Folic acid-mediated fibrosis is driven by C5a receptor 1-mediated activation of kidney myeloid cells.

We have previously reported that increased expression and activation of kidney cell complement components play an important role in the pathogenesis of renal scarring. Here, we used floxed green fluorescent protein (GFP)-C5a receptor 1 (C5aR1) knockin mice (GFP-) and the model of folic acid (FA)-induced kidney injury to define the cell types and potential mechanisms by which increased C5aR1 activation leads to fibrosis. Using flow cytometry and confocal microscopy, we identified macrophages as the major interstitial cell type showing increased expression of C5aR1 in FA-treated mice.

Parallel Lineage-Tracing Studies Establish Fibroblasts as the Prevailing In Vivo Adipocyte Progenitor.

Despite decades of studies suggesting that the in vivo adipocyte progenitor resides within the vascular niche, the exact nature of this progenitor remains controversial because distinct studies have attributed adipogenic properties to multiple vascular cell types. Using Cre recombinases labeling distinct vascular lineages, we conduct parallel lineage tracing experiments to assess their degree of contribution to de novo adipogenesis. Although we detect occasional adipocytes that were lineage traced by endothelial or mural recombinases, these are rare events.

The NG2 Proteoglycan in Pericyte Biology.

Studies of pericytes have been retarded by the lack of appropriate markers for identification of these perivascular mural cells. Use of antibodies against the NG2 proteoglycan as a pericyte marker has greatly facilitated recent studies of pericytes, emphasizing the intimate spatial relationship between pericytes and endothelial cells, allowing more accurate quantification of pericyte/endothelial cell ratios in different vascular beds, and revealing the participation of pericytes throughout all stages of blood vessel formation. The functional importance of NG2 in pericyte biology has been established via NG2 knockdown (in vitro) and knockout (in vivo) strategies that reveal significant deficits in blood vessel formation when NG2 is absent from pericytes.

Lgl1 controls NG2 endocytic pathway to regulate oligodendrocyte differentiation and asymmetric cell division and gliomagenesis.

Oligodendrocyte progenitor cells (OPC) undergo asymmetric cell division (ACD) to generate one OPC and one differentiating oligodendrocyte (OL) progeny. Loss of pro-mitotic proteoglycan and OPC marker NG2 in the OL progeny is the earliest immunophenotypic change of unknown mechanism that indicates differentiation commitment. Here, we report that expression of the mouse homolog of Drosophila tumor suppressor Lethal giant larvae 1 (Lgl1) is induced during OL differentiation.

Dissecting the multifactorial nature of demyelinating disease.

Chondroitin sulfate proteoglycan-4 (CSPG4) is a surface component of two key cell types (oligodendrocyte progenitor cells (OPCs) and myeloid cells) present in lysolecithin-induced lesions in mouse spinal cord. Two types of CSPG4 manipulations have been used to study the roles of these cells in myelin damage and repair: (1) OPC and myeloid-specific ablation of CSPG4, and (2) transplantation of enhanced green fluorescent protein (EGFP)-labeled progenitors to distinguish between bone marrow-derived macrophages and resident microglia. Ablation of CSPG4 in OPCs does not affect myelin damage, but decreases myelin repair, due to reduced proliferation of CSPG4-null OPCs that diminishes generation of mature oligodendrocytes for remyelination.

Cell surface chondroitin sulphate proteoglycan 4 (CSPG4) binds to the basement membrane heparan sulphate proteoglycan, perlecan, and is involved in cell adhesion.

Chondroitin sulphate proteoglycan 4 (CSPG4) is a cell surface proteoglycan highly expressed by tumour, perivascular and oligodendrocyte cells and known to be involved cell adhesion and migration. This study showed that CSPG4 was present as a proteoglycan on the cell surface of two melanoma cell lines, MM200 and Me1007, as well as shed into the conditioned medium. CSPG4 from the two melanoma cell lines differed in the amount of chondroitin sulphate (CS) decoration, as well as the way the protein core was fragmented.

Effects of chondroitin sulfate proteoglycan 4 (NG2/CSPG4) on soft-tissue sarcoma growth depend on tumor developmental stage.

Sarcomas, and the mesenchymal precursor cells from which they arise, express chondroitin sulfate proteoglycan 4 (NG2/CSPG4). However, NG2/CSPG4's function and its capacity to serve as a therapeutic target in this tumor type are unknown. Here, we used cells from human tumors and a genetically engineered autochthonous mouse model of soft-tissue sarcomas (STSs) to determine NG2/CSPG4's role in STS initiation and growth.

Distinct NG2 proteoglycan-dependent roles of resident microglia and bone marrow-derived macrophages during myelin damage and repair.

