Yulia Kiyan

oxLDL induces inflammatory responses in vascular smooth muscle cells via urokinase receptor association with CD36 and TLR4.

The pathogenesis of atherosclerosis involves an imbalanced lipid metabolism and a deregulated immune response culminating in chronic inflammation of the arterial wall. Recent studies show that endogenous ligands, such as modified plasma lipoproteins, can trigger pattern recognition receptors (PRR) of innate immunity for cellular and humoral reactions. The underlying molecular pathways remain less explored. In this study, we investigated the mechanisms of inflammatory effects of oxidized low-density lipoproteins (oxLDL) on human primary coronary artery smooth muscle cells (VSMC). We show that already low concentration of oxLDL initiated atherogenic signals triggering VSMC transition to proinflammatory phenotype. oxLDL impaired the expression of contractile proteins and myocardin in VSMC and initiated changes in cell functional responses, including expression of proinflammatory molecules. The effects of oxLDL were abolished by downregulation of the multifunctional urokinase receptor (uPAR). In response to oxLDL uPAR associated with CD36 and TLR4, the two main PRR for both pathogen and endogenous ligands. We demonstrate that uPAR association with CD36 and TLR4 mediated oxLDL-induced and NF-κB-dependent G-CSF and GM-CSF expression and changes in VSMC contractile proteins. uPAR-mediated release of G-CSF and GM-CSF by VSMC affected macrophage behavior and production of MCP-1. We provide evidence for functional relevance of our in vitro findings to in vivo human atherosclerotic tissues. Our data imply uPAR as a part of a PRR cluster interfering structurally and functionally with CD36 and TLR4 and responding to endogenous atherogenic ligands. They further point to specific function of each component of this cluster in mediating the ultimate signaling pattern.

Dual Inhibition of Classical Protein Kinase C-α and Protein Kinase C-β Isoforms Protects Against Experimental Murine Diabetic Nephropathy

Activation of protein kinase C (PKC) has been implicated in the pathogenesis of diabetic nephropathy with proteinuria and peritubular extracellular matrix production. We have previously shown that the PKC isoforms α and β mediate different cellular effects. PKC-β contributes to hyperglycemia-induced renal matrix production, whereby PKC-α is involved in the development of albuminuria. We further tested this hypothesis by deletion of both isoforms and used a PKC inhibitor. We analyzed the phenotype of nondiabetic and streptozotocin (STZ)-induced diabetic homozygous PKC-α/β double-knockout mice (PKC-α/β−/−). After 8 weeks of diabetes mellitus, the high-glucose–induced renal and glomerular hypertrophy as well as transforming growth factor-β1) and extracellular matrix production were diminished in the PKC-α/β−/− mice compared with wild-type controls. Urinary albumin/creatinine ratio also was significantly reduced, however, it was not completely abolished in diabetic PKC-α/β−/− mice. Treatment with CGP41252, which inhibits PKC-α and PKC-β, is able to prevent the development of albuminuria and to reduce existing albuminuria in type 1 (STZ model) or type 2 (db/db model) diabetic mice. These results support our hypothesis that PKC-α and PKC-β contribute to the pathogenesis of diabetic nephropathy, and that dual inhibition of the classical PKC isoforms is a suitable therapeutic strategy in the prevention and treatment of diabetic nephropathy.

Urokinase receptor mediates mobilization, migration, and differentiation of mesenchymal stem cells

AIMS Multipotent mesenchymal stem cells (MSCs) have regenerative properties and are recognized as putative players in the pathogenesis of cardiovascular diseases. The underlying molecular mechanisms remain, however, sparsely explored. Our study was designed to elucidate a probable role for the multifunctional urokinase (uPA)/urokinase receptor (uPAR) system in MSC regulation. Though uPAR has been implicated in a broad spectrum of pathophysiological processes, nothing is known about uPAR in MSCs. METHODS AND RESULTS uPAR was required to mobilize MSCs from the bone marrow (BM) of mice stimulated with granulocyte colony-stimulating factor (G-CSF) in vivo. An insignificant amount of MSCs was mobilized in uPAR(-/-) C57BL/6J mice, whereas in wild-type animals G-CSF induced an eight-fold increase of mobilized MSCs. uPAR(-/-) mice revealed up-regulated expression of G-CSF and stromal cell-derived factor 1 (CXCR4) receptors in BM. uPAR down-regulation leads to inhibition of human MSC migration, as shown in different migration assays. uPAR down- or up-regulation resulted in inhibition or stimulation of MSC differentiation into vascular smooth muscle cells (VSMCs) correspondingly, as monitored by changes in cell morphology and expression of specific marker proteins. Injection of fluorescently labelled MSCs in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice after femoral artery wire injury demonstrated impaired engraftment of uPAR-deficient MSCs at the place of injury. CONCLUSIONS These data suggest a multifaceted function of uPAR in MSC biology contributing to vascular repair. uPAR might guide and control the trafficking of MSCs to the vascular wall in response to injury or ischaemia and their differentiation towards functional VSMCs at the site of arterial injury.

