiated in which accumulation of oxidized matrix is increased.

Although a number of studies have used synthetic MMP inhibitors to show an improvement in cardiac function in chronic heart failure (CHF), the specificity of these inhibitors and their role in oxidative stress was unclear. There are four TIMPs, and, although all TIMPs bind to active MMPs, TIMP-4 is cardiac specific. The administration of CIMP is important for two reasons: 1) it creates a condition of high CIMP levels in the heart, which decreases MMP activity; and 2) CIMP contains residues that are capable of sequestering ROS. To determine whether the activation of latent resident myocardial MMP is associated with decreased levels of CIMP, it is important to administer CIMP post-aortic-vena cava fistula (AVF). There are more than 20 MMPs. Interstitial collagen is degraded by interstitial collagenase (MMP-1). However, unlike humans, rodents do not contain a typical MMP-1. Instead, they contain MMP-13, which is equivalent to MMP-1. On the other hand, MMP-2 degrades interstitial collagen as well as elastin and is induced in CHF. In addition, MMP-2 (gelatinase a) is expressed across the species, and MMP-9 at 92 kDa (gelatinase b) is robustly increased in CHF. Because myocardial stromas are rare, the contribution of stromelysin (MMP-3) to the heart is minimal. MMP-7 is a putative metalloproteinase (putative metalloproteinase 1) from high-molecular-weight MMPs such as MMP-2 and -9. Therefore, MMP-2 and -9 were measured in this study.

The proteinase-activated receptor (PAR) is a new class of extracellular receptors that regulate cell shape, growth, and differentiation in part by activating G protein-coupled receptors (2). There are four PARs: PAR-1, -2, -3, and -4. Primarily, PARs are activated by serine proteinases (6, 22). On the basis of signaling studies, it was suggested that PAR-1 was expressed in endothelial cells and modulated the permeability and growth of smooth muscle cells (3). Cardiomyocytes expressed PAR-1 and promoted cardiac hypertrophy (21). Although CIMP ameliorated oxidative and proteolytic stress, it was unclear whether nitro generation and MMP activation induced PAR-1 because CIMP induced NO in endocardial endothelial cells (EECs). We hypothesized that CIMP treatment normalizes PAR-1 expression and ameliorates endothelial-myocyte uncoupling by decreasing oxidative and proteolytic stresses. Alternatively, it is also possible that by administering CIMP and subsequently reducing MMP activity, adverse left ventricular (LV) remodeling could be reversed. A reduction in oxidative stress and decreases in PAR-1 would improve endothelial-myocyte coupling in CHF.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Experimental design. Wild-type, C57BL/6J, male mice (8–10 wk old) were obtained from Jackson Laboratories. In tribromoethanol-anesthetized (100 mg/kg ip) mice (5), a fistula was created between the aorta and the caudal vena cava (AVF) ~1 cm below the kidney using a 30-gauge needle. The consistency of AVF was confirmed by observing red blood flowing through the vena cava. In a previous study (4), we used age-matched control groups. The goal of this study was to determine temporal changes post-AVF; therefore, 0 wk was used as a control. Mice were grouped into 0-, 2-, and 8-wk groups (n = 6 mice/group). To regress cardiac remodeling, after 8 wk, other mice received CIMP and were grouped at 12 and 16 wk post-AVF (n = 6 mice/group). The experimental protocols for this study were approved by the Institutional Animal Care and Use Committee of the University of Louisville School of Medicine. All animal care and use programs were carried out according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Pub. No. 86-23, Revised 1985) and the regulations of the Animal Welfare Act.

CIMP was purified from mouse hearts (25) and infused into the intraperitoneal cavity after 8 wk of AVF by an Alzet microosmotic pump (model 1002) at 0.25 µl/h, 10 µg/day. The blood levels of CIMP reached steady state within 24 h (4). Because the binding constant between MMP and TIMP is in the nanomolar range, this amount of CIMP saturates all the binding sites in MMPs. LV variables were measured by a pressure-tipped Millar catheter inserted into the right common carotid artery and advanced to the LV, and arterial pressure was measured with a Micro-Med pressure transducer in anesthetized mice. After 10 min of stabilization, LV pressure (LVP), maximum LVP, LVP peak time derivatives, maximum LVP first derivatives, and LV end-diastolic pressure were recorded. Hearts were excised under deep anesthesia. The lung-to-body weight ratio was measured. The LV and right ventricle were separated and weighed, and LV rings were prepared. Fresh LV tissue was homogenized and used to measure NO and ROS.

