Faculty Bibliography

Mycobacterium tuberculosis (Mtb) survives in macrophages by evading delivery to the lysosome and promoting the accumulation of lipid bodies, which serve as a bacterial source of nutrients. We found that by inducing the microRNA (miRNA) miR-33 and its passenger strand miR-33*, Mtb inhibited integrated pathways involved in autophagy, lysosomal function and fatty acid oxidation to support bacterial replication. Silencing of miR-33 and miR-33* by genetic or pharmacological means promoted autophagy flux through derepression of key autophagy effectors (such as ATG5, ATG12, LC3B and LAMP1) and AMPK-dependent activation of the transcription factors FOXO3 and TFEB, which enhanced lipid catabolism and Mtb xenophagy. These data define a mammalian miRNA circuit used by Mtb to coordinately inhibit autophagy and reprogram host lipid metabolism to enable intracellular survival and persistence in the host.

OBJECTIVE: Cholesterol homeostasis is fundamental to human health and is, thus, tightly regulated. MicroRNAs exert potent effects on biological pathways, including cholesterol metabolism, by repressing genes with related functions. We reasoned that this mode of pathway regulation could be exploited to identify novel genes involved in cholesterol homeostasis. APPROACH AND RESULTS: Here, we identify oxysterol-binding protein-like 6 (OSBPL6) as a novel target of 2 miRNA hubs regulating cholesterol homeostasis: miR-33 and miR-27b. Characterization of OSBPL6 revealed that it is transcriptionally regulated in macrophages and hepatocytes by liver X receptor and in response to cholesterol loading and in mice and nonhuman primates by Western diet feeding. OSBPL6 encodes the OSBPL-related protein 6 (ORP6), which contains dual membrane- and endoplasmic reticulum-targeting motifs. Subcellular localization studies showed that ORP6 is associated with the endolysosomal network and endoplasmic reticulum, suggesting a role for ORP6 in cholesterol trafficking between these compartments. Accordingly, knockdown of OSBPL6 results in aberrant clustering of endosomes and promotes the accumulation of free cholesterol in these structures, resulting in reduced cholesterol esterification at the endoplasmic reticulum. Conversely, ORP6 overexpression enhances cholesterol trafficking and efflux in macrophages and hepatocytes. Moreover, we show that hepatic expression of OSBPL6 is positively correlated with plasma levels of high-density lipoprotein cholesterol in a cohort of 200 healthy individuals, whereas its expression is reduced in human atherosclerotic plaques. CONCLUSIONS: These studies identify ORP6 as a novel regulator of cholesterol trafficking that is part of the miR-33 and miR-27b target gene networks that contribute to the maintenance of cholesterol homeostasis.

RATIONALE: Several lines of evidence indicate that the regulation of microRNA levels by different stimuli may contribute to the modulation of stimulus-induced responses. The microRNA-17~92 (miR-17~92) cluster has been linked to tumor development and angiogenesis, but its role in VEGF-induced endothelial cell (EC) functions is unclear and its regulation is unknown. OBJECTIVE: The purpose of this study was to elucidate the mechanism by which VEGF regulates the expression of miR-17~92 cluster in ECs and determine its contribution to the regulation of endothelial angiogenic functions, both in vitro and in vivo. This was done by analyzing the effect of postnatal inactivation of miR-17~92 cluster in the endothelium (miR-17~92 iEC-KO mice) on developmental retinal angiogenesis, VEGF-induced ear angiogenesis, and tumor angiogenesis. METHODS AND RESULTS: Here we show that Erk/Elk1 activation upon VEGF stimulation of ECs is responsible for Elk-1-mediated transcription activation (ChIP analysis) of the miR-17~92 cluster. Furthermore, we demonstrate that VEGF-mediated upregulation of the miR-17~92 cluster in vitro is necessary for EC proliferation and angiogenic sprouting. Lastly, we provide genetic evidence that miR-17~92 iEC-KO mice have blunted physiological retinal angiogenesis during development and diminished VEGF-induced ear angiogenesis and tumor angiogenesis. Computational analysis and rescue experiments show that PTEN is a target of the miR-17~92 cluster and is a crucial mediator of miR-17-92-induced endothelial cell proliferation. However, the angiogenic transcriptional program is reduced when miR-17~92 is inhibited. CONCLUSIONS: Taken together, our results indicate that VEGF-induced miR-17~92 cluster expression contributes to the angiogenic switch of ECs and participates in the regulation of angiogenesis.

