Isolation of Functional Cardiac Immune Cells

McLarty, Jennifer L.; Meléndez, Giselle C.; Spencer, William J.; Levick, Scott P.; Brower, Gregory L.; Janicki, Joseph S.

doi:10.3791/3020

Cited by 8 publications

(5 citation statements)

References 14 publications

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“…Then, either a syringe filled with 30-50uL of HBSS with a 24-gauge Vialon angiocath (BD Insyte) tip was inserted into the pericardial sac between the sternum and heart, the pericardial space was gently filled with the buffer and witnessed for expansion and this was then removed and was repeated up to at least five times or an insulin syringe was inserted into the pericardial sac after removing excess fluid from the surrounding space with a kim-wipe and 300uL was injected into the sac and as much volume was retrieved as possible. These approaches were adapted for mice from a similar procedure used in rats (McLarty et al, 2011).…”

Section: Methods Detailsmentioning

confidence: 99%

A Stromal Niche Defined by Expression of the Transcription Factor WT1 Mediates Programming and Homeostasis of Cavity-Resident Macrophages

Buechler¹,

et al. 2019

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Tissue-resident macrophages require specific milieus for the maintenance of defining gene-expression programs. Expression of the transcription factor GATA6 is required for the homeostasis, function and localization of peritoneal cavity-resident macrophages. Gata6 expression is maintained in a non-cell autonomous manner and is elicited by the vitamin A metabolite, retinoic acid. Here, we found that the GATA6 transcriptional program is a common feature of macrophages residing in all visceral body cavities. Retinoic acid-dependent and -independent hallmark genes of GATA6 + macrophages were induced by mesothelial and fibroblastic stromal cells that express the transcription factor Wilms' Tumor 1 (WT1), which drives the expression of two rate-limiting enzymes in retinol metabolism. Depletion of Wt1 + stromal cells reduced the frequency of GATA6 + macrophages in the peritoneal, pleural and pericardial cavities. Thus, Wt1 + mesothelial and fibroblastic stromal cells constitute essential niche components supporting the tissue-specifying transcriptional landscape and homeostasis of cavity-resident macrophages.

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Section: Methods Detailsmentioning

confidence: 99%

A Stromal Niche Defined by Expression of the Transcription Factor WT1 Mediates Programming and Homeostasis of Cavity-Resident Macrophages

Buechler¹,

et al. 2019

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“…Cardiac inflammatory cells were isolated as described previously33 This isolation method results in collection of a mixed population of inflammatory cells, including T-lymphocytes (about 70%), macrophages (about 12%), and mast cells (about 12%) 34. Briefly, a thoracotomy exposed the intact pericardial sac.…”

Section: Methodsmentioning

confidence: 99%

Estrogen modulates the influence of cardiac inflammatory cells on function of cardiac fibroblasts

McLarty¹,

Li²,

Levick³

et al. 2013

JIR

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BackgroundInflammatory cells play a major role in the pathology of heart failure by stimulating cardiac fibroblasts to regulate the extracellular matrix in an adverse way. In view of the fact that inflammatory cells have estrogen receptors, we hypothesized that estrogen provides cardioprotection by decreasing the ability of cardiac inflammatory cells to influence fibroblast function.MethodsMale rats were assigned to either an untreated or estrogen-treated group. In the treated group, estrogen was delivered for 2 weeks via a subcutaneous implanted pellet containing 17β-estradiol. A mixed population of cardiac inflammatory cells, including T-lymphocytes (about 70%), macrophages (about 12%), and mast cells (about 12%), was isolated from each rat and cultured in a Boyden chamber with cardiac fibroblasts from untreated adult male rats for 24 hours. To examine if tumor necrosis factor-alpha (TNF-α) produced by inflammatory cells represents a mechanism contributing to the stimulatory effects of inflammatory cells on cardiac fibroblasts, inflammatory cells from the untreated group were incubated with cardiac fibroblasts in a Boyden chamber system for 24 hours in the presence of a TNF-α-neutralizing antibody. Cardiac fibroblasts were also incubated with 5 ng/mL of TNF-α for 24 hours. Fibro-blast proliferation, collagen synthesis, matrix metalloproteinase activity, β1 integrin protein levels, and the ability of fibroblasts to contract collagen gels were determined in all groups and statistically compared via one-way analysis of variance.ResultsInflammatory cells from the untreated group resulted in: 1) an increased fibroblast proliferation, collagen production and matrix metalloproteinase activity; and 2) a loss of β1 integrin protein and a reduced ability to contract collagen gels. In contrast, inflammatory cells from the treated group resulted in: 1) an attenuated fibroblast proliferation; 2) a nonsignificant reduction in collagen production; 3) the prevention of matrix metalloproteinase activation and the loss of β1 integrin by fibroblasts and 4) a preservation of the fibroblasts’ ability to contract collagen gels. The TNF-α neutralizing antibody attenuated or prevented the untreated inflammatory cell-induced fibroblast proliferation, collagen production, matrix metalloproteinase activation and loss of β1 integrin protein as well as preserved fibroblast contractile ability. Incubation with TNF-α yielded changes in the cardiac fibroblast parameters that were directionally similar to the results obtained with untreated inflammatory cells.ConclusionThese results and those of our previous in vivo studies suggest that a major mechanism by which estrogen provides cardioprotection is its ability to modulate synthesis of TNF-α by inflammatory cells, thereby preventing inflammatory cell induction of cardiac fibroblast events that contribute to adverse extracellular matrix remodeling.