In vivo echocardiography and LV function. Echocardiography was performed at 0, 2, 8, 12, and 16 wk (SONO-5500) using a 12-MHz probe in anesthetized mice. M-mode views were analyzed to measure end-systolic and end-diastolic diameters and LV wall thickness. To determine LV muscle function, norepinephrine (NE) was injected into a jugular vein, and LVP responses were measured as described (16). To normalize the effect of afterload, LVP was normalized by carotid arterial pressure.

Ex vivo endothelial-myocyte coupling. The "deli"-shaped LV rings were mounted between two wires in a tissue myobath as previously described (4, 5). The rings were free of any mechanical or hypoxic injuries under these conditions (4, 5). Contractile responses to cardiotonic agents [acetylcholine (ACh), bradykinin (BK), and nitroprusside] were measured in endothelin (ET)-1-contracted rings. The percent relaxation was based on 100% ET-1 contraction.

LV levels of NO and ROS. Total LV NO concentration was estimated by measurements of total nitrate/nitrite. Oxidative stress was assessed by measuring LV ROS by incubating LV tissue homogenates with 2,7'-dichlorofluorescein (DCFH; Sigma Chemical; St. Louis, MO). Although generation of O2–· is transient, O2– and H2O2 (2OH–) are stable. DCFH acquires fluorescence properties upon reaction with ROS and yields the fluorescent product dichloroflyorescein. This product was detected by a 530-nm emission when excited at 485 nm (11). A standard curve using oxidized DCFH was generated, and concentrations of ROS (in nmol/l) in the samples were determined.

Western blot analysis of PAR-1, nitrotyrosine, and TIMP-1, -3, and -4. LV tissue homogenates were prepared, analyzed on 10% SDS-PAGE, and transferred to polyvinylidene difluoride membranes. Twenty-five micrograms of total protein were loaded onto each lane. The membranes were blotted with anti-PAR-1, nitrotyrosine, and TIMP monoclonal antibodies (Chemicon) as previously described (5, 10). Secondary IgG-alkaline phosphatase was used for detection. Actin blots were used as loading controls. The bands were scanned and normalized with actin intensity. The gels were stained with Commassie blue for protein.

MMP-2 and -9 activities. Zymography using 1% gelatin gels was performed on LV tissue homogenates as previously described (30).

In situ CD-31 and histological labeling. To determine the number of endothelial cells, LV tissue was immunolabeled using FITC-CD-31 (an endothelial cell marker). Trichrome staining was used to measure collagenous material in the LV. LV wall, diameter, and myocyte size were measured by a digital micrometer. Fibrotic collagen was measured by estimating trichrome blue stain and expressing it as arbitrary units per centimeter.

CIMP induces EECs. To determine whether CIMP induces NO directly in EECs, we cultured serum-free EECs with 10 µM CIMP as previously described (29). The levels of NO were measured in the medium after CIMP treatment.

Statistical analysis. Values are given as means ± SD; n = 6 mice/group. Differences between groups were evaluated by ANOVA, followed by the Bonferroni post hoc test, focusing on the effects of volume overload (0-wk AVF mice vs. 2- and 8-wk AVF mice, indicated by *) and treatment (AVF + CIMP-treated mice compared with AVF mice, indicated by **). P < 0.05 was considered significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
PAR-1. Levels of PAR-1 were increased at 2 wk and remained elevated up to 8 wk post-AVF. Treatment with CIMP decreased the levels of PAR-1 to control levels at 12 and 16 wk (Fig. 1).



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  		Fig. 1. A: Western blot analysis of left ventricular (LV) proteinase-activated receptor 1 (PAR-1) at 0, 2, and 8 wk after aortic-vena cava fistula (AVF) and at 12 and 16 wk after AVF plus cardiac-specific inhibitor of metalloproteinase (CIMP) administration. Corresponding {beta}-actin blots are shown. Identical amounts of total protein were loaded in each lane. B: histographic presentation of scanned values [in arbitrary units (AU)] of Western blots normalized with actin (n = 6 in each group). *Significant compared with 0 wk; **significant compared with 8 wk.