Urokinase Receptor Associates With Myocardin to Control Vascular Smooth Muscle Cells Phenotype in Vascular Disease

Objective— The urokinase-type plasminogen activator (uPA) and its specific receptor (uPAR) are a potent multifunctional system involved in vascular remodeling. The goal of the study was to unravel the mechanisms of uPA/uPAR-directed vascular smooth muscle cell (VSMC) differentiation. Methods and Results— Using cultured human primary VSMCs, we identified a new molecular mechanism controlling phenotypic modulation in vitro and in vivo. We found that the urokinase-type plasminogen activator receptor (uPAR) acts together with the transcriptional coactivator myocardin to regulate the VSMC phenotype. uPAR, a glycosylphosphatidylinositol-anchored cell-surface receptor family member, undergoes ligand-induced internalization and nuclear transport in VSMCs. Platelet-derived growth factor receptor &bgr; and SUMOylated RanGAP1 mediate this trafficking. Nuclear uPAR associates with myocardin, which is then recruited from the promoters of serum response factor target genes and undergoes proteasomal degradation. This chain of events initiates the synthetic VSMC phenotype. Using mouse carotid artery ligation model, we show that this mechanism contributes to adverse vascular remodeling after injury in vivo. We then cultured cells on a microstructured biomaterial and found that substrate topography induced uPAR-mediated VSMC differentiation. Conclusion— These findings reveal the transcriptional activity of uPAR, controlling the differentiation of VSMCs in a vascular disease model. They also suggest a new role for uPAR as a therapeutic target and as a marker for VSMC phenotyping on prosthetic biomaterials.

Urokinase Receptor Mediates Osteoclastogenesis via M-CSF Release From Osteoblasts and the c-Fms/PI3K/Akt/NF-κB Pathway in Osteoclasts

Bone remodeling is a dynamic process based on a fine‐tuned balance between formation and degradation of bone. Osteoblasts (OBLs) are responsible for bone formation and bone resorption is mediated by osteoclasts (OCLs). The mechanisms regulating the OBL‐OCL balance are critical in health and disease; however, they are still far from being understood. We reported recently that the multifunctional urokinase receptor (uPAR) mediates osteogenic differentiation of mesenchymal stem cells (MSCs) to OBLs and vascular calcification in atherosclerosis. Here, we address the question of whether uPAR may also be engaged in regulation of osteoclastogenesis. We show that uPAR mediates this process in a dual fashion. Thus, uPAR affected OBL‐OCL interplay. We observed that osteoclastogenesis was significantly impaired in co‐culture of monocyte‐derived OCLs and in OBLs derived from MSCs lacking uPAR. We show that expression and release, from OBLs, of macrophage colony‐stimulating factor (M‐CSF), which is indispensable for OCL differentiation, was inhibited by uPAR loss. We further found that uPAR, on the other hand, controlled formation, differentiation, and functional properties of macrophage‐derived OCLs. Expression of osteoclastogenic markers, such as tartrate‐resistant acid phosphatase (TRAP) and cathepsin K, was impaired in OCLs derived from uPAR‐deficient macrophages. The requirement of uPAR for osteoclastogenesis was further confirmed by immunocytochemistry and in bone resorption assay. We provide evidence that the underlying signaling mechanisms involve uPAR association with the M‐CSF binding receptor c‐Fms followed by c‐Fms phosphorylation and activation of the PI3K/Akt/NF‐κB pathway in OCLs. We further show that uPAR uses this pathway to regulate a balance between OCL differentiation, apoptosis, and cell proliferation. Our study identified uPAR as an important and multifaceted regulator of OBL‐OCL molecular interplay that may serve as an attractive target in bone disease and ectopic calcification.

Urokinase Receptor Mediates Doxorubicin-Induced Vascular Smooth Muscle Cell Senescence via Proteasomal Degradation of TRF2

The anthracycline doxorubicin is a widely used effective anti-cancer drug. However, its application and dosage are severely limited due to its cardiotoxicity. The exact mechanisms of doxorubicin-induced cardiotoxic side effects remain poorly understood. Even less is known about the impact of doxorubicin treatment on vascular damage. We found that low doses of doxorubicin induced a senescent response in human primary vascular smooth muscle cells (VSMC). We observed that expression of urokinase receptor (uPAR) was upregulated in response to doxorubicin. Furthermore, the level of uPAR expression played a decisive role in developing doxorubicin-induced senescence. uPAR silencing in human VSMC by means of RNA interference as well as uPAR knockout in mouse VSMC resulted in abrogation of doxorubicin-induced cellular senescence. On the contrary, uPAR overexpression promoted VSMC senescence. We further found that proteasomal degradation of telomeric repeat binding factor 2 (TRF2) mediates doxorubicin-induced VSMC senescence. Our results demonstrate that uPAR controls the ubiquitin-proteasome system in VSMC and regulates doxorubicin-induced TRF2 ubiquitination and proteasomal degradation via this mechanism. Therefore, VSMC senescence induced by low doses of doxorubicin may contribute to vascular damage upon doxorubicin treatment. uPAR-mediated TRF2 ubiquitination and proteasomal degradation are further identified as a molecular mechanism underlying this process.

