Natarajan Sivasubramanian

Homocysteine Induces Expression and Secretion of Monocyte Chemoattractant Protein-1 and Interleukin-8 in Human Aortic Endothelial Cells Implications for Vascular Disease

Background—Proinflammatory cytokines play key roles in atherogenesis and disease progression. Because hyperhomocysteinemia is an independent risk factor for cardiovascular disease, we hypothesized that homocysteine could be atherogenic by altering the expression of specific cytokines in vascular endothelial cells. Methods and Results—Northern blot and RNase protection assays showed that dl-homocysteine induced mRNA expression of the proinflammatory cytokines monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8) in cultured human aortic endothelial cells (HAECs). Homocysteine had no effect on expression of other cytokines, namely tumor necrosis factor-&agr;, granulocyte-macrophage colony-stimulating factor, interleukin-1&bgr;, and transforming growth factor-&bgr;. MCP-1 mRNA expression increased 1 hour after homocysteine treatment, reached a maximum within 2 to 4 hours, and declined to basal levels over the next 24 hours. Induction of mRNA expression for both chemokines was observed with as little as 10 &mgr;mol/L dl-homocysteine, and maximal expression was achieved with 50 &mgr;mol/L dl-homocysteine. Homocysteine also triggered the release of MCP-1 and IL-8 protein from HAECs into the culture medium. The induction was specific for homocysteine, because equimolar concentrations of l-homocystine, l-cysteine, and l-methionine had no effect on mRNA levels and protein release. Furthermore, l-homocysteine induced chemokine expression, but d-homocysteine did not, thus demonstrating enantiomeric specificity. The culture medium from homocysteine-treated HAECs promoted chemotaxis in human peripheral blood monocytes and U937 cells. Anti-human recombinant MCP-1 antibody blocked the migration. Conclusions—Pathophysiological levels of l-homocysteine alter endothelial cell function by upregulating MCP-1 and IL-8 expression and secretion. This suggests that l-homocysteine may contribute to the initiation and progression of vascular disease by promoting leukocyte recruitment.

In Vivo Expression of Proinflammatory Mediators in the Adult Heart after Endotoxin Administration: The Role of Toll-Like Receptor–4

Tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and nitric oxide (NO) may play a role in lipopolysaccharide (LPS)-induced cardiac depression. Toll-like receptor-4 (TLR-4) mediates the cytokine response to LPS in immune cells. TLR-4 also is expressed in human and murine myocardial tissue. Therefore, the hypothesis that LPS induces proinflammatory cytokines in the heart via TLR-4 was tested. C3H/HeJ (TLR-4 deficient) and C3HeB/FeJ mice were studied. LPS induced a robust increase in myocardial TNF-alpha and IL-1beta mRNA in C3HeB/FeJ mice. The response in C3H/HeJ mice was blunted and delayed. Myocardial TNF-alpha and IL-1beta protein levels were higher in C3HeB/FeJ mice, as were inducible NO synthase protein and NO production. Activation of myocardial NF-kappaB was observed within 30 min in C3HeB/FeJ mice but not in C3H/HeJ mice. These findings suggest that myocardial TLR-4 is involved in signaling cytokine production within the heart during endotoxic shock.

Cardiac-Specific Overexpression of Tumor Necrosis Factor-α Causes Oxidative Stress and Contractile Dysfunction in Mouse Diaphragm

