Felicitas Escher

Toll-Like Receptor-4 Modulates Survival by Induction of Left Ventricular Remodeling after Myocardial Infarction in Mice

Left ventricular (LV) remodeling is known to contribute to morbidity and mortality after myocardial infarction (MI). Because LV remodeling is strongly associated with an inflammatory response, we investigated whether or not TLR-4 influences LV remodeling and survival in a mice model of MI. Six days after MI induction, TLR4 knockout (KO)-MI mice showed improved LV function 32 and reduced LV remodeling as indexed by reduced levels of atrial natriuretic factor and total collagen as well as by a reduced heart weight to body weight ratio when compared with WT-MI mice. This was associated with a reduction of protein levels of the intracellular TLR4 adapter protein MyD88 and enhanced protein expression of the anti-hypertrophic JNK in KO-MI mice when compared with wild-type (WT)-MI mice. In contrast, protein activation of the pro-hypertrophic kinases protein kinase Cδ and p42/44 were not regulated in KO-MI mice when compared with WT-MI mice. Improved LV function, reduced cardiac remodeling, and suppressed intracellular TLR4 signaling in KO-MI mice were associated with significantly improved survival compared with WT-MI mice (62 vs 23%; p < 0.0001). TLR4 deficiency led to improved survival after MI mediated by attenuated left ventricular remodeling.

Renin Inhibition Improves Cardiac Function and Remodeling After Myocardial Infarction Independent of Blood Pressure

Pharmacological renin inhibition with aliskiren is an effective antihypertensive drug treatment, but it is currently unknown whether aliskiren is able to attenuate cardiac failure independent of its blood pressure–lowering effects. We investigated the effect of aliskiren on cardiac remodeling, apoptosis, and left ventricular (LV) function after experimental myocardial infarction (MI). C57J/bl6 mice were subjected to coronary artery ligation and were treated for 10 days with vehicle or aliskiren (50 mg/kg per day via an SC osmopump), whereas sham-operated animals served as controls. This dose of aliskiren, which did not affect systemic blood pressure, improved systolic and diastolic LV function, as measured by the assessment of pressure-volume loops after MI. Furthermore, after MI LV dilatation, cardiac hypertrophy and lung weights were decreased in mice treated with aliskiren compared with placebo-treated mice after MI. This was associated with a normalization of the mitogen-activated protein kinase P38 and extracellular signal-regulated kinases 1/2, AKT, and the apoptotic markers bax and bcl-2 (all measured by Western blots), as well as the number of TUNEL-positive cells in histology. LV dilatation, as well as the associated upregulation of gene expression (mRNA abundance) and activity (by zymography) of the cardiac metalloproteinase 9 in the placebo group after MI, was also attenuated in the aliskiren-treated group. Aliskiren improved LV dysfunction after MI in a dose that did not affect blood pressure. This was associated with the amelioration of cardiac remodelling, hypertrophy, and apoptosis.

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

The mechanisms contributing to diabetic cardiomyopathy, as well as the protective pathways of the kallikrein‐kinin‐system (KKS), are incompletely understood. In a kallikrein‐overexpressing rat model of streptozotocin (STZ)‐induced diabetic cardiomyopathy, we investigated the involvement of inflammatory pathways, endothelial dysfunction, and oxidative stress. Six weeks after STZ injection, impairment of left ventricular (LV) function parameters measured by a Millar‐tip catheter (peak LV systolic pressure; dP/dtmax; dP/dtmin) was accompanied by a significant increment of ICAM‐1 and VCAM‐1 (CAMs) expression, as well as of β2‐leukocyte‐integrins+ (CD18+, CD11a+, CD11b+) and cytokine (TNF‐α and IL‐1β)‐expressing infiltrates in male Sprague‐Dawley (SD‐STZ) rats compared with normoglycemic littermates. Furthermore, SD‐STZ rats demonstrated a significant impairment of endothelium‐dependent relaxation evoked by acetylcholine and significantly increased plasma TBARS (plasma thiobarbituric acid reactive substances) levels as a measure of oxidative stress. These diabetic cardiomyopathy‐associated alterations were significantly attenuated (P<0.05) in diabetic transgenic rats expressing the human kallikrein 1 (hKLK1) gene with STZ‐induced diabetes. CAMs expression, β2‐leukocyte‐integrins+, and cytokine‐expressing infiltrates correlated significantly with all evaluated LV function parameters. The multiple protective effects of the KKS in experimental diabetic cardiomyopathy comprise the inhibition of intramyocardial inflammation (CAMs expression, β2‐leukocyte‐integrins+ infiltration and cytokine expression), an improvement of endothelium‐dependent relaxation and the attenuation of oxidative stress. These insights might have therapeutic implications also for human diabetic cardiomyopathy.