Cellular metabolism is increasingly recognized as a controller of immune cell fate and function. MicroRNA-33 (miR-33) regulates cellular lipid metabolism and represses genes involved in cholesterol efflux, HDL biogenesis, and fatty acid oxidation. Here, we determined that miR-33-mediated disruption of the balance of aerobic glycolysis and mitochondrial oxidative phosphorylation instructs macrophage inflammatory polarization and shapes innate and adaptive immune responses. Macrophage-specific Mir33 deletion increased oxidative respiration, enhanced spare respiratory capacity, and induced an M2 macrophage polarization-associated gene profile. Furthermore, miR-33-mediated M2 polarization required miR-33 targeting of the energy sensor AMP-activated protein kinase (AMPK), but not cholesterol efflux. Notably, miR-33 inhibition increased macrophage expression of the retinoic acid-producing enzyme aldehyde dehydrogenase family 1, subfamily A2 (ALDH1A2) and retinal dehydrogenase activity both in vitro and in a mouse model. Consistent with the ability of retinoic acid to foster inducible Tregs, miR-33-depleted macrophages had an enhanced capacity to induce forkhead box P3 (FOXP3) expression in naive CD4+ T cells. Finally, treatment of hypercholesterolemic mice with miR-33 inhibitors for 8 weeks resulted in accumulation of inflammation-suppressing M2 macrophages and FOXP3+ Tregs in plaques and reduced atherosclerosis progression. Collectively, these results reveal that miR-33 regulates macrophage inflammation and demonstrate that miR-33 antagonism is atheroprotective, in part, by reducing plaque inflammation by promoting M2 macrophage polarization and Treg induction.

Atherosclerosis (also known as arteriosclerotic vascular disease) is a chronic inflammatory disease of the arterial wall, characterized by the formation of lipid-laden lesions. The activation of endothelial cells at atherosclerotic lesion-prone sites in the arterial tree results in the up-regulation of cell adhesion molecules and chemokines, which mediate the recruitment of circulating monocytes. Accumulation of monocytes and monocyte-derived phagocytes in the wall of large arteries leads to chronic inflammation and the development and progression of atherosclerosis. The lesion experiences the following steps: foam cell formation, fatty streak accumulation, migration and proliferation of vascular smooth muscle cells, and fibrous cap formation. Finally, the rupture of the unstable fibrous cap causes thrombosis in complications of advanced lesions that leads to unstable coronary syndromes, myocardial infarction and stroke. MicroRNAs have recently emerged as a novel class of gene regulators at the post-transcriptional level. Several functions of vascular cells, such as cell differentiation, contraction, migration, proliferation and inflammation that are involved in angiogenesis, neointimal formation and lipid metabolism underlying various vascular diseases, have been found to be regulated by microRNAs and are described in the present review as well as their potential therapeutic application.

MicroRNAs are negative regulators of gene expression that have been shown to be essential elements in the coordination of complex regulatory pathways. One of these short non-coding RNAs, microRNA-126, is highly enriched in the vascular endothelium and was shown to play distinct roles in angiogenesis, vasculogenesis and endothelial inflammation. Abrogation of this microRNA leads to severe complications in the response in vascular development as well as vital repair mechanisms carried out by endothelial cells. Interestingly, recent data suggest that the homeostatic role of microRNA-126 may reach far beyond its endothelial functions as this microRNA was also found to be present in cells of the hematopoietic system and in microvesicles or 'free-form' in the periphery. MicroRNA-126 is controlling the fate and/or function of a variety of cells differentiating from the hematopoietic lineage, including megakaryocytes and erythrocytes. Recent studies identified circulating microRNA-126 as a biomarker for myocardial injury and vascular damage in diabetes. Furthermore, reports have suggested a protective role of circulating microRNA-126 in murine models of organ ischemia. Here, we review current insights in the role of microRNA-126 in vascular homeostasis and conclude that this microRNA may serve to integrate and facilitate both local as well as systemic functions in vascular maintenance and repair.

Ischemia/reperfusion injury (IRI) is a central phenomenon in kidney transplantation and AKI. Integrity of the renal peritubular capillary network is an important limiting factor in the recovery from IRI. MicroRNA-126 (miR-126) facilitates vascular regeneration by functioning as an angiomiR and by modulating mobilization of hematopoietic stem/progenitor cells. We hypothesized that overexpression of miR-126 in the hematopoietic compartment could protect the kidney against IRI via preservation of microvascular integrity. Here, we demonstrate that hematopoietic overexpression of miR-126 increases neovascularization of subcutaneously implanted Matrigel plugs in mice. After renal IRI, mice overexpressing miR-126 displayed a marked decrease in urea levels, weight loss, fibrotic markers, and injury markers (such as kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin). This protective effect was associated with a higher density of the peritubular capillary network in the corticomedullary junction and increased numbers of bone marrow-derived endothelial cells. Hematopoietic overexpression of miR-126 increased the number of circulating Lin(-)/Sca-1(+)/cKit(+) hematopoietic stem and progenitor cells. Additionally, miR-126 overexpression attenuated expression of the chemokine receptor CXCR4 on Lin(-)/Sca-1(+)/cKit(+) cells in the bone marrow and increased renal expression of its ligand stromal cell-derived factor 1, thus favoring mobilization of Lin(-)/Sca-1(+)/cKit(+) cells toward the kidney. Taken together, these results suggest overexpression of miR-126 in the hematopoietic compartment is associated with stromal cell-derived factor 1/CXCR4-dependent vasculogenic progenitor cell mobilization and promotes vascular integrity and supports recovery of the kidney after IRI.