 
Oxidative stress/nitrotyrosine. Because generation of nitrotyrosine is a hallmark of increased oxidative stress, we measured the protein content of nitrotyrosine, and it was detected at ~66 kDa. The levels of nitrotyrosine were increased with time at 2 and 8 wk post-AVF (Fig. 2). Treatment with CIMP reduced the nitrotyrosine levels and oxidative stress (Fig. 2). Tissue levels of ROS showed a similar trend. However, levels of NO were inversely related to ROS (Table 1).



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  		Fig. 2. A: LV Western blot analysis of nitrotyrosine (NO2-T) at 0, 2, and 8 wk after AVF and at 12 and 16 wk after AVF plus CIMP administration. Corresponding actin blots are shown. B: scanned values, normalized with actin, of nitrotyrosine are shown by histograms (n = 6 in each group). *P < 0.001 compared with 0 wk; **P < 0.005 compared with 8 wk.

 

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  		Table 1. Parameters of mice after 0, 2, and 8 wk of AVF and after 12 and 16 wk of AVF + CIMP treatment

 
MMPs/TIMPs. Levels of MMP-2 increased at 2 and 8 wk. However, MMP-9 increased significantly only at 8 wk post-AVF. Treatment with CIMP decreased MMP-2 and -9 to basal levels (Fig. 3). TIMPs were regulated differentially in AVF. TIMP-1 was present at 0, 2, and 8 wk of AVF. However, TIMP-3 was induced at 2 and 8 wk of AVF. Treatment with CIMP decreased TIMP-3 to basal levels (Fig. 4, A and B). TIMP-4 was constitutively present at 0 wk, but at 2 and 8 wk the levels dwindled. Treatment with CIMP increased the levels of TIMP-4 to control values (Fig. 4, A and B).



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  		Fig. 3. A: gelatin-gel-zymographic analysis of matrix metalloproteinase (MMP) activity in LV homogenates at 0, 2, and 8 wk after AVF and at 12 and 16 wk after AVF plus CIMP administration. Corresponding actin blots are shown. B: scanned values for MMP-2 and -9, normalized with actin, are shown by histograms (n = 6 in each group). *P < 0.01 compared with 0 wk; **P < 0.01 compared with 8 wk.

 


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  		Fig. 4. A: LV Western blot analysis of tissue inhibitor of metalloproteinase (TIMP)-1, -3, and -4 at 0, 2, and 8 wk after AVF and at 12 and 16 wk after AVF plus CIMP administration. Corresponding actin blots are shown. B: scanned values, normalized with actin, of TIMP-1, -3, and -4 are shown by histograms (n = 6 in each group). *P < 0.005 compared with 0 wk; **P < 0.001 compared with 8 wk.

 
LVH/fibrosis. There was significant fibrosis, collagen deposition, and LV hypertrophy (LVH) at 2 and 8 wk of AVF in hearts. However, treatment with CIMP ameliorated the collagen content and myocyte hypertrophy after AVF (Fig. 5).



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  		Fig. 5. A: histological analysis of the LV at 0 and 8 wk after AVF and at 16 wk after AVF plus CIMP administration. Serial tissue sections were labeled with trichrome for collagen. Magnification, x40. B: levels of collagen were estimated based on hydroxyproline contents. Myocyte size was measured by micrometer. Each bar represents the mean ± SD from 6 independent experiments. *P < 0.005 compared with 0 wk; **P < 0.01 compared with 8 wk.

 
Endothelial-myocyte coupling. Labeling with CD31/PECAM-1 antibody demonstrated organized capillaries in sham and AVF mice that were treated with CIMP. Endothelial cell density was significantly lower in the LV of AVF mice than in sham controls (Fig. 6). Treatment with CIMP ameliorated the decrease in endothelial cell density (Fig. 6). To determine whether decreased endothelial cell density was associated with impairment of endothelial-myocyte coupling and cardiac relaxation, we measured responses to ACh, BK, and nitroprusside in cardiac rings from AVF mice at 0, 2, and 8 wk before CIMP treatment and at 12 and 16 wk post-CIMP treatment. ACh and BK responses were attenuated at 2 and 8 wk compared with 0 wk post-AVF. Interestingly, at 8 wk, BK induced a paradoxic contraction that was attenuated by treatment with CIMP. Except at 8 wk, there was no significant attenuation of the nitroprusside response (Fig. 7).