Cardioprotective and Anti-Inflammatory Effects of Interleukin Converting Enzyme Inhibition in Experimental Diabetic Cardiomyopathy

OBJECTIVE—We investigated the effect of pharmacological inhibition of the interleukin converting enzyme (ICE) on cardiac inflammation, apoptosis, fibrosis, and left ventricular function in an animal model of diabetes. RESEARCH DESIGN AND METHODS—Diabetes was induced in 24 Sprague-Dawley rats by injection of streptozotozin (STZ) (70 mg/kg). Diabetic animals were treated with the interleukin converting enzyme (ICE) inhibitor (ICEI) (n = 12) or with a placebo (n = 12). Nondiabetic rats served as controls (n = 12). Left ventricular function was documented 6 weeks after induction of diabetes. Cardiac tissue was analyzed for the expression of cytokines, intracellular adhesion molecule-1 and vascular cell adhesion molecule-1, leukocyte and macrophage integrins, and collagen. Phosphorylation of Akt was analyzed by Western blot and apoptosis by Blc-2 and Bax measurements. RESULTS—Left ventricular function was significantly impaired in diabetic animals. This was accompanied by a significant increase of cytokines, cell adhesion molecules, leukocytes and macrophages, and collagen content. In addition, the phosphorylation state of Akt was reduced. These changes were significantly attenuated in the diabetic group treated with ICEI. CONCLUSIONS—Cardiac dysfunction is associated with cardiac inflammation in experimental diabetic cardiomyopathy. Both of these—cardiac dysfunction and inflammation—are attenuated after treatment with ICEI. These data suggest that anticytokine-based therapies might be beneficial in diabetic cardiomyopathy.

Prevention of cardiac fibrosis and left ventricular dysfunction in diabetic cardiomyopathy in rats by transgenic expression of the human tissue kallikrein gene

Diabetic cardiomyopathy includes fibrosis. Kallikrein (KLK) can inhibit collagen synthesis and promote collagen breakdown. We investigated cardiac fibrosis and left ventricular (LV) function in transgenic rats (TGR) expressing the human kallikrein 1 (hKLK1) gene in streptozotocin (STZ) ‐induced diabetic conditions. Six weeks after STZ injection, LV function was determined in male Sprague‐Dawley (SD) rats and TGR(hKLK1) (n=10/group) by a Millar tip catheter. Total collagen content (Sirius Red staining) and expression of types I, III, and VI collagen were quantified by digital image analysis. SD‐STZ hearts demonstrated significantly higher total collagen amounts than normoglycemic controls, reflected by the concomitant increment of collagen types I, III, and VI. This correlated with a significant reduction of LV function vs. normoglycemic controls. In contrast, surface‐specific content of the extracellular matrix, including collagen types I, III, and VI expression, was significantly lower in TGR(hKLK1)‐STZ, not exceeding the content of SD and TGR(hKLK1) controls. This was paralleled by a preserved LV function in TGR(hKLK1)‐STZ animals. The kallikrein inhibitor aprotinin and the bradykinin (BK) B2 receptor antagonist icatibant reduced the beneficial effects on LV function and collagen content in TGR(hKLK1)‐STZ animals. Transgenic expression of hKLKl counteracts the progression of LV contractile dysfunction and extracellular matrix remodeling in STZ‐induced diabetic cardiomyopathy via a BK B2 receptor‐dependent pathway.—Tschope, C., Walther, T., Königer, J., Spillmann, F., Westermann, D., Escher, F., Pauschinger, M., Pesquero, J. B., Bader, M., Schultheiss, H.‐P., Noutsias, M. Prevention of cardiac fibrosis and left ventricular dysfunction in diabetic cardiomyopathy in rats by transgenic expression of the human tissue kallikrein gene. FASEB J. 18, 828–835 (2004)