We used a bone marrow transplantation approach to distinguish the activities of bone marrow-derived macrophages from the activities of central nervous system-resident microglia in phenomena associated with axon demyelination and remyelination. We transplanted wild type or germline NG2 null beta-actin-EGFP expressing bone marrow into irradiated wild type or NG2 null recipient mice, followed by analysis of lysolecithin-induced spinal cord demyelination and remyelination and quantification of Iba-1+/ F4/80+/ EGFP+ macrophages and Iba-1+/ F4/80+/ EGFP- microglia. One week after microinjection of 1% lysolecithin into the spinal cord, wild type recipients receiving NG2 null bone marrow exhibit greatly reduced infiltration of macrophages into lesions, compared to wild type recipients receiving wild type bone marrow.

Localization of VEGF to Vascular ECM Is an Important Aspect of Tumor Angiogenesis.

Our research has identified several examples in which reduced VEGF-A binding to deficient vascular extracellular matrix leads to deficits in tumor vascularization and tumor growth: (1) germline ablation of collagen VI in the stroma of intracranial B16F10 melanomas; (2) knockdown of the Tks5 scaffolding protein in MDA-MB-231 mammary tumor cells; (3) germline ablation of NG2 proteoglycan in the stroma of MMTV-PyMT mammary tumors; and (4) myeloid-specific ablation of NG2 in the stroma of intracranial B16F10 melanomas. Tumor hypoxia is increased in each of the four types of experimental mice, accompanied by increases in total VEGF-A. However, while VEGF-A is highly associated with tumor blood vessels in control mice, it is much more diffusely distributed in tumors in all four sets of experimental mice, likely due to reduced extent of the vascular extracellular matrix.

NG2 Proteoglycan Enhances Brain Tumor Progression by Promoting Beta-1 Integrin Activation in both Cis and Trans Orientations.

By physically interacting with beta-1 integrins, the NG2 proteoglycan enhances activation of the integrin heterodimers. In glioma cells, co-localization of NG2 and 31 integrin in individual cells (cis interaction) can be demonstrated by immunolabeling, and the NG2-integrin interaction can be confirmed by co-immunoprecipitation. NG2-dependent integrin activation is detected via use of conformationally sensitive monoclonal antibodies that reveal the activated state of the beta-1 subunit in NG2-positive versus NG2-negative cells.

Pericytes of Multiple Organs Do Not Behave as Mesenchymal Stem Cells In Vivo.

Pericytes are widely believed to function as mesenchymal stem cells (MSCs), multipotent tissue-resident progenitors with great potential for regenerative medicine. Cultured pericytes isolated from distinct tissues can differentiate into multiple cell types in vitro or following transplantation in vivo. However, the cell fate plasticity of endogenous pericytes in vivo remains unclear.

Pericytes and immune cells contribute to complement activation in tubulointerstitial fibrosis.

We have examined the pathogenic role of increased complement expression and activation during kidney fibrosis. Here, we show that PDGFRβ-positive pericytes isolated from mice subjected to obstructive or folic acid injury secrete C1q. This was associated with increased production of proinflammatory cytokines, extracellular matrix components, collagens, and increased Wnt3a-mediated activation of Wnt/β-catenin signaling, which are hallmarks of myofibroblast activation.

Frizzled proteins are colonic epithelial receptors for C. difficile toxin B.

Clostridium difficile toxin B (TcdB) is a critical virulence factor that causes diseases associated with C. difficile infection. Here we carried out CRISPR-Cas9-mediated genome-wide screens and identified the members of the Wnt receptor frizzled family (FZDs) as TcdB receptors.

Tumor-Targeted Multimodal Optical Imaging with Versatile Cadmium-Free Quantum Dots.

The rapid development of fluorescence imaging technologies requires concurrent improvements in the performance of fluorescent probes. Quantum dots have been extensively used as an imaging probe in various research areas because of their inherent advantages based on unique optical and electronic properties. However, their clinical translation has been limited by the potential toxicity especially from cadmium.

Role of NG2 proteoglycan in macrophage recruitment to brain tumors and sites of CNS demyelination.

Macrophage infiltration is a factor in most if not all inflammatory pathologies. Understanding molecular interactions that underlie this process is therefore important for our ability to modulate macrophage behavior for therapeutic purposes. Our studies show that cell surface expression of the nerveglial antigen 2 (NG2) proteoglycan is important for the ability of macrophages to colonize both brain tumors and sites of central nervous system (CNS) demyelination.

NG2 Proteoglycan Ablation Reduces Foam Cell Formation and Atherogenesis via Decreased Low-Density Lipoprotein Retention by Synthetic Smooth Muscle Cells.

Objectives: Obesity and hyperlipidemia are critical risk factors for atherosclerosis. Because ablation of NG2 proteoglycan in mice leads to hyperlipidemia and obesity, we investigated the impact of NG2 ablation on atherosclerosis in apoE null mice.

Approach And Results: Immunostaining indicates that NG2 expression in plaque, primarily by synthetic smooth muscle cells, increases during atherogenesis.

