Ivan G. Gomez

Macrophage expression of active MMP-9 induces acute plaque disruption in apoE-deficient mice

The majority of acute clinical manifestations of atherosclerosis are due to the physical rupture of advanced atherosclerotic plaques. It has been hypothesized that macrophages play a key role in inducing plaque rupture by secreting proteases that destroy the extracellular matrix that provides physical strength to the fibrous cap. Despite reports detailing the expression of multiple proteases by macrophages in rupture-prone regions, there is no direct proof that macrophage-mediated matrix degradation can induce plaque rupture. We aimed to test this hypothesis by retrovirally overexpressing the candidate enzyme MMP-9 in macrophages of advanced atherosclerotic lesions of apoE-/- mice. Despite a greater than 10-fold increase in the expression of MMP-9 by macrophages, there was only a minor increase in the incidence of plaque fissuring. Subsequent analysis revealed that macrophages secrete MMP-9 predominantly as a proform, and this form is unable to degrade the matrix component elastin. Expression of an autoactivating form of MMP-9 in macrophages in vitro greatly enhances elastin degradation and induces significant plaque disruption when overexpressed by macrophages in advanced atherosclerotic lesions of apoE-/- mice in vivo. These data show that enhanced macrophage proteolytic activity can induce acute plaque disruption and highlight MMP-9 as a potential therapeutic target for stabilizing rupture-prone plaques.

MMP-9 Sheds the β2 Integrin Subunit (CD18) from Macrophages

Activated macrophages are essential effectors of immunity and a rich source of matrix metalloproteinase-9 (MMP-9; gelatinase B). To search for cellular substrates of the enzyme, we subjected wild-type macrophages and macrophages expressing an autoactivating form of pro-MMP-9 (M9A macrophages) to proteomics analysis. Two-dimensional liquid chromatography together with tandem mass spectrometry identified 467 proteins in medium conditioned by M9A and/or wild-type macrophages. Subtractive proteomics identified 18 candidate MMP-9 substrates. Biochemical studies confirmed that two transmembrane proteins, β2 integrin subunit (CD18) and amyloid protein precursor (APP), were enriched in the medium of M9A macrophages. To identify potential cleavage sites, we synthesized an overlapping library of peptides that spanned 60 residues of the ectodomain and transmembrane domain of β2 integrin. Active MMP-9 cleaved a single peptide, ECVKGPNVAAIVGGT, at residues corresponding to Ala705 and Ile706 of the β2 integrin. Peptides corresponding to this cleavage site were detected by tandem mass spectrometric analysis only in medium from M9A macrophages, strongly supporting the proposal that β2 integrin is shed by autoactivating MMP-9. Our observations indicate that subtractive proteomics in concert with peptide substrate mapping is a powerful approach for identifying proteolytic substrates and suggest that MMP-9 plays previously unsuspected roles in the regulation and shedding of β2 integrin.

Adam17-dependent shedding limits early neutrophil influx, but does not alter early monocyte recruitment to inflammatory sites

TNF-α-converting enzyme (TACE, herein denoted as Adam17) proteolytically sheds several cell-surface inflammatory proteins, but the physiologic importance of the cleavage of these substrates from leukocyte subsets during inflammation is incompletely understood. In this study, we show that Adam17-null neutrophils have a 2-fold advantage in their initial recruitment during thioglycollate-induced peritonitis, and they roll slower and adhere more readily in the cremaster model than wild-type neutrophils. Although CD44 and ICAM-1 are both in vitro substrates of Adam17, their surface levels are not altered on Adam17-null neutrophils. In contrast, L-selectin levels are elevated up to 10-fold in Adam17-null circulating neutrophils, and their accelerated peritoneal influx, slower rolling, and increased adhesion in the cremaster muscle are dependent on L-selectin. Analysis of mixed chimeras shows that enhanced L-selectin levels and accelerated influx were both cell-intrinsic properties of neutrophils lacking Adam17. In contrast, Adam17-null monocytes display no acceleration of infiltration into the peritoneum in spite of elevated L-selectin surface levels, and their peritoneal influx was independent of L-selectin. Therefore, our data demonstrate substrate and myeloid cell-type specificity of Adam17-mediated cleavage of its substrates, and show that neutrophils and monocytes use distinct mechanisms for infiltration of tissues.

