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Department of Cardiology and Pneumonology, Campus Benjamin Franklin, Charité-University Medicine, Free University of Berlin;
* Department of Biophysics, Universidade Federal de São Paulo, São Paulo, Brazil; and
Max-Delbrück-Center for Molecular Medicine, Berlin, Germany
1Correspondence: Charite-University Medicine, Campus Benjamin Franklin, Department of Cardiology and Pneumonology, Free University of Berlin, Hindenburgdamm 30, D-12220 Berlin, Germany. E-mail: ctschoepe{at}yahoo.com
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: diabetes mellitus • diabetic cardiomyopathy • kallikrein • pathogenesis • therapy • transgenic animal
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Primary myocardial injury has been shown even in the absence of hypertension and atherosclerosis in diabetic patients and so has been called "diabetic cardiomyopathy" (2)
. The histopathological changes found include diffuse myocardial degeneration with changes in the myocardial extracellular matrix. The composition of the extracellular matrix, a complex network of structural proteins including collagen types I and III, provides architectural support for the muscle cells and plays an important role in myocardial function (3
, 4)
. Several studies of diabetic cardiomyopathy have demonstrated an accumulation of myocardial collagen including types I, III, and VI, leading to interstitial and perivascular fibrosis which has been correlated with LV early diastolic and systolic dysfunction (5
6
7
8)
. The mechanisms involved in these changes are still not completely understood. Behind myocardial microischemia due to focal changes in microvessels, nonenzymatic glycosylation of collagens, down-regulation, or reduced activity of collagen degrading matrix metalloproteinases (MMP), increased transforming growth factor-ß (TGF-ß) activity or up-regulation in collagen mRNA expression are discussed as hallmarks for myocardial fibrosis in diabetic cardiomyopathy (5
, 7
8
9)
. Remodeling of the extracellular matrix is also regulated by profibrotic acting peptides (e.g., angiotensin II, aldosterone, endothelin) and antagonistically acting systems like the kallikrein-kinin system (KKS) (10
, 11)
.
Kinins are important peptides mediating a diverse range of physiological and pathological functions of the cardiovascular system (12)
. The effector peptides of the KKS like bradykinin (BK) are produced after enzymatic cleavage of kininogens by kallikrein (KLK). Several in vivo studies elucidated that the KKS is involved in extracellular matrix regulation (13
, 14)
. KLK itself can serve as a collagenase and may activate MMPs, promoting collagen breakdown (15
, 16)
. Locally expressed kinins can inhibit collagen synthesis in vivo by stimulating nitric oxide (NO) and the prostaglandin system via the BK B2 receptor (17)
. Thus, the KKS could be an ideal mediator for counteracting the mechanisms contributing to cardiac fibrosis in diabetic cardiomyopathy.
To further examine the antifibrotic effect of cardiac KKS during the development of diabetic cardiomyopathy, we investigated cardiac collagen accumulation and LV function in transgenic rats (TGR) expressing the human kallikrein 1 (hKLK1) gene (14)
under streptozotocin (STZ) -induced diabetic conditions.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. A 5.6 kb DNA fragment containing the entire (hKLK1) gene under the control of the heavy metal-responsive mouse metallothionein promoter was microinjected into SD rat oocytes. The resultant founders were bred to homozygosity. These transgenic rats are characterized by expression of the transgene in all organs, with a rank order of expression heart>kidney>brain>lung (14)
.
Diabetes mellitus was induced by a single injection of STZ (70 mg/kg; i.p.) prepared in 0.1 M sodium citrate buffer, pH 4.5 (Sigma, Munich, Germany) as described in detail elsewhere (18)
. Hyperglycemia was confirmed 48 h later by a reflectance meter (Acutrend, Hoffmann-La Roche, Grenzach-Wyhlen, Germany). Vehicle-treated SD and TGR(hKLK1) served as controls (n=10). To investigate whether a KLK- and/or BK B2 receptor-dependent pathway is responsible for differences observed in TGR(hKLK1)-STZ, we treated in a separate experiment TGR(hKLK1)-STZ with the KLK inhibitor aprotinin (19)
(Trasylol 500,000 KIE by Bayer, Leverkusen, Germany; n=9) or with the BK B2 receptor antagonist icatibant (HOE140) (500 µL/kg 24 h by Aventis, Frankfurt, Germany; n=6). Both drugs were administered 5 days after STZ injection for 6 wk.