NG2 Proteoglycan-Dependent Contributions of Pericytes and Macrophages to Brain Tumor Vascularization and Progression.

The NG2 proteoglycan promotes tumor growth as a component of both tumor and stromal cells. Using intracranial, NG2-negative B16F10 melanomas, we have investigated the importance of PC and Mac NG2 in brain tumor progression. Reduced melanoma growth in Mac-NG2ko and PC-NG2ko mice demonstrates the importance of NG2 in both stromal compartments.

Stonin1 mediates endocytosis of the proteoglycan NG2 and regulates focal adhesion dynamics and cell motility.

Cellular functions, ranging from focal adhesion (FA) dynamics and cell motility to tumour growth, are orchestrated by signals cells receive from outside via cell surface receptors. Signalling is fine-tuned by the exo-endocytic cycling of these receptors to control cellular responses such as FA dynamics, which determine cell motility. How precisely endocytosis regulates turnover of the various cell surface receptors remains unclear.

NG2-proteoglycan-dependent contributions of oligodendrocyte progenitors and myeloid cells to myelin damage and repair.

Background: The NG2 proteoglycan is expressed by several cell types in demyelinated lesions and has important effects on the biology of these cells. Here we determine the cell-type-specific roles of NG2 in the oligodendrocyte progenitor cell (OPC) and myeloid cell contributions to demyelination and remyelination.

Methods: We have used Cre-Lox technology to dissect the cell-type-specific contributions of NG2 to myelin damage and repair.

NG2 proteoglycan-dependent recruitment of tumor macrophages promotes pericyte-endothelial cell interactions required for brain tumor vascularization.

Early stage growth of intracranial B16F10 tumors is reduced by 87% in myeloid-specific NG2 null (Mac-NG2ko) mice and by 77% in pericyte-specific NG2 null (PC-NG2ko) mice, demonstrating the importance of the NG2 proteoglycan in each of these stromal compartments. In both genotypes, loss of pericyte-endothelial cell interaction results in numerous structural defects in tumor blood vessels, including decreased formation of endothelial cell junctions and decreased assembly of the vascular basal lamina. All vascular deficits are larger in Mac-NG2ko mice than in PC-NG2ko mice, correlating with the greater decrease in pericyte-endothelial cell interaction in Mac-NG2ko animals.

The invadopodia scaffold protein Tks5 is required for the growth of human breast cancer cells in vitro and in vivo.

The ability of cancer cells to invade underlies metastatic progression. One mechanism by which cancer cells can become invasive is through the formation of structures called invadopodia, which are dynamic, actin-rich membrane protrusions that are sites of focal extracellular matrix degradation. While there is a growing consensus that invadopodia are instrumental in tumor metastasis, less is known about whether they are involved in tumor growth, particularly in vivo.

Entrapment via synaptic-like connections between NG2 proteoglycan+ cells and dystrophic axons in the lesion plays a role in regeneration failure after spinal cord injury.

NG2 is purportedly one of the most growth-inhibitory chondroitin sulfate proteoglycans (CSPGs) produced after spinal cord injury. Nonetheless, once the severed axon tips dieback from the lesion core into the penumbra they closely associate with NG2+ cells. We asked if proteoglycans play a role in this tight cell-cell interaction and whether overadhesion upon these cells might participate in regeneration failure in rodents.

Shedding of NG2 by MMP-13 attenuates anoikis.

Disruption of cell-matrix interactions can lead to anoikis-apoptosis due to loss of matrix contacts. We previously showed that Nerve/glial antigen 2 (NG2) is a novel anoikis receptor. Specifically, overexpression of NG2 leads to anoikis propagation, whereas its suppression leads to anoikis attenuation.

Resident fibroblast lineages mediate pressure overload-induced cardiac fibrosis.

Activation and accumulation of cardiac fibroblasts, which result in excessive extracellular matrix deposition and consequent mechanical stiffness, myocyte uncoupling, and ischemia, are key contributors to heart failure progression. Recently, endothelial-to-mesenchymal transition (EndoMT) and the recruitment of circulating hematopoietic progenitors to the heart have been reported to generate substantial numbers of cardiac fibroblasts in response to pressure overload-induced injury; therefore, these processes are widely considered to be promising therapeutic targets. Here, using multiple independent murine Cre lines and a collagen1a1-GFP fusion reporter, which specifically labels fibroblasts, we found that following pressure overload, fibroblasts were not derived from hematopoietic cells, EndoMT, or epicardial epithelial-to-mesenchymal transition.

Loss of caveolin-1 causes blood-retinal barrier breakdown, venous enlargement, and mural cell alteration.

Blood-retinal barrier (BRB) breakdown and related vascular changes are implicated in several ocular diseases. The molecules and mechanisms regulating BRB integrity and pathophysiology are not fully elucidated. Caveolin-1 (Cav-1) ablation results in loss of caveolae and microvascular pathologies, but the role of Cav-1 in the retina is largely unknown.