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  		Fig. 6. A: CD31/PECAM-1 labeling for endothelial cell density. Tissue was fixed with 10% zinc-formalin after perfusion at LV end-diastolic pressure (in vivo end-diastolic pressure = 4 mmHg). The tissue was deparaffinized. Endothelial cells were labeled with CD31/PECAM-1 antibody (1:10, Sigma) conjugated with FITC. B: fluorescence images were captured. The counts of CD31-positive cells were normalized to a sham count in a fixed grid of tissue area. An average from 5 randomly selected grids per tissue was used. Endocardial tissue was from 0 and 8 wk after AVF and 12 and 16 wk after AVF plus CIMP administration. The numbers of endothelial (Endo) cell in a fixed-cm2 grid were counted and reported as a percentage of those at 0 wk. Each bar represents the mean ± SD from 6 independent measurements. *P < 0.001 compared with 0 wk; **P < 0.02 compared with 8 wk.

 


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  		Fig. 7. A: endocardial endothelial-myocyte responses to acetylcholine (ACh). LV rings from 0-wk mice were stretched, brought to resting tension, and contracted with 10 nM endothelin (ET)-1. Different doses of ACh were added to the ring in a tissue myobath. The relaxation to ACh was estimated as the percent ET-1 contraction. A typical contractile response to ET-1 and relaxation to 10–9, 10–8, and 10–7 M ACh is shown. B: ACh dose-response curves representing the best fit of the data. C: endocardial endothelial response to bradykinin (BK) doses. Curves represent the best fit of the data. D: endocardial response to nitroprusside (Npr) doses. Curves represent best fit of the data. Data are means ± SD from 6 animals in each group.

 
CIMP induces NO. To determine whether CIMP induced NO in endothelial cells, we cultured serum-free EECs with CIMP. The results suggested that CIMP selectively induced the production of NO in EECs (Fig. 8).



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  		Fig. 8. Cardiac microvascular endocardial endothelial (EE) cells were cultured with and without CIMP. Levels of nitrate/nitrite (NO) were measured. Bars represent EE cells, EE cells treated with CIMP (10 µM), EE cells treated with anti-TIMP-4 (1:100 dilution), and EE cells treated with CIMP plus anti-TIMP-4 antibody. Each bar represents the mean ± SD from 6 sets of EE cultures. *P < 0.001 compared with 0 wk; **P < 0.002 compared with 8 wk.

 
Cardiac muscle function. To determine whether the attenuation of the nitroprusside response at 8 wk post-AVF was, in part, due to a decrease in muscle strength, we measured the in vivo LVP induced by NE injection. The NE responses were attenuated at 2 and 8 wk post-AVF. Treatment with CIMP ameliorated this attenuation (Table 1). Echocardiographic data indicated an improvement in the coordination of endothelial-muscle coupling during systole and diastole and suggested regression of LV wall dilatation in AVF hearts after CIMP treatment (Fig. 9).



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  		Fig. 9. M-mode echocardiography of a sham (A) and an AVF mouse (B). C: cumulative data of LV wall/LV diameter. Each bar represents the average ± SD from 6 mice. *P < 0.002 compared with 0 wk; **P < 0.001 compared with 8 wk.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
PARs have been suggested to have a role in the ischemic myocardium due to the induction of inflammatory serine proteinases or thrombogenic pathways and subsequent elevation in intracellular calcium, which causes cardiac arrhythmias. This process promotes hypertrophic growth and/or influences cardiomyocyte survival (6, 22). The results of this study provide novel evidence that cardiac microvessel endothelial cells express functional PAR-1. Latent myocardial MMPs were activated by oxidative stress and induced PAR-1. Administration of CIMP ameliorated PAR-1 expression as well as oxidative and proteolytic stresses. Previously, we reported increases in the levels of ROS and NADH/NAD oxidase in CHF (4, 5, 10, 27). Others suggested increased inducible NO synthase levels in CHF (14). However, due to a robust increase in oxidative stress, the availability of NO was decreased and nitrotyrosine generation was increased [i.e., the equilibrium shifted toward nitrotyrosine and MMP activation (10)]. The present study demonstrated that increased oxidative stress and ROS were associated with a lower level of NO bioavailability and increased nitrotyrosine levels. Because oxidative stress and proteolytic activation occur concurrently, inhibitors of metalloproteinase can prevent oxidative stress. CIMP treatment inhibited NADH/NAD oxidase and ROS, therefore reversing the oxidative-stress mediated nitrotyrosine generation. This finding is consistent with the notion that CIMP is cardioprotective against oxidative stress in volume overload. Moreover, temporal changes in ROS, NO, and nitrotyrosine and amelioration of these changes by CIMP determined ROS as the inciting molecules causing oxidative stress. Treatment with CIMP increased NO and decreased ROS in EECs. These results suggest a role of CIMP in the modulation of oxidative stress in EECs.