BackgroundWe have developed a transgenic mouse with cardiac-restricted overexpression of tumor necrosis factor-&agr; (TNF-&agr;). These mice develop a heart failure phenotype characterized by left ventricular dysfunction and remodeling, pulmonary edema, and elevated levels of TNF-&agr; in the peripheral circulation from cardiac spillover. Given that TNF-&agr; causes atrophy and loss of function in respiratory muscle, we asked whether transgenic mice developed diaphragm dysfunction and whether contractile losses were caused by oxidative stress or tissue remodeling. Methods and ResultsMuscles excised from transgenic mice and littermate controls were studied in vitro with direct electrical stimulation. Cytosolic oxidant levels were measured with 2′,7′-dichlorofluorescin diacetate; emissions of the oxidized product were detected by fluorescence microscopy. Force generation by the diaphragm of transgenic animals was 47% less than control (13.2±0.8 [±SEM] versus 25.1±0.6 N/cm2;P <0.001); this weakness was associated with greater intracellular oxidant levels (P <0.025) and was partially reversed by 30-minute incubation with the antioxidant N-acetylcysteine 10 mmol/L (P <0.01). Exogenous TNF-&agr; 500 &mgr;mol/L increased oxidant production in diaphragm of wild-type mice and caused weakness that was inhibited by N-acetylcysteine, suggesting that changes observed in the diaphragm of transgenic animals were mediated by TNF-&agr;. There were no differences in body or diaphragm weights between transgenic and control animals, nor was there evidence of muscle injury or apoptosis. ConclusionsElevated circulating levels of TNF-&agr; provoke contractile dysfunction in the diaphragm through an endocrine mechanism thought to be mediated by oxidative stress.

THE ROLE OF CYTOKINES IN THE FAILING HUMAN HEART

Despite repeated attempts to develop a unifying hypothesis that explains the clinical syndrome of heart failure, no single conceptual paradigm has withstood the test of time. In this regard, recent studies have shown that a class of biologically active molecules, generically referred to as cytokines, are overexposed in heart failure. This article will review recent clinical and experimental material that suggest proinflammatory (stress activated) cytokines such as tumor necrosis factor-alpha (TFN-alpha), interleukin-1 (IL-1), and interleukin-6 (IL-6) may play a role in the pathogenesis of congestive heart failure. The scope of this article includes an overview of the biology of cytokines in the heart, as well as review of the clinical studies that have documented elevated levels of cytokines and cytokine receptors in patients with heart failure.

Nuclear Factor-κB Protects the Adult Cardiac Myocyte Against Ischemia-Induced Apoptosis in a Murine Model of Acute Myocardial Infarction

Background—Previous studies have shown that tumor necrosis factor (TNF) confers cytoprotective responses in cardiac myocytes. However, the mechanisms for the cytoprotective effects of TNF remain unknown. Given that TNF signals through nuclear factor &kgr;B (NF-&kgr;B) and given that NF-&kgr;B mediates cytoprotective responses, we asked whether NF-&kgr;B activation conferred cytoprotective responses in acute myocardial ischemia/infarction. Methods and Results—We examined infarct size and the prevalence of apoptosis in transgenic mice harboring cardiac-restricted expression of a mutated I&kgr;B&agr; protein (I&kgr;B&agr;&Dgr;N) that prevents nuclear translocation of NF-&kgr;B in cardiac myocytes. Triphenyltetrazolium chloride staining showed that infarct size was ≈50% greater (P <0.02) in the I&kgr;B&agr;&Dgr;N mice compared with littermate controls at 24 hours. The prevalence of cardiac myocyte apoptosis was significantly greater (P <0.008) in the I&kgr;B&agr;&Dgr;N mice compared with the littermate control mice 3 and 6 hours after left anterior descending occlusion. To explore the mechanism for these findings, we examined protein levels of c-IAP1, c-IAP2, and Bcl-2 as well as manganese superoxide dismutase and c-Jun NH2-terminal kinase activity. These studies showed that protein levels of c-IAP1 and Bcl-2 were significantly lower in the I&kgr;B&agr;&Dgr;N mice, whereas there was no change in c-IAP2 levels, manganese superoxide dismutase, or c-Jun NH2-terminal kinase activity. Conclusions—Transgenic mice with a defect in activation of NF-&kgr;B have increased susceptibility to tissue injury after acute left anterior descending occlusion. These studies suggest that the cytoprotective effects of NF-&kgr;B are mediated, at least in part, by Bcl-2 or c-IAP1.