Integrity of the capillary network in the kidney is essential in the recovery from ischemia/ reperfusion injury (IRI), a phenomenon central to kidney transplantation and acute kidney injury. MicroRNA- 126 (miR-126) is known to be important in maintaining vascular homeostasis by facilitating vascular regeneration and modulating the mobilization of vascular progenitor cells. Stromal cell-derived factor 1 (SDF-1), important in the mobilization of vascular progenitor cells, is a direct target of miR-126 and modulation of miR-126 was previously shown to affect the number of circulating Sca-1(+)/Lin(-) vascular progenitor cells in a mouse model for hind limb ischemia. Here, we assessed the in vivo contribution of miR-126 to progenitor cell mobilization and kidney function following IRI in mice. A three day follow up of blood urea levels following kidney IRI demonstrated that systemic antagomir silencing of miR-126 did not impact the loss or subsequent restoration of kidney function. However, whole kidney lysates displayed elevated gene expression levels of Sdf-1, Vegf-A and eNOS after IRI as a result of systemic silencing of miR-126. Furthermore, FACS-analysis on whole blood three days after surgery revealed a marked up regulation of the number of circulating Sca-1(+)/Lin(-) progenitor cells in the antagomir-126 treated mice, in an ischemia dependent manner. Our data indicate that silencing of miR-126 can enhance renal expression of Sdf-1 after IRI, leading to the mobilization of vascular progenitor cells into the circulation.

AIMS/OBJECTIVE:MicroRNA-126 (miR-126) facilitates angiogenesis and regulates endothelial cell function. Recent data suggest that miR-126 can serve as a biomarker for vascular disease. Although endothelial cells are enriched for miR-126, platelets also contain miR-126. In this paper, we investigated the contribution of platelets to the pool of miR-126 in plasma of patients with type 2 diabetes (DM2) and how this is affected by aspirin. METHODS AND RESULTS/RESULTS:In vitro platelet activation resulted in the transfer of miR-126 from the platelet to the plasma compartment, which was prevented by aspirin. In vivo platelet activation, monitored in patients with DM2 by measuring soluble P-selectin, correlated directly with circulating levels of miR-126. The administration of aspirin resulted both in platelet inhibition and concomitantly reduced circulating levels of platelet-derived microRNAs including miR-126. CONCLUSION/CONCLUSIONS:Platelets are a major source of circulating miR-126. Consequently, in patho-physiological conditions associated with platelet activation, such as diabetes type 2, the administration of aspirin may lead to reduced levels of circulating miR-126. Thus, the use of platelet inhibitors should be taken into account when using plasma levels of miR-126 as a biomarker.

RATIONALE/BACKGROUND:RNA-binding proteins are critical post-transcriptional regulators of RNA and can influence pre-mRNA splicing, RNA localization, and stability. The RNA-binding protein Quaking (QKI) is essential for embryonic blood vessel development. However, the role of QKI in the adult vasculature, and in particular in vascular smooth muscle cells (VSMCs), is currently unknown. OBJECTIVE:We sought to determine the role of QKI in regulating adult VSMC function and plasticity. METHODS AND RESULTS/RESULTS:We identified that QKI is highly expressed by neointimal VSMCs of human coronary restenotic lesions, but not in healthy vessels. In a mouse model of vascular injury, we observed reduced neointima hyperplasia in Quaking viable mice, which have decreased QKI expression. Concordantly, abrogation of QKI attenuated fibroproliferative properties of VSMCs, while potently inducing contractile apparatus protein expression, rendering noncontractile VSMCs with the capacity to contract. We identified that QKI localizes to the spliceosome, where it interacts with the myocardin pre-mRNA and regulates the splicing of alternative exon 2a. This post-transcriptional event impacts the Myocd_v3/Myocd_v1 mRNA balance and can be modulated by mutating the quaking response element in exon 2a of myocardin. Furthermore, we identified that arterial damage triggers myocardin alternative splicing and is tightly coupled with changes in the expression levels of distinct QKI isoforms. CONCLUSIONS:We propose that QKI is a central regulator of VSMC phenotypic plasticity and that intervention in QKI activity can ameliorate pathogenic, fibroproliferative responses to vascular injury.