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Transgenic activation of the kallikrein-kinin system inhibits intramyocardial inflammation, endothelial dysfunction and oxidative stress in experimental diabetic cardiomyopathy

Carsten Tschöpe¹, Thomas Walther, Felicitas Escher, Frank Spillmann, Jing Du, Christine Altmann^*, Ingolf Schimke, Michael Bader, Carlos F. Sanchez-Ferrer, Heinz-Peter Schultheiss and Michel Noutsias

Department of Cardiology and Pneumonology, Charité-University Medicine Berlin, Campus Benjamin Franklin and
^* Campus Mitte, Germany;
Max-Delbrück-Center for Molecular Medicine, Berlin, Germany; and
Department of Pharmacology and Therapeutics, Faculty of Medicine, Autonomous University of Madrid, Spain

¹Correspondence: Department of Cardiology and Pneumology, Charité-University Medicine Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, D-12220 Berlin, Germany. E-Mail: ctschoepe{at}yahoo.com

SPECIFIC AIMS

In light of the possible involvement of intramyocardial inflammation in diabetic cardiomyopathy, we investigated cardiac CAMs expression (ICAM-1 and VCAM-1) and ß₂-leukocyte integrins⁺ infiltrates as well as cytokine (TNF and IL-1ß) expression in STZ-induced diabetic wild type rats as well as the role of the cardioprotective role of the kallikein-kinin system (KKS) under this hyperglycemic condition by investigating transgenic diabetic rats harboring the human kallikrein 1 gene TGR(hKLK1). We also assessed endothelial function by measuring vascular tone in isolated mesenteric microvessels, and the degree of oxidative stress by determination of plasma thiobarbituric acid reactive substances (TBARS) in the wild-type (WT) and transgenic strains, respectively.

PRINCIPAL FINDINGS

1. Improved left ventricular performance in diabetic hKLK1-expressing transgenic rat
There were no adverse or beneficial effects of tissue hKLK1 gene expression in transgenic rats under euglycemic conditions. Left ventricular function (LV) characteristics were similar in nondiabetic WT and TGR(hKLK1). Six weeks after a single STZ injection, both diabetic WT and TGR(hKLK1) displayed severe hyperglycemia and a marked reduction in body weight. Analyses of LV function in anesthetized, intubated, and ventilated open-chest animals using a Millar tip catheter system revealed a marked LV dysfunction in diabetic WT. Thus, LV pressure (LVP) and the maximum rate of LV pressure rise (dP/dt_max) reached only 68% of the nondiabetic WT values. However, LV function of diabetic TGR(hKLK1) was improved compared with STZ rat hearts. Thus, targeting the cardiac KLK expression was beneficial for defective systolic and diastolic performance in diabetic cardiomyopathy.

2. Correlation between cardiac inflammation and LV function in diabetic cardiopathy
Immunohistology analysis showed that 6 wk after STZ injection, cardiac ICAM-1 and VCAM-1 (Fig. 1 ) (CAMs) expression as well as of ß₂-leukocyte integrins⁺ (CD18⁺, CD11a⁺, CD11b⁺) and cytokine (TNF and IL-1ß) expressing infiltrates was significantly increased in STZ-WT rats compared with normoglycemic littermates. The latter also correlated with the cardiac mRNA levels of IL1ß expression determined by a specific RNase protection assay. Above all, there was an inverse correlation between LV function parameters and CAMs expression as well as infiltrate density. Moreover, preservation on LV function of diabetic TGR(hKLK1)-STZ rats was associated with baseline CAMs expression and infiltrate densities comparable to normoglycemic TGR(hKLK1) and SD rats, which indicates that transgenic hKLK1 inhibits CAM induction and immunocompetent infiltration dependent on these CAMs.

View larger version (127K): [in this window] [in a new window]   Figure 1. Quantification by digital image analyses showed a significantly increased expression of VCAM-1 in the SD-STZ group (red staining) compared with either the nondiabetic SD and TGR(hKLK1) or the diabetic TGR(hKLK1)-STZ rat cardiac sections, whereby the sham-treated SD and TGR(hKLK1) did not statistically differ.