Hemodynamic characterization
Six weeks after injecting STZ or vehicle, heart rate (HR, in bpm), LV peak systolic pressure (LVP, in mmHg), the maximal rate of LV pressure rise (dP/dtmax., in mmHg/s) as a measure of LV systolic contraction, the minimal rate of LV pressure fall (dP/dtmin, in mmHg/s) as a measure of LV systolic relaxation and the LV end-diastolic pressure (LVEDP, in mmHg) were recorded via a Millar-tip catheter (2F) system in anesthetized (ketamine [50 mg/kg; i.p.], 2% xylasine [5 mg/kg; i.p.], ventilated, open-chest animals as described in detail elsewhere (20)
. After the experiment, the hearts were excised and 
0.2 cm-thick transverse sections of the hearts were rapidly frozen in liquid nitrogen and stored at –80°C for immunohistochemistry. Another section was paraffin embedded for Sirius Red staining.
Sirius Red staining, immunohistology, and quantification of collagen
Total collagen content of the Sirius Red (Polyscience, Inc., Warrington, PA, USA) stained sections was measured under circularly polarized light according to published methods (3)
. Representative aspects of Sirius Red stainings are illustrated in Fig. 1
. Serial 5 µm-thick transverse cryosections of Tissue Tec® embedded SD, SD-STZ, TGR(hKLK1), TGR(hKLK1)-STZ, TGR(hKLK1)-STZ-aprotinin and TGR(hKLK1)-STZ-icatibant (HOE 140) hearts were placed on poly-L-lysine precoated slides and fixed in cold acetone for 10 min. Immunohistochemistry with rabbit anti-collagen subtypes I, III, and VI specific antibodies (optimal working dilution: 1:500; kindly donated by Dr. Schuppan; ref 21
) and peroxidase-conjugated IgG (heavy and light chains) polyclonal anti-rabbit secondary antibody (working dilution: 1:500) was quantified by digital image analysis as described in detail elsewhere (22)
. All available fields (>30 fields) were measured, including the septum, right and left ventricles. Representative immunohistochemical stainings of collagen type III are demonstrated in Fig. 2
. Immunohistochemistry on collagen types I and VI provided comparable results.
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Statistical analysis
Statistical analysis was performed using JMP Statistical Discovery SoftwareTM Version 4.05 (SAS Institute, Cary, NC, USA). Since normal distribution was excluded regarding all parameters in conducting the Shapiro-Wilk W test (P<0.05), exclusively nonparametric tests were performed. Quantitative and qualitative data were compared conducting the Wilcoxon-Kruskal-Wallis test on rank sums. The honestly significant difference for multiple comparisons of all pairs was calculated by the Tukey-Kramer posthoc analysis. Multivariate (multiple bivariate) analysis was conducted for the Spearman Rho correlation coefficient. P< 0.05 was considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
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LV function did not differ between SD and TGR(hKLK1) controls. SD-STZ showed significant impairment in LVP, LVEDP, dP/dtmax. and dP/dtmin.. In contrast, LV function in TGR(hKLK1)-STZ was significantly improved compared with SD-STZ rats and showed no significant difference with the normoglycemic controls by Tukey-Kramer post hoc analysis. Administration of TGR(hKLK1)-STZ with the kallikrein inhibitor aprotinin led to a significant deterioration of LV function compared with untreated TGR(hKLK1)-STZ and there was a statistically nonsignificant trend toward improved LV function compared with SD-STZ rats (Table 1)
. Except for an increase in LVEDP, icatibant treatment did not lead to significant changes in the remaining hemodynamic parameters vs. untreated TGR(hKLK1)-STZ.