Dicer1 activity in the stromal compartment regulates nephron differentiation and vascular patterning during mammalian kidney organogenesis

MicroRNAs, activated by the enzyme Dicer1, control post-transcriptional gene expression. Dicer1 has important roles in the epithelium during nephrogenesis, but its function in stromal cells during kidney development is unknown. To study this we inactivated Dicer1 in renal stromal cells. This resulted in hypoplastic kidneys, abnormal differentiation of the nephron tubule and vasculature, and perinatal mortality. In mutant kidneys, genes involved in stromal cell migration and activation were suppressed as were those involved in epithelial and endothelial differentiation and maturation. Consistently, polarity of the proximal tubule was incorrect, distal tubule differentiation was diminished, and elongation of Henle’s loop attenuated resulting in lack of inner medulla and papilla in stroma-specific Dicer1 mutants. Glomerular maturation and capillary loop formation were abnormal while peritubular capillaries, with enhanced branching and increased diameter, formed later. In Dicer1-null renal stromal cells, expression of factors associated with migration, proliferation and morphogenic functions including α-smooth muscle actin, integrin-α8, -β1, and the WNT pathway transcriptional regulator LEF1 were reduced. Dicer1 mutation in stroma led to loss of expression of distinct microRNAs. Of these, miR-214, -199a-5p and -199a-3p regulate stromal cell functions ex vivo, including WNT pathway activation, migration and proliferation. Thus, Dicer1 activity in the renal stromal compartment regulates critical stromal cell functions that, in turn, regulate differentiation of the nephron and vasculature during nephrogenesis.

Metalloproteinase-mediated Shedding of Integrin β2 Promotes Macrophage Efflux from Inflammatory Sites

Background: Efflux of macrophages limits inflammation. Results: Macrophage integrin αMβ2 is cleaved during exiting from inflammatory sites, released αMβ2 retains ligand binding capabilities, and inhibition of its metalloproteinase-mediated cleavage impairs macrophage efflux. Conclusion: Metalloproteinase-mediated proteolysis of integrin β2 promotes macrophage efflux from inflammatory sites. Significance: Regulated proteolysis of integrin β2 during inflammation demonstrates the potential of this mechanism to contribute to resolution of inflammation. Macrophage exiting from inflammatory sites is critical to limit the local innate immune response. With tissue insult, resident tissue macrophages rapidly efflux to lymph nodes where they modulate the adaptive immune response, and inflammatory macrophages attracted to the site of injury then exit during the resolution phase. However, the mechanisms that regulate macrophage efflux are poorly understood. This study has investigated soluble forms of integrin β2 whose levels are elevated in experimental peritonitis at times when macrophages are exiting the peritoneum, suggesting that its proteolytic shedding may be involved in macrophage efflux. Both constitutive and inducible metalloproteinase-dependent shedding of integrin β2 from mouse macrophages are demonstrated. Soluble integrin β2 is primarily released as a heterodimeric complex with αM that retains its ability to bind its ligands intracellular adhesion molecule-1, fibrin, and collagen and thus may serve as a soluble antagonist. In a model of accelerated exiting, administration of a metalloproteinase inhibitor prevents macrophage efflux by 50% and impedes loss of macrophage integrin β2 from the cell surface. Exiting of peritoneal macrophages in mice lacking integrin β2 is accelerated, and antibody disruption of integrin β2-substrate interactions can reverse 50% of the metalloprotease inhibitor blockade of macrophage exiting. Thus, our study demonstrates the ability of metalloproteinase-mediated shedding of integrin β2 to promote macrophage efflux from inflammatory sites, and the release of soluble integrin heterodimers may also limit local inflammation.

The FOXD1 lineage of kidney perivascular cells and myofibroblasts: functions and responses to injury.

Recent studies have identified a poorly appreciated yet extensive population of perivascular mesenchymal cells in the kidney, which are derived from metanephric mesenchyme progenitor cells during nephrogenesis at which time they express the transcription factor FOXD1. Some studies have called these resident fibroblasts, whereas others have called them pericytes. Regardless of nomenclature, many are partially integrated into the capillary basement membrane and contribute in important ways to the homeostasis of peritubular capillaries. Fate-mapping studies using conditional CreER recombinase-mediated tracing of discrete cell cohorts have identified these pericytes and resident fibroblasts as the major precursor population of interstitial myofibroblasts in animal models of kidney disease. Here, we will review the evidence that they are the major population of myofibroblast precursors, highlight some critical functions in homeostasis, and focus on the cell signaling pathways that are important to their differentiation into, and persistence as myofibroblasts.

