Complex Relationship Between Cardiac Fibroblasts and Cardiomyocytes in Health and Disease

Cardiac fibroblasts are the primary cell type responsible for deposition of extracellular matrix in the heart, providing support to the contracting myocardium and contributing to a myriad of physiological signaling processes. Despite the importance of fibrosis in processes of wound healing, excessive fibroblast proliferation and activation can lead to pathological remodeling, driving heart failure and the onset of arrhythmias. Our understanding of the mechanisms driving the cardiac fibroblast activation and proliferation is expanding, and evidence for their direct and indirect effects on cardiac myocyte function is accumulating. In this review, we focus on the importance of the fibroblast‐to‐myofibroblast transition and the cross talk of cardiac fibroblasts with cardiac myocytes. We also consider the current use of models used to explore these questions.

DYNC1LI2 regulates localization of the chaperone-mediated autophagy-receptor LAMP2A and improves cellular homeostasis in cystinosis

SUMMARYThe dynein motor protein complex is required for retrograde transport but the functions of the intermediate-light chains that form the cargo-binding complex are not elucidated and the importance of individual subunits in the maintenance of cellular homeostasis is unknown. Here, using mRNA arrays and protein analysis, we show that the dynein subunit, intermediate chain 2 (DYNC1LI2) is downregulated in cystinosis, a lysosomal storage disorder caused by genetic defects in the lysosomal cystine transporter, cystinosin. Reconstitution of the expression of DYNC1LI2 in Ctns-/- cells re-established endolysosomal dynamics. Defective vesicular trafficking in cystinotic cells was rescued by DYNC1LI2 expression which correlated with decreased endoplasmic reticulum stress manifested as decreased expression levels of the chaperone Grp78. Mitochondrial fragmentation in cystinotic fibroblasts was also rescued by DYNC1LI2. Survival of cystinotic cells to oxidative stress insult was increased by DYNC1LI2 reconstitution but not by its paralog DYNC1LI1, which also failed to decrease ER stress levels and mitochondrial fragmentation. Restoring DYNC1LI2 expression rescued the localization of the chaperone-mediated autophagy receptor, LAMP2A, and restored cellular homeostasis of cystinotic proximal tubule cells, the primary cell type affected in cystinosis. DYNC1LI2 failed to rescue phenotypes in cystinotic cells when LAMP2A was downregulated or when co-expressed with dominant negative (DN) RAB7 or DN-RAB11, which impair LAMP2A trafficking. DYNC1LI2 emerges as a new target to repair underlying trafficking and CMA defects in cystinosis, a mechanism that is not restored by currently used lysosomal depletion therapies.

The cell biology of fat expansion

Adipose tissue is a complex, multicellular organ that profoundly influences the function of nearly all other organ systems through its diverse metabolite and adipokine secretome. Adipocytes are the primary cell type of adipose tissue and play a key role in maintaining energy homeostasis. The efficiency with which adipose tissue responds to whole-body energetic demands reflects the ability of adipocytes to adapt to an altered nutrient environment, and has profound systemic implications. Deciphering adipocyte cell biology is an important component of understanding how the aberrant physiology of expanding adipose tissue contributes to the metabolic dysregulation associated with obesity.

Abstract 310: Production of Basal Lamina Collagen IV by Cardiac Fibroblasts in 3-Dimensional Cultures

Cardiac fibroblasts are generally considered the primary cell type that controls extracellular matrix homeostasis in the heart. Distinct changes in amounts and composition of extracellular matrix occur from birth to old age. Accordingly, age-dependent alterations in cardiac extracellular matrix are an important factor governing response to injury and pathological remodeling. Whereas cardiac myocytes are surrounded by a basal lamina, cardiac fibroblasts do not assemble a cell-adjacent basal lamina. Evidence is presented that in addition to fibrillar collagen production, cardiac fibroblasts are also the primary cell type responsible for the production of collagen IV. First, patterns and abundance of collagen IV in murine heart was established in sections from neonate, adult, and aged mice. Second, production of collagen IV by fibroblasts grown in 3-D fibrin gels was assessed by confocal microscopy and quantification by immmunoblot analysis. Finally, co-cultures of cardiac fibroblasts with myocytes were performed to show that fibroblasts are the primary cell type producing collagen IV under these conditions. The basal lamina of the myocytes plays a critical role in aligning and tethering myocytes together as well as making connections to the interstitial collagen fibers of the heart. Hence, production of collagen IV by cardiac fibroblasts would provide a mechanism by which cardiac fibroblasts assist in aligning and securing cardiac myocyte alignment and structural integrity via basal lamina production.

