Faculty Bibliography

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.

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 lead 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.

Endothelial cells in different microvascular segments of the kidney have diverse functions and exhibit differential responsiveness to disease stimuli. The responsible molecular mechanisms are largely unknown. We previously showed that during hemorrhagic shock, VCAM-1 protein was expressed primarily in extraglomerular compartments of the kidney, while E-selectin protein was highly induced in glomeruli only (van Meurs M, Wulfert FM, Knol AJ, de Haes A, Houwertjes M, Aarts LPHJ, Molema G. Shock 29: 291-299, 2008). Here, we investigated the molecular control of expression of these endothelial cell adhesion molecules in mouse models of renal inflammation. Microvascular segment-specific responses to the induction of anti-glomerular basement membrane (anti-GBM), glomerulonephritis and systemic TNF-α treatment showed that E-selectin expression was transcriptionally regulated, with high E-selectin mRNA and protein levels preferentially expressed in the glomerular compartment. In contrast, VCAM-1 mRNA expression was increased in both arterioles and glomeruli, while VCAM-1 protein expression was limited in the glomeruli. These high VCAM-1 mRNA/low VCAM-1 protein levels were accompanied by high local microRNA (miR)-126 and Egfl7 levels, as well as higher Ets1 levels compared with arteriolar expression levels. Using miR-reporter constructs, the functional activity of miR-126 in glomerular endothelial cells could be demonstrated. Moreover, in vivo knockdown of miR-126 function unleashed VCAM-1 protein expression in the glomeruli upon inflammatory challenge. These data imply that miR-126 has a major role in the segmental, heterogenic response of renal microvascular endothelial cells to systemic inflammatory stimuli.

Endothelial to mesenchymal transition (EndoMT) has been proposed to be involved in the loss of microvascular capillaries in the pathophysiology of fibrosis and organ failure. In EndoMT, endothelial cells (EC) undergo a mesenchymal transition associated with the loss of cell-cell contacts and the acquisition of a synthetic, contractile phenotype. Here, we sought to identify microRNAs (miRNAs) that could play a central role in regulating EndoMT. In a TGF-β dependent in vitro model for EndoMT, we identified miRNAs that were differentially expressed in normoxic and hypoxic conditions. These studies identified miR-155 to be significantly upregulated in EndoMT, an effect that was enhanced under hypoxia, which further augments EndoMT. Silencing of miR-155 directly increased RhoA expression and activity in endothelial cells and affected phosphorylation of downstream LIMK. In contrast, overexpression of miR-155 counteracted RhoA function. Using a selective Rho kinase inhibitor, we could partly suppress EndoMT, strengthening the notion that RhoA plays a central role in EndoMT. Forced overexpression of miR-155 completely suppressed EndoMT, as evidenced by the maintenance of EC characteristics and blocking the acquisition of a mesenchymal phenotype, as compared to control cells. Our data demonstrate that miRNA-155 functions as a negative regulator of RhoA signaling in TGF-β-induced endothelial to mesenchymal transition.

AIMS/OBJECTIVE:MicroRNA-126 (miR-126), which is enriched in endothelial cells, plays a role in angiogenesis. Based on the seed sequence, miR-126 can also be predicted to regulate vasculogenesis by modulating the endothelial expression of stromal cell-derived factor-1 (SDF-1). METHODS AND RESULTS/RESULTS:Using miR-reporter constructs, we first validated that miR-126 inhibits SDF-1 expression in endothelial cells in vitro. Next, we investigated the potential relevance of this observation with respect to the mobilization of progenitor cells. For this, we studied the migration of human CD34+ progenitor cells towards chemotactic factors present in endothelial cell-conditioned medium. Antagomir-induced silencing of miR-126 elevated SDF-1 expression by human umbilical vein endothelial cells and enhanced migration of the CD34+ cells. In a murine model of hind limb ischaemia, a striking increase in the number of circulating Sca-1(+)/Lin(-) progenitor cells in antagomir-126-treated mice was observed when compared with scramblemir-treated controls. Immunohistochemical staining of capillaries in the post-ischaemic gastrocnemius muscle of miR-126-silenced mice revealed elevated SDF-1 expressing CD31-positive capillaries, whereas a mobilizing effect of miR-126 inhibition was not detected in healthy control animals. CONCLUSION/CONCLUSIONS:miR-126 can regulate the expression of SDF-1 in endothelial cells. In the context of an ischaemic event, systemic silencing of miR-126 leads to the mobilization of Sca-1(+)/Lin(-) progenitor cells into the peripheral circulation, potentially in response to elevated SDF-1 expression by endothelial cells present in the ischaemic tissue.

MicroRNAs are negative regulators of gene expression that play a key role in cell-type specific differentiation and modulation of cell function and have been proposed to be involved in neovascularization. Previously, using an extensive cloning and sequencing approach, we identified miR-126 to be specifically and highly expressed in human endothelial cells (EC). Here, we demonstrate EC-specific expression of miR-126 in capillaries and the larger vessels in vivo. We therefore explored the potential role of miR-126 in arteriogenesis and angiogenesis. Using miR-reporter constructs, we show that miR-126 is functionally active in EC in vitro and that it could be specifically repressed using antagomirs specifically targeting miR-126. To study the consequences of miR-126 silencing on vascular regeneration, mice were injected with a single dose of antagomir-126 or a control 'scramblemir' and exposed to ischemia of the left hindlimb by ligation of the femoral artery. Although miR-126 was effectively silenced in mice treated with a single, high dose (HD) of antagomir-126, laser Doppler perfusion imaging did not show effects on blood flow recovery. In contrast, quantification of the capillary density in the gastrocnemius muscle revealed that mice treated with a HD of antagomir-126 had a markedly reduced angiogenic response. Aortic explant cultures of the mice confirmed the role of miR-126 in angiogenesis. Our data demonstrate a facilitary function for miR-126 in ischemia-induced angiogenesis and show the efficacy and specificity of antagomir-induced silencing of EC-specific microRNAs in vivo.