All tissues are surrounded by a mixture of noncellular matrix components, that not only provide physical and mechanical support to cells, but also mediate biochemical signaling between cells. The... Show moreAll tissues are surrounded by a mixture of noncellular matrix components, that not only provide physical and mechanical support to cells, but also mediate biochemical signaling between cells. The extracellular matrix (ECM) of endothelial cells, also known as the perivascular matrix, forms an organ specific vascular niche that orchestrates mechano-, growth factor, and angiocrine signaling required for tissue homeostasis and organ repair. This concise review describes how this perivascular ECM functions as a signaling platform and how this knowledge can impact the field of regenerative medicine, for example, when designing artificial matrices or using decellularized scaffolds from organs. Stem Cells Translational Medicine 2019;8:375-382 Show less
There is an increasing prevalence of chronic kidney disease (CKD), which associates with the development of interstitial fibrosis. Pericytes (perivascular fibroblasts) provide a major source of... Show moreThere is an increasing prevalence of chronic kidney disease (CKD), which associates with the development of interstitial fibrosis. Pericytes (perivascular fibroblasts) provide a major source of alpha-SMA-positive myofibroblasts that are responsible for the excessive deposition of extracellular matrix. In order to identify pericyte long non-coding RNAs (lncRNAs) that could serve as a target to decrease myofibroblast formation and counteract the progression of kidney fibrosis we employed two models of experimental kidney injury, one focused on kidney fibrosis (unilateral ureteral obstruction; UUO), and one focused on acute kidney injury that yields kidney fibrosis in the longer term (unilateral ischemia-reperfusion injury; IRI). This was performed in FoxD1-GC;tdTomato stromal cell reporter mice that allowed pericyte fate tracing. Tomato red-positive FoxD1-derivative cells of control and injured kidneys were FACS-sorted and used for lncRNA and mRNA profiling yielding a distinctive transcriptional signature of pericytes and myofibroblasts with 244 and 586 differentially expressed lncRNAs (> twofold, P < 0.05), in the UUO and IRI models, respectively. Next, we selected two differentially expressed and conserved lncRNAs, Rian (RNA imprinted and accumulated in nucleus) and Miat (Myocardial infarction associated transcript), and explored their potential regulatory role in myofibroblast formation through knockdown of their function with gapmers. While Miat was upregulated in myofibroblasts of UUO and IRI in mice, gapmer silencing of Miat attenuated myofibroblast formation as evidenced by decreased expression of alpha-SMA, col1 alpha 1, SMAD2, and SMAD3, as well as decreased alpha-SMA and pro-collagen-1 alpha 1 protein levels. In contrast, silencing Rian, which was found to be downregulated in kidney myofibroblast after IRI and UUO, resulted in increased myofibroblast formation. In addition, we found microRNAs that were previously linked to Miat (miR-150) and Rian (14q32 miRNA cluster), to be dysregulated in the FoxD1-derivative cells, suggesting a possible interaction between miRNAs and these lncRNAs in myofibroblast formation. Taken together, lncRNAs play a regulatory role in myofibroblast formation, possibly through interacting with miRNA regulation, implicating that understanding their biology and their modulation may have the potential to counteract the development of renal fibrosis. Show less
Kidney fibrosis is the common pathophysiological mechanism in end-stage renal disease characterized by excessive accumulation of myofibroblast-derived extracellular matrix. Natriuretic peptides... Show moreKidney fibrosis is the common pathophysiological mechanism in end-stage renal disease characterized by excessive accumulation of myofibroblast-derived extracellular matrix. Natriuretic peptides have been demonstrated to have cyclic guanosine monophosphate (cGMP)-dependent anti-fibrotic properties likely due to interference with pro-fibrotic tissue growth factor beta (TGF-beta) signaling. However, in vivo, natriuretic peptides are rapidly degraded by neutral endopeptidases (NEP). In a unilateral ureteral obstruction (UUO) mouse model for kidney fibrosis we assessed the anti-fibrotic effects of SOL1, an orally active compound that inhibits NEP and endothelin-converting enzyme (ECE). Mice (n= 10 per group) subjected to UUO were treated for 1 week with either solvent, NEP-/ECE-inhibitor SOL1 (two doses), reference NEP-inhibitor candoxatril or the angiotensin II receptor type 1 (AT1)-antagonist losartan. While NEP-inhibitors had no significant effect on blood pressure, they did increase urinary cGMP levels as well as endothelin-1 (ET-1) levels. Immunohistochemical staining revealed a marked decrease in renal collagen (similar to 55% reduction, P< 0.05) and alpha-smooth muscle actin (alpha-SMA; similar to 40% reduction, P< 0.05). Moreover, the number of alpha-SMA positive cells in the kidneys of SOL1-treated groups inversely correlated with cGMP levels consistent with a NEP-dependent anti-fibrotic effect. To dissect the molecular mechanisms associated with the anti-fibrotic effects of NEP inhibition, we performed a 'deep serial analysis of gene expression (Deep SAGE)' transcriptome and targeted metabolomics analysis of total kidneys of all treatment groups. Pathway analyses linked increased cGMP and ET-1 levels with decreased nuclear receptor signaling (peroxisome proliferator-activated receptor [PPAR] and liver X receptor/retinoid X receptor [LXR/RXR] signaling) and actin cytoskeleton organization. Taken together, although our transcriptome and metabolome data indicate metabolic dysregulation, our data support the therapeutic potential of NEP inhibition in the treatment of kidney fibrosis via cGMP elevation and reduced myofibroblast formation. Show less
Neutrophil extracellular traps (NETs) are immunogenic extracellular DNA structures that can be released by neutrophils upon a wide variety of triggers. NETs have been demonstrated to serve as an... Show moreNeutrophil extracellular traps (NETs) are immunogenic extracellular DNA structures that can be released by neutrophils upon a wide variety of triggers. NETs have been demonstrated to serve as an important host defense mechanism that traps and kills microorganisms. On the other hand, they have been implicated in diverse systemic autoimmune diseases. NETs are immunogenic and toxic structures that contain a pool of relevant autoantigens including anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) and systemic lupus erythematosus (SLE). Different forms of NETs can be induced depending on the stimulus. The amount of NETs can be quantified using different techniques including measuring DNA release in supernatants, measuring DNA-complexed with NET-molecules like myeloperoxidase (MPO) or neutrophil elastase (NE), measuring the presence of citrullinated histones by fluorescence microscopy, or flow cytometric detection of NET-components which all have different features regarding their specificity, sensitivity, objectivity, and quantity. Here is a protocol to quantify ex vivo NET formation in a highly-sensitive, high-throughput manner by using three-dimensional immunofluorescence confocal microscopy. This protocol can be applied to address various research questions about NET formation and degradation in health and disease. Show less
Witjas, F.M.R.; Berg, B.M. van den; Berg, C.W. van den; Engelse, M.A.; Rabelink, T.J. 2018
All tissues are surrounded by a mixture of noncellular matrix components, that not only provide physical and mechanical support to cells, but also mediate biochemical signaling between cells. The... Show moreAll tissues are surrounded by a mixture of noncellular matrix components, that not only provide physical and mechanical support to cells, but also mediate biochemical signaling between cells. The extracellular matrix (ECM) of endothelial cells, also known as the perivascular matrix, forms an organ specific vascular niche that orchestrates mechano-, growth factor, and angiocrine signaling required for tissue homeostasis and organ repair. This concise review describes how this perivascular ECM functions as a signaling platform and how this knowledge can impact the field of regenerative medicine, for example, when designing artificial matrices or using decellularized scaffolds from organs. Show less