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“…Collagen, a fibrous protein constituting approximately one-third of the total protein in the body, is synthesized by fibroblasts and is a major component of the extracellular matrix (ECM) in multiple tissues, including those in the heart, and collagen deposition in the heart is significantly higher in SHRs than in normal control rats (19,43). Collagen I and III represent 80% and ϳ10% of collagen protein in the heart, respectively, and high levels of collagen I and III expression are found in SHRs (41,43).…”

Section: Discussionmentioning

confidence: 99%

Chronic administration of hexarelin attenuates cardiac fibrosis in the spontaneously hypertensive rat

Fan

Pang

et al. 2012

American Journal of Physiology-Heart and Circulatory Physiology

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Cardiac fibrosis is a hallmark of heart disease and plays a vital role in cardiac remodeling during heart diseases, including hypertensive heart disease. Hexarelin is one of a series of synthetic growth hormone secretagogues (GHSs) possessing a variety of cardiovascular effects via action on GHS receptors (GHS-Rs). However, the role of hexarelin in cardiac fibrosis in vivo has not yet been investigated. In the present study, spontaneously hypertensive rats (SHRs) were treated with hexarelin alone or in combination with a GHS-R antagonist for 5 wk from an age of 16 wk. Hexarelin treatment significantly reduced cardiac fibrosis in SHRs by decreasing interstitial and perivascular myocardial collagen deposition and myocardial hydroxyproline content and reducing mRNA and protein expression of collagen I and III in SHR hearts. Hexarelin treatment also increased matrix metalloproteinase (MMP)-2 and MMP-9 activities and decreased myocardial mRNA expression of tissue inhibitor of metalloproteinase (TIMP)-1 in SHRs. In addition, hexarelin treatment significantly attenuated left ventricular (LV) hypertrophy, LV diastolic dysfunction, and high blood pressure in SHRs. The effect of hexarelin on cardiac fibrosis, blood pressure, and cardiac function was mediated by its receptor, GHS-R, since a selective GHS-R antagonist abolished these effects and expression of GHS-Rs was upregulated by hexarelin treatment. In summary, our data demonstrate that hexarelin reduces cardiac fibrosis in SHRs, perhaps by decreasing collagen synthesis and accelerating collagen degradation via regulation of MMPs/TIMP. Hexarelin-reduced systolic blood pressure may also contribute to this reduced cardiac fibrosis in SHRs. The present findings provided novel insights and underscore the therapeutic potential of hexarelin as an antifibrotic agent for the treatment of cardiac fibrosis.

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Isolation of Functional Cardiac Immune Cells

Cited by 8 publications

References 14 publications

A Stromal Niche Defined by Expression of the Transcription Factor WT1 Mediates Programming and Homeostasis of Cavity-Resident Macrophages

A Stromal Niche Defined by Expression of the Transcription Factor WT1 Mediates Programming and Homeostasis of Cavity-Resident Macrophages

Estrogen modulates the influence of cardiac inflammatory cells on function of cardiac fibroblasts

Chronic administration of hexarelin attenuates cardiac fibrosis in the spontaneously hypertensive rat

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