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In JoVE (1)
Other Publications (5)
Articles by Jennifer L. McLarty in JoVE
JoVE Immunology and Infection
Isolation of Functional Cardiac Immune Cells
Department of Cell Biology and Anatomy, University of South Carolina- School of Medicine
This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.
Other articles by Jennifer L. McLarty on PubMed
Cardiac Mast Cells Mediate Left Ventricular Fibrosis in the Hypertensive Rat Heart
Correlative data suggest that cardiac mast cells are a component of the inflammatory response that is important to hypertension-induced adverse myocardial remodeling. However, a causal relationship has not been established. We hypothesized that adverse myocardial remodeling would be inhibited by preventing the release of mast cell products that may interact with fibroblasts and other inflammatory cells. Eight-week-old male spontaneously hypertensive rats were treated for 12 weeks with the mast cell stabilizing compound nedocromil (30 mg/kg per day). Age-matched Wistar-Kyoto rats served as controls. Nedocromil prevented left ventricular fibrosis in the spontaneously hypertensive rat independent of hypertrophy and blood pressure, despite cardiac mast cell density being elevated. The mast cell protease tryptase was elevated in the spontaneously hypertensive rat myocardium and was normalized by nedocromil. Treatment of isolated adult spontaneously hypertensive rat cardiac fibroblasts with tryptase induced collagen synthesis and proliferation, suggesting this as a possible mechanism of mast cell-mediated fibrosis. In addition, nedocromil prevented macrophage infiltration into the ventricle. The inflammatory cytokines interferon-gamma and interleukin (IL)-4 were increased in the spontaneously hypertensive rat and normalized by nedocromil, whereas IL-6 and IL-10 were decreased in the spontaneously hypertensive rat, with nedocromil treatment normalizing IL-6 and increasing IL-10 above the control. These results demonstrate for the first time a causal relationship between mast cell activation and fibrosis in the hypertensive heart. Furthermore, these results identify several mechanisms, including tryptase, inflammatory cell recruitment, and cytokine regulation, by which mast cells may mediate hypertension-induced left ventricular fibrosis.
Interleukin 6 Mediates Myocardial Fibrosis, Concentric Hypertrophy, and Diastolic Dysfunction in Rats
Although there is a correlation between hypertension and levels of interleukin (IL) 6, the exact role this cytokine plays in myocardial remodeling is unknown. This is complicated by the variable tissue and circulating levels of IL-6 reported in numerous experimental models of hypertension. Accordingly, we explored the hypothesis that elevated levels of IL-6 mediate adverse myocardial remodeling. To this end, adult male Sprague-Dawley rats were infused with IL-6 (2.5 microg . kg(-1) . h(-1), IP) for 7 days via osmotic minipump and compared with vehicle-infused, aged-matched controls. Left ventricular function was evaluated using a blood-perfused isolated heart preparation. Myocardial interstitial collagen volume fraction and isolated cardiomyocyte size were also assessed. Isolated adult cardiac fibroblast experiments were performed to determine the importance of the soluble IL-6 receptor in mediating cardiac fibrosis. IL-6 infusions in vivo resulted in concentric left ventricular hypertrophy, increased ventricular stiffness, a marked increase in collagen volume fraction (6.2% versus 1.7%; P<0.001), and proportional increases in cardiomyocyte width and length, all independent of blood pressure. The soluble IL-6 receptor in combination with IL-6 was found to be essential to producing increased collagen concentration by isolated cardiac fibroblasts and also played a role in mediating a phenotypic conversion to myofibroblasts. These novel observations demonstrate that IL-6 induces a myocardial phenotype almost identical to that of the hypertensive heart, identifying IL-6 as potentially important in this remodeling process.