Pericyte MyD88 and IRAK4 control inflammatory and fibrotic responses to tissue injury

Fibrotic disease is associated with matrix deposition that results in the loss of organ function. Pericytes, the precursors of myofibroblasts, are a source of pathological matrix collagens and may be promising targets for treating fibrogenesis. Here, we have shown that pericytes activate a TLR2/4- and MyD88-dependent proinflammatory program in response to tissue injury. Similarly to classic immune cells, pericytes activate the NLRP3 inflammasome, leading to IL-1&bgr; and IL-18 secretion. Released IL-1&bgr; signals through pericyte MyD88 to amplify this response. Unexpectedly, we found that MyD88 and its downstream effector kinase IRAK4 intrinsically control pericyte migration and conversion to myofibroblasts. Specific ablation of MyD88 in pericytes or pharmacological inhibition of MyD88 signaling by an IRAK4 inhibitor in vivo protected against kidney injury by profoundly attenuating tissue injury, activation, and differentiation of myofibroblasts. Our data show that in pericytes, MyD88 and IRAK4 are key regulators of 2 major injury responses: inflammatory and fibrogenic. Moreover, these findings suggest that disruption of this MyD88-dependent pathway in pericytes might be a potential therapeutic approach to inhibit fibrogenesis and promote regeneration.

MicroRNAs as potential therapeutic targets in kidney disease

One cornerstone of chronic kidney disease (CKD) is fibrosis, as kidneys are susceptible due to their high vascularity and predisposition to ischemia. Presently, only therapies targeting the angiotensin receptor are used in clinical practice to retard the progression of CKD. Thus, there is a pressing need for new therapies designed to treat the damaged kidney. Several independent laboratories have identified a number of microRNAs that are dysregulated in human and animal models of CKD. This review will explore the evidence suggesting that by blocking the activity of such dysregulated microRNAs, new therapeutics could be developed to treat the progression of CKD.

TWEAK-Fn14 Signaling Activates Myofibroblasts to Drive Progression of Fibrotic Kidney Disease

The identification of the cellular origins of myofibroblasts has led to the discovery of novel pathways that potentially drive myofibroblast perpetuation in disease. Here, we further investigated the role of innate immune signaling pathways in this process. In mice, renal injury-induced activation of pericytes, which are myofibroblast precursors attached to endothelial cells, led to upregulated expression of TNF receptor superfamily member 12a, also known as fibroblast growth factor-inducible 14 (Fn14), by these cells. In live rat kidney slices, administration of the Fn14 ligand, TNF-related weak inducer of apoptosis (TWEAK), promoted pericyte-dependent vasoconstriction followed by pericyte detachment from capillaries. In vitro, administration of TWEAK activated and differentiated pericytes into cytokine-producing myofibroblasts, and further activated established myofibroblasts in a manner requiring canonical and noncanonical NF-κB signaling pathways. Deficiency of Fn14 protected mouse kidneys from fibrogenesis, inflammation, and associated vascular instability after in vivo injury, and was associated with loss of NF-κB signaling. In a genetic model of spontaneous CKD, therapeutic delivery of anti-TWEAK blocking antibodies attenuated disease progression, preserved organ function, and increased survival. These results identify the TWEAK-Fn14 signaling pathway as an important factor in myofibroblast perpetuation, fibrogenesis, and chronic disease progression.

Connective Tissue Growth Factor Domain 4 Amplifies Fibrotic Kidney Disease through Activation of LDL Receptor–Related Protein 6

Connective tissue growth factor (CTGF), a matrix-associated protein with four distinct cytokine binding domains, has roles in vasculogenesis, wound healing responses, and fibrogenesis and is upregulated in fibroblasts and myofibroblasts in disease. Here, we investigated the role of CTGF in fibrogenic cells. In mice, tissue-specific inducible overexpression of CTGF by kidney pericytes and fibroblasts had no bearing on nephrogenesis or kidney homeostasis but exacerbated inflammation and fibrosis after ureteral obstruction. These effects required the WNT receptor LDL receptor-related protein 6 (LRP6). Additionally, pericytes isolated from these mice became hypermigratory and hyperproliferative on overexpression of CTGF. CTGF is cleaved in vivo into distinct domains. Treatment with recombinant domain 1, 1+2 (N terminus), or 4 (C terminus) independently activated myofibroblast differentiation and wound healing responses in cultured pericytes, but domain 4 showed the broadest profibrotic activity. Domain 4 exhibited low-affinity binding to LRP6 in in vitro binding assays, and inhibition of LRP6 or critical signaling cascades downstream of LRP6, including JNK and WNT/β-catenin, inhibited the biologic activity of domain 4. Administration of blocking antibodies specifically against CTGF domain 4 or recombinant Dickkopf-related protein-1, an endogenous inhibitor of LRP6, effectively inhibited inflammation and fibrosis associated with ureteral obstruction in vivo Therefore, domain 4 of CTGF and the WNT signaling pathway are important new targets in fibrosis.