Induction of coxsackievirus-adenovirus-receptor expression during myocardial tissue formation and remodeling: Identification of a cell-to-cell contact-dependent regulatory mechanism

Background—The coxsackievirus-adenovirus receptor (CAR) was cloned as a receptor for both viruses, but its primary biological functions and regulatory mechanisms are unknown. CAR was low in healthy adult myocardium, whereas strong CAR reexpression was observed in human dilated cardiomyopathy. The molecular mechanisms of CAR induction in cardiomyocytes are unknown. Methods and Results—We report on CAR regulation during development, CAR induction after myocardial infarction, and cell-to-cell contact–dependent CAR regulation in the rat. The high CAR expression during development in various organs decreased up to 190-fold after birth. After infarction resulting in severe cardiac dysfunction (dP/dtmax, −53%; dP/dtmin, −58%; left ventricular pressure, −45%), CAR was induced locally in cardiomyocytes of the infarct zone, where it was also expressed by capillary-like CD31+ structures and CD18+ interstitial cells, whereas it remained confined to subendothelial layers of arterioles and venules. In cultured cardiomyocytes, endothelin-1, cardiotrophin-1, leukemia-inhibiting factor, and cyclic stretch had no effect on CAR, whereas at high versus low cell density, CAR was suppressed up to 10-fold (P =0.006). Conditioned media from low- or high-density cardiomyocytes or cardiofibroblasts had no effect. Conclusions—The locally confined CAR upregulation after infarction makes induction by various humoral factors unlikely, because cardiac dysfunction results in high activities of sympathetic and renin-angiotensin systems and cytokines. The cell culture experiments identify a cell-to-cell contact–dependent mechanism of CAR regulation. Further characterization of the signals linking cell-to-cell interactions to CAR gene expression may provide insight into mechanisms and functional consequences of the generalized CAR induction in dilated cardiomyopathy, and of its local induction after myocardial infarction.

Human Apolipoprotein A-I Gene Transfer Reduces the Development of Experimental Diabetic Cardiomyopathy

Background— The hallmarks of diabetic cardiomyopathy are cardiac oxidative stress, intramyocardial inflammation, cardiac fibrosis, and cardiac apoptosis. Given the antioxidative, antiinflammatory, and antiapoptotic potential of high-density lipoprotein (HDL), we evaluated the hypothesis that increased HDL via gene transfer (GT) with human apolipoprotein (apo) A-I, the principal apolipoprotein of HDL, may reduce the development of diabetic cardiomyopathy. Methods and Results— Intravenous GT with 3×1012 particles/kg of the E1E3E4-deleted vector Ad.hapoA-I, expressing human apoA-I, or Ad.Null, containing no expression cassette, was performed 5 days after streptozotocin (STZ) injection. Six weeks after apoA-I GT, HDL cholesterol levels were increased by 1.6-fold (P<0.001) compared with diabetic controls injected with the Ad.Null vector (STZ-Ad.Null). ApoA-I GT and HDL improved LV contractility in vivo and cardiomyocyte contractility ex vivo, respectively. Moreover, apoA-I GT was associated with decreased cardiac oxidative stress and reduced intramyocardial inflammation. In addition, compared with STZ-Ad.Null rats, cardiac fibrosis and glycogen accumulation were reduced by 1.7-fold and 3.1-fold, respectively (P<0.05). Caspase 3/7 activity was decreased 1.2-fold (P<0.05), and the ratio of Bcl-2 to Bax was upregulated 1.9-fold (P<0.005), translating to 2.1-fold (P<0.05) reduced total number of cardiomyocytes with apoptotic characteristics and 3.0-fold (P<0.005) reduced damaged endothelial cells compared with STZ-Ad.Null rats. HDL supplementation ex vivo reduced hyperglycemia-induced cardiomyocyte apoptosis by 3.4-fold (P<0.005). The apoA-I GT-mediated protection was associated with a 1.6-, 1.6-, and 2.4-fold induction of diabetes-downregulated phospho to Akt, endothelial nitric oxide synthase, and glycogen synthase kinase ratio, respectively (P<0.005). Conclusion— ApoA-I GT reduced the development of streptozotocin-induced diabetic cardiomyopathy.