Angiotensin II Induces Tumor Necrosis Factor Biosynthesis in the Adult Mammalian Heart Through a Protein Kinase C–Dependent Pathway

Background—Previous studies suggest that angiotensin II (Ang II) upregulates the expression of tumor necrosis factor (TNF) in nonmyocyte cell types; however, the effect of Ang II on TNF expression in the adult mammalian heart is not known. Methods and Results—To determine whether Ang II was sufficient to provoke TNF biosynthesis in the adult heart, we examined the effects of Ang II in isolated buffer-perfused Langendorff feline hearts. Ang II (10−7 mol/L) treatment resulted in a time- and dose-dependent increase in myocardial TNF mRNA and protein biosynthesis in the heart as well as in cultured adult cardiac myocytes. The effects of Ang II on myocardial TNF mRNA and protein synthesis were mediated through the angiotensin type 1 receptor (AT1R), insofar as an AT1R antagonist (AT1a) blocked the effects of Ang II, whereas an angiotensin type 2 receptor (AT2R) antagonist (AT2a) had no effect. Stimulation with Ang II led to the activation of nuclear factor-&kgr;B and activator protein-1 (AP-1), two transcription factors that are important for TNF gene expression. Nuclear factor-&kgr;B activation was accompanied by phosphorylation of I&kgr;B&agr; on serine 32 as well as degradation of I&kgr;B&agr;, suggesting that the effects of Ang II were mediated through an I&kgr;B&agr;-dependent pathway. The important role of protein kinase C (PKC) was suggested by studies in which a phorbol ester triggered TNF biosynthesis, and a PKC inhibitor abrogated Ang II–induced TNF biosynthesis. Conclusions—These studies suggest that Ang II provokes TNF biosynthesis in the adult mammalian heart through a PKC-dependent pathway.

Load-Dependent and -Independent Regulation of Proinflammatory Cytokine and Cytokine Receptor Gene Expression in the Adult Mammalian Heart

Background—Although previous studies have examined the effects of acute hemodynamic pressure overload on proinflammatory cytokine gene expression, the effects of sustained hemodynamic overloading have not been examined. Methods and Results—Sustained hemodynamic pressure overloading was produced in mice by transverse constriction of the aorta. Proinflammatory cytokine and cytokine receptor gene expression were determined by ribonuclease protection assays (RPA) at 6 hours and at 3, 7, 14 and 35 days after banding. M-mode echocardiography was used to assess left ventricular structure and function at identical time points. RPA showed that tumor necrosis factor (TNF), interleukin (IL)-1&bgr;, and IL-6 mRNA levels were maximal at 6 hours and returned to baseline levels within 72 hours. There was a significant increase in IL-1RII and IL-6R&agr; receptor mRNA levels after overloading but no significant increase in TNFR1, TNFR2, IL-1RI, or gp130 mRNA levels. The transient increase in expression of proinflammatory cytokine gene expression was not explained by changes in left ventricular loading conditions, left ventricular wall stress, desensitization of proinflammatory genes, or decreased nuclear factor-&kgr;B activation. It is interesting that transverse constriction of the aorta provoked an increase in the expression of tristetraprolin, a homeostatic zinc finger protein that is known to destabilize TNF mRNA. Conclusion—Sustained hemodynamic overloading provokes a transient increase in proinflammatory cytokine and cytokine receptor gene expression; however, the decrease in proinflammatory cytokine gene expression occurred in the absence of changes in loading conditions, suggesting that the expression of proinflammatory cytokines in the heart is regulated, at least in part, by load-dependent and load-independent mechanisms.

Nitric Oxide Provokes Tumor Necrosis Factor-α Expression in Adult Feline Myocardium Through a cGMP-Dependent Pathway

BackgroundThe mechanism(s) responsible for the persistent coexpression of tumor necrosis factor-&agr; (TNF-&agr;) and nitric oxide (NO) in the failing heart is unknown. Methods and ResultsTo determine whether NO was sufficient to provoke TNF-&agr; biosynthesis, we examined the effects of an NO donor, S-nitroso-N-acetyl penicillamine (SNAP), in buffer-perfused Langendorff hearts. SNAP (1 &mgr;mol/L) treatment resulted in a time- and dose-dependent increase in myocardial TNF-&agr; mRNA and protein biosynthesis in adult cat hearts. The effects of SNAP were completely abrogated by a NO quenching agent, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (C-PTIO), and mimicked by sodium nitroprusside. Electrophoretic mobility shift assays demonstrated that SNAP treatment led to the rapid induction of nuclear factor kappa-beta (NF-&kgr;B) but not AP-1. The importance of the cGMP pathway in terms of mediating NO-induced TNF-&agr; biosynthesis was shown by studies that demonstrated that 8-bromo-cGMP mimicked the effects of SNAP and that the effects of SNAP could be completely abrogated using a cGMP antagonist, 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ), or protein kinase G antagonist (Rp-8-Br-cGMPS). SNAP and 8-Br-cGMP were both sufficient to lead to the site-specific phosphorylation (serine 32) and degradation of I&kgr;B&agr; in isolated cardiac myocytes. Finally, protein kinase G was sufficient to directly phosphorylate I&kgr;B&agr; on serine 32, a critical step in the activation of NF-&kgr;B. ConclusionsThese studies show that NO provokes TNF-&agr; biosynthesis through a cGMP-dependent pathway, which suggests that the coincident expression of TNF-&agr; and NO may foster self-sustaining positive autocrine/paracrine feedback inflammatory circuits within the failing heart.

