scanning-electron-microscopy-macerated-tissue-to-visualize

Scanning Electron Microscopy of Macerated Tissue to Visualize the Extracellular Matrix

Fibrosis is a component of all forms of heart disease regardless of etiology, and while much progress has been made in the field of cardiac matrix ...

Vanderbilt University Medical Center

Ig and T cell receptor (TCR) genes consist of separate genomic elements, which must undergo rearrangement and joining before a functional protein can be expressed. Considerable plasticity in the genomic arrangement of these elements has occurred during the evolution of the immune system. In tetrapods, all Ig and TCR chain elements are arranged as translocons. In teleosts, the Ig heavy and TCR chains are translocons, but light chain genes may occur as clusters. However, in chondrichthyes, all of the Ig light and heavy chain genes are arranged as clusters. These clusters vary in number from <10 to several hundred, depending on isotype and species. Here, we report that the germ-line gene for the TCR gamma chain in a chondrichthyan, the sandbar shark (Carcharhinus plumbeus), is present as a single locus arranged in a classic translocon pattern. Thus, the shark utilizes 2 types of genomic arrangements, the unique cluster organization for Ig genes and the "conventional" translocon organization for TCR genes. The TCR gamma translocon contains at least 5 V region genes, 3 J segment genes, and 1 C segment. As expected, the third hypervariable segment (CDR3), formed by the rearrangement of the Vgamma and Jgamma segments, contributed the major variability in the intact V region structure. Our data also suggest that diversity may be generated by mutation in the V regions.

Characterization of arrangement and expression of the T cell receptor gamma locus in the sandbar shark.

Atherosclerosis is a chronic inflammatory disease characterized by dyslipidemia and accumulation of lipids in the arterial intima, with activation of both innate and adaptive immunity. Reciprocally, dyslipidemia associated with atherosclerosis can perturb normal immune function. Natural killer T (NKT) cells are a specialized group of immune cells that share characteristics with both conventional T cells and natural killer cells. However, unlike these cells, NKT cells recognize glycolipid antigens and produce both pro- and anti-inflammatory cytokines upon activation. Because of these unique characteristics, NKT cells have recently been ascribed a role in the regulation of immunity and inflammation, including cardiovascular disease. In addition, NKT cells represent a bridge between dyslipidemia and immune regulation. This review summarizes the current knowledge of NKT cells and discusses the interplay between dyslipidemia and the normal functions of NKT cells and how this might modulate inflammation and atherosclerosis.

Natural killer T cells and atherosclerosis: form and function meet pathogenesis.

In this study, we investigated whether dyslipidemia-associated perturbed invariant natural killer T (iNKT) cell function is due to intrinsic changes in iNKT cells or defects in the ability of antigen-presenting cells to activate iNKT cells.

Development of spontaneous anergy in invariant natural killer T cells in a mouse model of dyslipidemia.

Atherosclerosis is widely accepted as an inflammatory disease involving both innate and adaptive immunity. B cells and/or antibodies have previously been shown to play a protective role against atherosclerosis. Aside from their ability to bind to antigens, antibodies can influence inflammatory responses by interacting with various Fcγ receptors on the surface of antigen presenting cells. Although studies in mice have determined that stimulatory Fcγ receptors contribute to atherosclerosis, the role of the inhibitory Fcγ receptor IIb (FcγRIIb) has only recently been investigated.

The inhibitory FcγRIIb modulates the inflammatory response and influences atherosclerosis in male apoE(-/-) mice.

Expression of molecules involved in lipid homeostasis such as the low density lipoprotein receptor (LDLr) on antigen presenting cells (APCs) has been shown to enhance invariant natural killer T (iNKT) cell function. However, the contribution to iNKT cell activation by other lipoprotein receptors with shared structural and ligand binding properties to the LDLr has not been described. In this study, we investigated whether a structurally related receptor to the LDLr, known as LDL receptor-related protein (LRP), plays a role in iNKT cell activation. We found that, unlike the LDLr which is highly expressed on all immune cells, the LRP was preferentially expressed at high levels on F4/80+ macrophages (MΦ). We also show that CD169+ MΦs, known to present antigen to iNKT cells, exhibited increased expression of LRP compared to CD169- MΦs. To test the contribution of MΦ LRP to iNKT cell activation we used a mouse model of MΦ LRP conditional knockout (LRP-cKO). LRP-cKO MΦs pulsed with glycolipid alpha-galactosylceramide (αGC) elicited normal IL-2 secretion by iNKT hybridoma and in vivo challenge of LRP-cKO mice led to normal IFN-γ, but blunted IL-4 response in both serum and intracellular expression by iNKT cells. Flow cytometric analyses show similar levels of MHC class-I like molecule CD1d on LRP-cKO MΦs and normal glycolipid uptake. Survey of the iNKT cell compartment in LRP-cKO mice revealed intact numbers and percentages and no homeostatic disruption as evidenced by the absence of programmed death-1 and Ly-49 surface receptors. Mixed bone marrow chimeras showed that the inability iNKT cells to make IL-4 is cell extrinsic and can be rescued in the presence of wild type APCs. Collectively, these data demonstrate that, although MΦ LRP may not be necessary for IFN-γ responses, it can contribute to iNKT cell activation by enhancing early IL-4 secretion.