The levels of CIMP were found to be decreased in animal models of heart failure (4, 5). Here, we demonstrated that CIMP was decreased in the LV of AVF mice compared with 0-wk AVF. Oxidative stress activates MMPs (32). The present results suggest that in vivo oxidative stress increases MMP-2 and -9 activities in AVF mice. The differential expression of TIMP-3 and -4 suggests that TIMP-3 may induce apoptosis in synergism with oxidative stress, whereas TIMP-4 is cardioprotective during overt heart failure by preventing oxidative and proteolytic stresses.

ACh stimulates NO release in the isolated heart preparation (8). Because ROS abrogates NO, we demonstrated that the magnitude of endothelial-myocyte coupling and the ACh response is significantly depressed in the endocardium of AVF mice compared with sham controls. Because treatment with CIMP sequesters ROS, it elicits an increase in NO accumulation. The numbers of endothelial cells are decreased in the endocardium of AVF mice compared with sham controls and the treatment with CIMP ameliorated the endothelial cell apoptosis. Therefore, endothelial-myocyte uncoupling was ameliorated in fistula mice treated with CIMP.

These results suggest that the decrease in endothelial NO availability and attenuation of endothelial function in the LV of AVF hearts were due to disruption of endothelium-myocyte connection and NO transport to the muscle. BK induces relaxation via EDHF pathways (17). The paradoxic contraction-induced by BK may suggest an imbalance in epoxyeicosatrienoic acids (EETs) and hydroeicosatrienoic acids (HETEs) in CHF. Oxidative and proteolytic stresses play a pivotal role in the modulation of the BK effect. Treatment with CIMP reversed this paradoxic effect of BK, indicating a role of CIMP in the regulation of HETE/EET. The response to endothelium-independent nitroprusside is also attenuated in AVF mice and suggests that underlying cardiac muscle and nonendothelial cells were also affected by oxidative stress in AVF mice. CIMP repressed the oxidative stress and ROS, and treatment with CIMP normalized the endothelial-myocyte coupling to the control value.

The oxidized matrix between the endothelium and myocyte is increased in chronic volume overload in AVF, contributing to endothelial myocyte uncoupling and decreased bioavailability of endothelial NO. The results of this study suggest that the decreased endothelial NO was due to inactivation of CIMP by ROS, causing MMP activation or, alternatively, due to the direct effect of ROS and NO on CIMP. NO keeps MMP latent. However, during increased ROS via a S-nitrosoperoxynitrite intermediate, latent MMP is activated. Others have suggested increased MMP activity after N-nitro-L-arginine methyl ester treatment (20). CIMP inhibits MMP and sequesters ROS by oxidative modification.

The results from this study suggest that oxidative stress is reduced by CIMP administration in a model of CHF. CIMP also decreases MMP and hence PAR-1 and improves endothelial function, with the latter effect being responsible for the reduced cardiac cavity size and increased function. This conclusion is based on the present in vitro data showing that CIMP induces NO in endothelial cells. However, it is still possible that by administering CIMP and subsequently reducing MMP activity, adverse LV remodeling would be reversed. The consequence would be a reduction in oxidative stress and hence a decrease in PAR-1 and improved endothelial-myocyte coupling. If this were the case, then PAR-1 and NO effects are the consequence and not the cause of the increased cardiac cavity size and decreased function.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported in part by National Heart, Lung, and Blood Institute Grants HL-71010 and HL-74185.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. C. Tyagi, Univ. of Louisville School of Medicine, A-1115, Dept. of Physiology and Biophysics, 500 S. Preston St., Louisville, KY 40202 (E-mail: s0tyag01{at}louisville.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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