Cyclooxygenase-2 Inhibitor Treatment Improves Left Ventricular Function and Mortality in a Murine Model of Doxorubicin-Induced Heart Failure

Background—Progression of heart failure after initial myocardial injury is mediated in part by various redundant inflammatory mediators, including the widely expressed cyclooxygenase-2 (COX-2). Because COX-2 inhibitors are useful in treating many inflammation-mediated diseases, we asked whether COX-2 inhibition can attenuate heart failure progression. Methods and Results—Heart failure was experimentally induced in 100 mice by administration of doxorubicin (4 mg · kg−1 · wk−1 for 6 weeks). Beginning at day 42, mice were fed daily with either COX-2 inhibitor–containing mice chow (n=50) or plain mice chow (controls; n=50). Left ventricular ejection fraction was evaluated as a measure of heart failure by a novel method of transthoracic echocardiography (with intravascular ultrasound catheters) at baseline and on days 42, 56, and 70. From baseline to study termination, left ventricular ejection fraction in COX-2 inhibitor–treated mice decreased significantly less than in control mice (9% versus 29%, P <0.01). Mortality was significantly lower for COX-2 inhibitor–treated mice than for control mice (18% versus 38%, P <0.01). These results were confirmed in a revalidation study in COX-2 inhibitor–treated mice (n=25) and controls (n=25). That study revealed that the hearts from control mice weighed roughly the same as hearts from COX-2 inhibitor–treated mice but showed more extensive signs of cardiomyopathy (as determined by pathological analysis by an independent, blinded observer) and higher levels of COX-2 proteins (as determined by immunoblotting [6442±1635 versus 4300±2408 arbitrary units, P <0.022]). Conclusions—COX-2 inhibitors can attenuate the progression of heart failure in a murine model of doxorubicin-induced heart failure.

Targeted Overexpression of Noncleavable and Secreted Forms of Tumor Necrosis Factor Provokes Disparate Cardiac Phenotypes

Background—Recent studies suggest that posttranslation processing or “shedding” (ie, secretion) of tumor necrosis factor (TNF) by tumor necrosis factor-&agr; converting enzyme (TACE) may contribute to the left ventricular (LV) remodeling that occurs in the failing human heart. Methods and Results—To address the functional significance of TNF shedding, we generated lines of transgenic mice with targeted overexpression of secreted wild-type (MHCsTNF2) TNF and overexpression of a mutated noncleavable transmembrane form of TNF (MHCmTNF). Both lines of mice had overlapping levels of myocardial TNF protein; however, the phenotypes of the MHCsTNF2 and MHCmTNF mice were strikingly disparate. Whereas the MHCmTNF mice developed a concentric LV hypertrophy phenotype, the MHCsTNF2 mice developed a dilated LV phenotype. The fibrillar collagen weave in MHCmTNF mice with concentric hypertrophy was characterized by thick collagen fibrils and increased collagen content, whereas the fibrillar collagen weave in the MHCsTNF2 mice with LV dilation was characterized by a diminished collagen content. Inhibition of matrix metalloproteinases with a broad-based matrix metalloproteinase inhibitor prevented LV dilation in the MHCsTNF2 mice. Conclusions—These findings suggest that posttranslational processing of TNF, as opposed to TNF expression per se, is responsible for the adverse cardiac remodeling that occurs after sustained TNF overexpression.