Histochemical quantification of collagen content
Quantification of collagen content by digital image analysis revealed significantly increased total collagen content (Sirius Red staining; Fig. 1
and Table 1
) and immunohistochemically detected collagen types I, III, and VI (Fig. 2
, Figs. 3
, 4
and Table 2
) in the SD-STZ group compared with nondiabetic SD and TGR(hKLK1) and diabetic TGR(hKLK1)-STZ littermates, with the TGR(hKLK1) not being statistically different from the SD rats. Aprotinin- and icatibant-treated TGR(hKLK1)-STZ hearts demonstrated significantly raised total collagen content compared with untreated TGR(hKLK1)-STZ hearts, but still significantly lower than the SD-STZ rat group (Fig. 1
and Table 1
).
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Multivariate analysis of LV function parameters and collagen expression
Multivariate analysis of collagen expression and LV function parameters revealed that total collagen content (Sirius Red staining) and collagen types I, III, and VI expression were significantly (P<0.01) interrelated with parameters of LV function (Table 3
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Collagen types I, III, IV, and VI, in addition to type V collagen and elastin, constitute the main components of the extracellular cardiac matrix. Changes in the extracellular matrix, especially in the collagen composition, decisively influence the passive mechanical properties of the myocardium and thus are important for cardiac hemodynamics (4)
.
Massive interstitial and perivascular fibrosis with focal collagen accumulation and fibrillar fragmentation has been reported in diabetic patients (9)
. Apart from replacement fibrosis, hearts from diabetic patients display reactive fibrosis including thickening of the basal vascular and cellular membrane. The etiology of this collagen accumulation is multifactorial. Increased glucose levels stimulate fibroblasts. Findings on increased TGF-ß levels in diabetic hearts indicate that cytokines are also involved in the collagen accumulation under diabetic conditions (8)
. Moreover, collagen digestion by collagenases is impaired due to the increased glycosylation level (7)
. Increased cardiac hydroxyproline content, an index of total collagen, was described in diabetic patients and animals depending on the severity and duration of the disease (5)
. This finding was confirmed in STZ diabetic rats after 6 wk and is consistent with the significantly increased Sirius Red staining observed in the hearts of SD-STZ rats. Similar to findings in hearts of diabetic patients, we found an increase in cardiac types I, III, and VI collagen in the extracellular matrix of SD-STZ rats (7
, 9)
. While an increase of type I and III collagen probably contributes to changed cardiac hemodynamics in SD-STZ animals due to increased stiffness and loss of elasticity, the observed additional perivascular increase of type III and VI collagen may impede the exchange of cellular substrate. This might further intensify oxidative stress and endothelial dysfunction.
Although both SD-STZ and TGR(hKLK1)-STZ diabetic strains developed comparable values of hyperglycemia and weight loss, STZ diabetic rats with transgenic expression of the hKLK1 gene showed preserved LV function and significantly lower levels of the three investigated collagen types than wild-type SD-STZ animals. Collagen content of TGR(hKLK1)-STZ did not differ significantly from normoglycemic TGR(hKLK1) and SD rats. Hemodynamically, the latter corresponds especially with the improvement in LVEDP. Chronic treatment with the KLK inhibitor aprotinin blunted these effects in TGR(hKLK1)-STZ, but not in SD-STZ rats (data not shown), confirming the KLK dependency of our findings. However, apart from KLK-inhibition, aprotinin exerts a variety of KLK-independent effects. We therefore investigated the BK B2 receptor-specific inhibitor icatibant, which significantly blunted the effects on collagen content in TGR(hKLK1)-STZ. As to the LV function parameters, only LVEDP, the hemodynamic parameter for passive LV stiffness, was impaired in diabetic TGR(hKLK1) after B2 receptor-specific inhibition. This agrees with data by Cheng et al. (23)
showing that icatibant treatment led to an increase in LVEDP without influencing dP/dtmax and dP/dtmin in a dog heart failure model. Thus, the BK B2 receptor contributes to the cardioprotective effects of TGR(hKLK1) under STZ diabetic conditions. Since we recently found an up-regulation in cardiac BK B2 and BK-B1 receptor mRNA expression in SD-STZ diabetic rats (24)
, it is possible that the different effects between aprotinin and icatibant on LVP, dP/dtmax, and dP/dtmin in diabetic TGR(hKLK1) depends on the more comprehensive degree of KKS inhibition after aprotinin treatment in our model.