Cardiac Mast Cells: the Centrepiece in Adverse Myocardial Remodelling
Increased numbers of mast cells have been reported in explanted human hearts with dilated cardiomyopathy and in animal models of experimentally induced hypertension, myocardial infarction, and chronic volume overload secondary to aortocaval fistula and mitral regurgitation. Accordingly, mast cells have been implicated to have a major role in the pathophysiology of these cardiovascular disorders. In vitro studies have verified that mast cell proteases are capable of activating collagenase, gelatinases and stromelysin. Recent results have shown that with chronic ventricular volume overload, there is an elevation in mast cell density, which is associated with a concomitant increase in matrix metalloproteinase (MMP) activity and extracellular matrix degradation. However, the role of the cardiac mast cell is not one dimensional, with evidence from hypertension and cardiac transplantation studies suggesting that they can also assume a pro-fibrotic phenotype in the heart. These adverse events do not occur in mast cell deficient rodents or when cardiac mast cells are pharmacologically prevented from degranulating. This review is focused on the regulation and dual roles of cardiac mast cells in: (i) activating MMPs and causing myocardial fibrillar collagen degradation and (ii) causing fibrosis in the stressed, injured or diseased heart. Moreover, there is strong evidence that premenopausal female cardioprotection may at least partly be due to gender differences in cardiac mast cells. This too will be addressed.
Tryptase/Protease-activated Receptor 2 Interactions Induce Selective Mitogen-activated Protein Kinase Signaling and Collagen Synthesis by Cardiac Fibroblasts
The mast cell product, tryptase, has recently been implicated to mediate fibrosis in the hypertensive heart. Tryptase has been shown to mediate noncardiac fibroblast function via activation of protease-activated receptor 2 and subsequent activation of the mitogen-activated protein kinase pathway, including extracellular signal-regulated kinase 1/2. Therefore, we hypothesized that this pathway may be a mechanism leading to fibrosis in the hypertensive heart. Isolated adult cardiac fibroblasts were treated with tryptase, which induced activation of extracellular signal-regulated kinase 1/2 via protease-activated receptor 2. Blockade of protease activated receptor 2 with FSLLRY (10 μmol/L) and inhibition of the extracellular signal-regulated kinase pathway with PD98059 (10 μmol/L) prevented collagen synthesis in isolated cardiac fibroblasts stimulated with tryptase. In contrast, p38 mitogen-activated protein kinase and stress-activated protein/c-Jun N-terminal kinase were not activated by tryptase. Cardiac fibroblasts isolated from spontaneously hypertensive rats showed this same pattern of activation. Treatment of spontaneously hypertensive rats with FSLLRY prevented fibrosis in these animals, indicating the in vivo applicability of the cultured fibroblast findings. Also, tryptase induced a myofibroblastic phenotype indicated by elevations in α-smooth muscle actin and extra type III domain A (ED-A) fibronectin. Thus, the results from this study demonstrate the importance of tryptase for inducing a cardiac myofibroblastic phenotype, ultimately leading to the development of cardiac fibrosis. Specifically, tryptase causes cardiac fibroblasts to increase collagen synthesis via a mechanism involving activation of protease-activated receptor 2 and subsequent induction of extracellular signal-regulated kinase signaling.
Estrogenic Modulation of Inflammation-related Genes in Male Rats Following Volume Overload
Our laboratory has previously reported significant increases of the proinflammatory cytokine TNF-α in male hearts secondary to a sustained volume overload. These elevated levels of TNF-α are accompanied by left ventricular (LV) dilatation and cardiac dysfunction. In contrast, estrogen has been shown to protect against this adverse cardiac remodeling in both female and male rats. The purpose of this study was to determine whether estrogen has an effect on inflammation-related genes that contribute to this estrogen-mediated cardioprotection. Myocardial volume overload was induced by aortocaval fistula in 8 week old male Sprague Dawley rats (N=30) and genes of interest were identified using an inflammatory PCR array in Sham, Fistula, and Fistula + Estrogen treated (0.02 mg/kg per day beginning two-weeks prior to fistula) groups. A total of 55 inflammatory genes which were modified (≥ 2-fold change) at three days post-fistula was reduced to 20 genes by estrogen treatment, whereas 13 genes were comparably modulated in both fistula groups. The most notable were: TNF-α, which was downregulated by estrogen; and the TNF-α receptors, which were differentially regulated by estrogen. Specific genes related to arachidonic acid metabolism were downregulated by estrogen, including cyclooxygenase-1 and -2. Finally, gene expression for the β1 integrin cell adhesion subunit was significantly upregulated in the LV of estrogen treated animals. Protein levels reflected the changes observed at the gene level. These data suggest that estrogen provides its cardioprotective effects, at least in part, via genomic modulation of numerous inflammation-related genes.