Specific deletion of LDL receptor-related protein on macrophages has skewed in vivo effects on cytokine production by invariant natural killer T cells.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes atherosclerosis by increasing low-density lipoprotein (LDL) cholesterol levels through degradation of hepatic LDL receptor (LDLR). Studies have described the systemic effects of PCSK9 on atherosclerosis, but whether PCSK9 has local and direct effects on the plaque is unknown. To study the local effect of human PCSK9 (hPCSK9) on atherosclerotic lesion composition, independently of changes in serum cholesterol levels, we generated chimeric mice expressing hPCSK9 exclusively from macrophages, using marrow from hPCSK9 transgenic (hPCSK9tg) mice transplanted into apoE(-/-) and LDLR(-/-) mice, which were then placed on a high-fat diet (HFD) for 8 weeks. We further characterized the effect of hPCSK9 expression on the inflammatory responses in the spleen and by mouse peritoneal macrophages (MPM) in vitro. We found that MPMs from transgenic mice express both murine (m) Pcsk9 and hPCSK9 and that the latter reduces macrophage LDLR and LRP1 surface levels. We detected hPCSK9 in the serum of mice transplanted with hPCSK9tg marrow, but did not influence lipid levels or atherosclerotic lesion size. However, marrow-derived PCSK9 progressively accumulated in lesions of apoE(-/-) recipient mice, while increasing the infiltration of Ly6C(hi) inflammatory monocytes by 32% compared with controls. Expression of hPCSK9 also increased CD11b- and Ly6C(hi) -positive cell numbers in spleens of apoE(-/-) mice. In vitro, expression of hPCSK9 in LPS-stimulated macrophages increased mRNA levels of the pro-inflammatory markers Tnf and Il1b (40% and 45%, respectively) and suppressed those of the anti-inflammatory markers Il10 and Arg1 (30% and 44%, respectively). All PCSK9 effects were LDLR-dependent, as PCSK9 protein was not detected in lesions of LDLR(-/-) recipient mice and did not affect macrophage or splenocyte inflammation. In conclusion, PCSK9 directly increases atherosclerotic lesion inflammation in an LDLR-dependent but cholesterol-independent mechanism, suggesting that therapeutic PCSK9 inhibition may have vascular benefits secondary to LDL reduction.

Local effects of human PCSK9 on the atherosclerotic lesion.

Following myocardial infarction, purinergic nucleotides and nucleosides are released via non-specific and specific mechanisms in response to cellular activation, stress, or injury. These extracellular nucleotides are potent mediators of physiologic and pathologic responses, contributing to the inflammatory and fibrotic milieu within the injured myocardium. Via autocrine or paracrine signaling, cell-specific effects occur through differentially expressed purinergic receptors of the P2X, P2Y, and P1 families. Nucleotide activation of the ionotropic (ligand-gated) purine receptors (P2X) and several of the metabotropic (G-protein-coupled) purine receptors (P2Y) or adenosine activation of the P1 receptors can have profound effects on inflammatory cell function, fibroblast function, and cardiomyocyte function. Extracellular nucleotidases that hydrolyze released nucleotides regulate the magnitude and duration of purinergic signaling. While there are numerous studies on the role of the purinergic signaling pathway in cardiovascular disease, the extent to which the purinergic signaling pathway modulates cardiac fibrosis is incompletely understood. Here we provide an overview of the current understanding of how the purinergic signaling pathway modulates cardiac fibroblast function and myocardial fibrosis.

Article	Year
Characterization of arrangement and expression of the T cell receptor gamma locus in the sandbar shark. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 19439654	2009
Natural killer T cells and atherosclerosis: form and function meet pathogenesis. Journal of innate immunity\| PubMed ID: 20375560	2010
Development of spontaneous anergy in invariant natural killer T cells in a mouse model of dyslipidemia. Arteriosclerosis, thrombosis, and vascular biology\| PubMed ID: 20539017	2010
The inhibitory FcγRIIb modulates the inflammatory response and influences atherosclerosis in male apoE(-/-) mice. Atherosclerosis\| PubMed ID: 21084088	2011
Specific deletion of LDL receptor-related protein on macrophages has skewed in vivo effects on cytokine production by invariant natural killer T cells. PloS one\| PubMed ID: 25050824	2014
Local effects of human PCSK9 on the atherosclerotic lesion. The Journal of pathology\| PubMed ID: 26333678	2016
Extracellular nucleotide regulation and signaling in cardiac fibrosis. Journal of molecular and cellular cardiology\| PubMed ID: 26891859	2016

Roman Covarrubias

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