The present evidence for kinins as antifibrotic agents is to some extent contradictory. In the absence of NO providing endothelial cells, kinins may act as growth factors on cultured cardiomyocytes and smooth muscle cells via the extracellular signal-regulated/mitogen-activated (ERK/MAP) kinase cascade (25)
. However, Cellier et al. reported the opposite effect after B2 receptor stimulation in isolated glomeruli under high glucose conditions (26)
. BK reduces type I and III collagen and fibronectin mRNA expression in cultured fibroblasts, probably by a NO/prostaglandin-dependent pathway (13
, 17)
. The latter agrees with data showing that the antifibrotic effects of angiotensin-converting enzyme inhibition are reduced after cotreatment with icatibant in a model of LV hypertrophy (27)
as well as in a model of myocardial infarction using B2 receptor knockout mice (28)
. Accordingly, we showed recently that cardiac fibrosis is less severe in TGR(hKLK1) after isoproterenol-induced stress; similar to the present results in diabetic cardiomyopathy, this effect was B2 receptor dependent (14)
. By pharmacological B2 receptor blockade, genetic ablation of the B2 receptor, and transgenic expression of the KLK gene, Schanstra et al. recently demonstrated a B2 receptor antifibrotic effect in the unilateral ureteral obstruction animal model (29)
. Thus, several in vivo experiments, including our investigations, clearly show the importance of kinins in reducing extracellular matrix accumulation under pathophysiological conditions. IKLK itself increases collagen breakdown by its own collagenase activity and by increasing MMP activity (15
, 16
, 30)
. Since we and others have shown that cardiac KLK activity is reduced under severe STZ diabetic conditions (18
, 31)
, it is reasonable to suggest that direct KLK-dependent effects might have contributed to the prohibiting effect on collagen accumulation in TGR(hKLK1)-STZ.
The antifibrotic effects of kinins have been elucidated by prostaglandin- and NO-dependent pathways (17
, 32)
. Several studies have shown that transient expression of hKLK in rats by somatic gene transfer can attenuate hypertension, cardiac remodeling, renal injury and induce angiogenesis in a hindlimb ischemic model and normoperfused skeletal muscle (33
34
35
36)
; the beneficial effect on hypertension was blunted by the KLK inhibitor aprotinin (33)
. More important, local KLK gene transfer protects from diabetes-induced microangiopathy by attenuating apoptosis of endothelial cells and promoting collateralization, thus ensuring improved hemodynamic recovery in case of supervening vascular occlusion (37)
. In this setting, insulin supplementation did not exert an additive effect in preventing diabetic microangiopathy. Kinins can increase coronary perfusion (38)
and stimulate myocardial glucose uptake (39
40
41)
. It is therefore conceivable that apart from the primary reduction of collagen abundance, additional kinin-dependent cardioprotective effects might have contributed to the preserved LV function under STZ-induced diabetic conditions. However, direct antifibrotic effects of the KKS and the above mentioned additional effects of KLK are not mutually exclusive, since the latter might ultimately exert secondary antifibrotic effects via inhibition of cell death mechanisms and improved supply of nutrients and oxygen.
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ACKNOWLEDGMENTS |
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Received for publication August 4, 2003. Accepted for publication November 21, 2003.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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