Toll‐like receptor‐4 deficiency attenuates doxorubicin‐induced cardiomyopathy in mice

Cardiac inflammation and generation of oxidative stress are known to contribute to doxorubicin (Dox)‐induced cardiomyopathy. Toll‐like receptors (TLRs) are a part of the innate immune system and are involved in cardiac stress reactions. Since TLR4 might play a relevant role in cardiac inflammatory signalling, we investigated whether or not TLR4 is involved in Dox‐induced cardiotoxicity.

Reduced Degradation of the Chemokine MCP-3 by Matrix Metalloproteinase-2 Exacerbates Myocardial Inflammation in Experimental Viral Cardiomyopathy

Background— Myocarditis is an important cause for cardiac failure, especially in younger patients, followed by the development of cardiac dysfunction and death. The present study investigated whether gene deletion of matrix metalloproteinase-2 influences cardiac inflammation and function in murine coxsackievirus B3 (CVB3)–induced myocarditis. Methods and Results— Matrix metalloproteinase-2 knockout mice (MMP-2−/−) and their wild-type controls (WT) were infected with CVB3 to induce myocarditis. Three days after infection, an increased invasion of CD4+-activated T cells into the myocardium was documented, followed by an excess of inflammatory cells 7 days after infection, which was significantly higher in the MMP-2−/−animals compared with the WT animals. Moreover, cardiac apoptosis, remodeling, viral load, and function were deteriorated in MMP-2−/− animals after CVB3 infection. This overwhelming inflammation was followed by 100% mortality after 15 days. This was associated with increased levels of MCP-3 in the cardiac tissue of MMP-2−/− mice. Because MMP-2 cleaves the chemokine MCP-3, the loss of this cleavage lead to an overreaction of the immune system with pronounced myocardial damage mediated by the inflammatory cells. When a neutralizing antibody against MCP-3 was given to MMP-2−/− mice, this exaggerated reaction of the immune system could be normalized to levels similar to WT-CVB3 animals. Conclusions— Loss of MMP-2 increased the inflammatory response after CVB3 infection, which impaired cardiac function and survival during CVB3-induced myocarditis. Matrix metalloproteinase-2–mediated chemokine cleavage has an important role in cardiac inflammation as a negative feedback mechanism.

Circulating Rather Than Cardiac Angiotensin-(1-7) Stimulates Cardioprotection After Myocardial Infarction

Background—Angiotensin (Ang)-(1-7) attenuates the development of heart failure. In addition to its local effects on cardiovascular tissue, Ang-(1-7) also stimulates bone marrow, which harbors cells that might complement the therapeutic effect of Ang-(1-7). We studied the effects of Ang-(1-7) either produced locally in the heart or subcutaneously injected during the development of heart failure induced by myocardial infarction (MI) and explored the role of cardiovascular progenitor cells in promoting the effects of this heptapeptide. Methods and Results—Effects of Ang-(1-7) on bone marrow–derived mononuclear cells in rodents, particularly endothelial progenitor cells, were investigated in vitro and in vivo in rats, in mice deficient for the putative Ang-(1-7) receptor Mas, and in mice overexpressing Ang-(1-7) exclusively in the heart. Three weeks after MI induction through permanent coronary artery occlusion, effects of Ang-(1-7) either produced locally in the heart or injected into the subcutaneous space were investigated. Ang-(1-7) stimulated proliferation of endothelial progenitor cells isolated from sham or infarcted rodents. The stimulation was blunted by A779, a Mas receptor blocker, or by Mas deficiency. Infusion of Ang-(1-7) after MI increased the number of c-kit– and vascular endothelial growth factor–positive cells in infarcted hearts, inhibited cardiac hypertrophy, and improved cardiac function 3 weeks after MI, whereas cardiomyocyte-derived Ang-(1-7) had no effect. Conclusions—Our data suggest circulating rather than cardiac Ang-(1-7) to be beneficial after MI. This beneficial effect correlates with a stimulation of cardiac progenitor cells in vitro and in vivo. This characterizes the heptapeptide as a promising new tool in stimulating cardiovascular regeneration under pathophysiological conditions.