3. Vascular function and oxidative stress in STZ-induced wild-type rats and TGR(hKLK1)
To investigate whether changes in adhesion molecule regulation of cardiac microvessels can be associated with alterations of endothelial function, vascular reagibility of isolated microvessels was studied ex vivo. Third branch mesenteric arteries were removed and placed on a small vessel myograph capable of measuring isometric tension and containing a physiological solution. Their passive tension and internal circumference were determined; the mean internal diameter ranged between 200 and 400 µm, with nonsignificant differences among the different groups of rats, then endothelium-dependent relaxations to acetylcholine (ACh) were recorded. Endothelium-dependent relaxations evoked by ACh were similar in nondiabetic SD and TGR(hKLK1) rats, indicating unaltered endothelial function under basal conditions in the transgenic animals. The impairment of endothelium-dependent relaxations was significantly lower in diabetic TGR(hKLK1)-STZ than in diabetic SD-STZ rats, suggesting a lower level of endothelial dysfunction due to KLK overexpression under hyperglycemic conditions (Fig. 2 ).

View larger version (17K): [in this window] [in a new window]   Figure 2. Relaxation responses to acetylcholine (ACh) in isolated mesenteric arteries from SD, TGR(hKLK1), SD-STZ, and TGR(hKLK1)-STZ rats. The responses are expressed as the percentage of a previous contraction elicited by norepinephrine (1 µmol/L). The values of these previous contractions were 10.01 ± 0.36, 11.20 ± 0.80, 11.07 ± 0.51, and 11.04 ± 0.63 mNewtons (nonstatistical differences) for SD, TGR(hKLK1), SD-STZ, and TGR(hKLK1)-STZ, respectively.

Thiobarbituric acid reactive substances (TBARS) were measured as an indicator of lipid peroxidation. Both diabetic groups (SD-STZ and TGR(hKLK1-STZ)) revealed significantly increased serum TBARS levels compared with the normoglycemic littermates. SD-STZ rats depicted significantly higher TBARS levels compared with all remaining groups. Plasma TBARS levels in TGR(hKLK1-STZ) rats, although significantly increased compared with normoglycemic TGR(hKLK1) and SD-CO littermates, were still significantly lower than in SD-STZ rats.

CONCLUSIONS AND SIGNIFICANCE

Multiple, but incompletely understood, mechanisms are involved in the pathogenesis of diabetic cardiomyopathy. The important findings of this study are the involvement of intramyocardial inflammation under STZ-induced diabetic conditions (CAMs expression, immunocompetent infiltrates, and cytokine expression), and the protective anti-inflammatory effect of hKLK1 gene transfer on LV function under hyperglycemic conditions. Given the specific inhibition of kininogens with the interactions of ICAM-1 with ß₂-leukocyte integrins, it is conceivable that the transgenic expression of hKLK1 inhibited CAMs integrins interactions in our model and thereby transendothelial migration of immunocompetent cells, which in turn promote local expression of cytokines and further enhance the intramyocardial inflammatory reaction by providing an additional stimulus for sustained endothelial CAMs expression in terms of a vicious circle comparable to human inflammatory cardiomyopathy. These anti-inflammatory effects found in diabetic TGR(hKLK1) belong to several vascular/cardioprotective effects, including the improvement of the deranged extracellular matrix regulation as well as the improvement of the defective sarcoplasmic reticulum Ca²⁺ transport in this animal model, as recently shown by us (Fig. 3 ).

View larger version (22K): [in this window] [in a new window]   Figure 3. Hypothetical schematic diagram showing multiple synergistic protective effects of the kallikrein-kinin system (KKS) leading to cardiovascular protection under diabetic conditions. Activation of the KKS induces several nitric oxidative-dependent effects, including vasodilation as well as anti-inflammatory effects, as shown in our study. These effects, directly or indirectly induced by the KKS, are known to be able to reduce oxidative stress, profibrotic effects as well as have a protective effect of the intracellular Ca2+ handling under STZ diabetic conditions.

In conclusion, our data reveal multiple synergistic protective effects of the transgenic expression of the hKLK1 by inhibition of intramyocardial inflammation (CAMs expression, immunocompetent infiltration, and cytokine expression), and an improvement of microvascular reagibility, contributing to a reduction of oxidative stress. Therefore, the stimulation of the KKS under diabetic conditions could be a promising target in the treatment end-organ failure and of vascular pathology in diabetes mellitus.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4095fje;

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