Abstract 1592: Electrical Stimulation of LV Fibroblasts Causes Frequency-Dependent Modulation of Matrix Metalloproteinase Release and Increase in Tissue Inhibitors of Metalloproteinases

LV fibroblasts (LVMFs), which form a cellular netwrork throughout the myocardium including electrical connectivity to myocytes, are a primary cell type that contributes to remodfeling of the extracellular matrix through the synthesis of matrix metalloproteinases (MMP) and tissue inhibitors of the MMPs (TIMPs). While cytokines and other signaling molecules can induce MMP and TIMP synthesis and release from LVMFs, whether and to what degree LVMFs respond to changes in electrical stimulation with respect to modulation of MMP/TIMP release remains unknown. Porcine LVMFs were grown to confluence, serum deprived for 24 hours, and then randomized to electrical stimulation (6x10 5 cells/well, 130V 5ms pulses) for 24 hours at 1, 2, and 4 Hz (n=6 wells/frequency). Unstimulated cells (0 Hz) served as controls. Electrical stimulation had no effect on LVMF morphology. Cell media and pellets were collected for levels of MMP-2, MMP-9 (zymography), MT1-MMP, and TIMP-4 (immunoblotting). Changes in MMP and TIMP levels with stimulation are reported as a percentage of unstimulated values (TABLE ). MMP-2, MMP-9, and TIMP-1 levels were higher than unstimulated levels at 2 and 4 Hz. MT1-MMP levels were lower than unstimulated levels at 1 and 2 Hz. Ratios for each of the MMPs to TIMP-1 were computed (TABLE ). While the MMP-2/TIMP-1 and MMP-9/TIMP-1 ratios remained similar to unstimulated values, the MT1-MMP/TIMP-1 ratio was lower than unstimulated values at 1 and 2 Hz. The unique findings of this study are two-fold. First, in vitro electrical stimulation of LVMFs, which are generally considered to be electrically inert, resulted in demonstrable changes in the release of specific MMP and TIMP species. Second, the reduced MT1-MMP to TIMP-1 ratio suggests electrical stimulation of LVMFs caused a shift in the stoichiometric balance between MMPs and TIMPs that may favor accumulation of extracellular matrix components.

Experimental therapeutic approaches to peripheral nerve tumors

✓Discovery that the Schwann cell is the primary cell type responsible for both the neurofibroma as well as the schwannoma has proven to represent a crucial milestone in understanding the pathogenesis of peripheral nerve tumor development. This information and related findings have served as a nidus for research aimed at more fully characterizing this family of conditions. Recent discoveries in the laboratory have clarified an understanding of the molecular mechanisms underlying the pathogenesis of benign peripheral nerve tumors. Similarly, the mechanisms whereby idiopathic and syndromic (NF1- and NF2-associated) nerve sheath tumors progress to malignancy are being elucidated. This detailed understanding of the molecular pathogenesis of peripheral nerve tumors provides the information necessary to create a new generation of therapies tailored specifically to the prevention, cessation, or reversal of pathological conditions at the fundamental level of dysfunction. The authors review the data that have helped to elucidate the molecular pathogenesis of this category of conditions, explore the current progress toward exploitation of these findings, and discuss potential therapeutic avenues for future research.

Siglec-H is an IPC-specific receptor that modulates type I IFN secretion through DAP12

Natural interferon (IFN)-producing cells are the primary cell type responsible for production of type I IFN in response to viruses. Herein we report the identification of the first molecular marker of mouse natural interferon-producing cells (IPCs), a novel member of the sialic acid-binding immunoglobulin (Ig)-like lectin (Siglec) family termed Siglec-H. Siglec-H is expressed exclusively on IPCs and is unique among Siglec proteins in that it associates with the adaptor protein DAP12. Moreover, we show that DAP12 modulates the type I IFN response of IPCs to a Toll-like receptor 9 (TLR9) agonist. This observation explains our previous finding that stimulation of IPCs with 440c, a Siglec-H-specific antibody, reduces IPC secretion of type I IFN. Moreover, it supports a model in which engagement of DNAX-activation protein 12 (DAP12)-associated receptors with antibodies or low avidity endogenous ligands interferes with TLR-mediated cellular activation. (Blood. 2006;107:2474-2476)

Perivascular Macrophages Are the Primary Cell Type Productively Infected by Simian Immunodeficiency Virus in the Brains of Macaques

The macrophage is well established as a target of HIV and simian immunodeficiency virus (SIV) infection and a major contributor to the neuropathogenesis of AIDS. However, the identification of distinct subpopulations of monocyte/macrophages that carry virus to the brain and that sustain infection within the central nervous system (CNS) has not been examined. We demonstrate that the perivascular macrophage and not the parenchymal microglia is the primary cell productively infected by SIV. We further demonstrate that although productive viral infection of the CNS occurs early, thereafter it is not easily detectable until terminal AIDS. The biology of perivascular macrophages, including their rate of turnover and replacement by peripheral blood monocytes, may explain the timing of neuroinvasion, disappearance, and reappearance of virus in the CNS, and questions the ability of the brain to function as a reservoir for productive infection by HIV/SIV.