Urokinase-Type Plasminogen Activator Downregulates Paraoxonase 1 Expression in Hepatocytes by Stimulating Peroxisome Proliferator–Activated Receptor-γ Nuclear Export

Objective—The atherosclerotic lesion is characterized by lipid peroxide accumulation. Paraoxonase 1 (PON1) reduces atherosclerotic lesion oxidative stress, whereas urokinase-type plasminogen activator (uPA) increases oxidative stress in atherosclerotic lesions and contributes to the progression and complications of atherosclerosis. We hypothesized that uPA may promote oxidative stress in the arterial wall via modulation of PON1 activity. Because the liver is the main site for PON1 production, in the present study, we tested whether uPA influences PON1 expression in hepatocytes. Methods and Results—HuH7 hepatocytes were incubated in culture with increasing concentrations of uPA. uPA decreased PON1 gene expression and activity in a dose-dependent manner and accordingly suppressed PON1 secretion from hepatocytes. This effect required uPA/uPA receptor interaction. uPA downregulated PON1 gene expression via inactivation of peroxisome proliferator–activated receptor-&ggr; (PPAR&ggr;) activity, and this effect was dependent on uPA-mediated mitogen-activated protein kinase kinase activation. Mechanistic studies showed that uPA enhanced mitogen-activated protein kinase kinase–PPAR&ggr; interaction, resulting in PPAR&ggr; nuclear export to the cytosol. Conclusion—This study provides the first evidence that uPA interferes with PPAR&ggr; transcriptional activity in hepatocytes, resulting in downregulation of PON1 expression and its secretion to the medium. This may explain, at least in part, the prooxidative effect of uPA in the vascular wall.

Loss of Urokinase Receptor Sensitizes Cells to DNA Damage and Delays DNA Repair

DNA damage induced by numerous exogenous or endogenous factors may have irreversible consequences on the cell leading to cell cycle arrest, senescence and cell death. The DNA damage response (DDR) is powerful signaling machinery triggered in response to DNA damage, to provide DNA damage recognition, signaling and repair. Most anticancer drugs induce DNA damage, and DNA repair in turn attenuates therapeutic efficiency of those drugs. Approaches delaying DNA repair are often used to increase efficiency of treatment. Recent data show that ubiquitin-proteasome system is essential for signaling and repair of DNA damage. However, mechanisms providing regulation of proteasome intracellular localization, activity, and recruitment to DNA damage sites are elusive. Even less investigated are the roles of extranuclear signaling proteins in these processes. In this study, we report the involvement of the serine protease urokinase-type plasminogen activator receptor (uPAR) in DDR-associated regulation of proteasome. We show that in vascular smooth muscle cells (VSMC) uPAR activates DNA single strand break repair signaling pathway. We provide evidence that uPAR is essential for functional assembly of the 26S proteasome. We further demonstrate that uPAR mediates DNA damage-induced phosphorylation, nuclear import, and recruitment of the regulatory subunit PSMD6 to proteasome. We found that deficiency of uPAR and PSMD6 delays DNA repair and leads to decreased cell survival. These data may offer new therapeutic approaches for diseases such as cancer, cardiovascular and neurodegenerative disorders.

Urokinase Receptor Counteracts Vascular Smooth Muscle Cell Functional Changes Induced by Surface Topography

Current treatments for human coronary artery disease necessitate the development of the next generations of vascular bioimplants. Recent reports provide evidence that controlling cell orientation and morphology through topographical patterning might be beneficial for bioimplants and tissue engineering scaffolds. However, a concise understanding of cellular events underlying cell-biomaterial interaction remains missing. In this study, applying methods of laser material processing, we aimed to obtain useful markers to guide in the choice of better vascular biomaterials. Our data show that topographically treated human primary vascular smooth muscle cells (VSMC) have a distinct differentiation profile. In particular, cultivation of VSMC on the microgrooved biocompatible polymer E-shell induces VSMC modulation from synthetic to contractile phenotype and directs formation and maintaining of cell-cell communication and adhesion structures. We show that the urokinase receptor (uPAR) interferes with VSMC behavior on microstructured surfaces and serves as a critical regulator of VSMC functional fate. Our findings suggest that microtopography of the E-shell polymer could be important in determining VSMC phenotype and cytoskeleton organization. They further suggest uPAR as a useful target in the development of predictive models for clinical VSMC phenotyping on functional advanced biomaterials.

CHK1 and RAD51 activation after DNA damage is regulated via urokinase receptor/TLR4 signaling

Mechanisms of DNA damage and repair signaling are not completely understood that hinder the efficiency of cancer therapy. Urokinase-type plasminogen activator receptor (PLAUR) is highly expressed in most solid cancers and serves as a marker of poor prognosis. We show that PLAUR actively promotes DNA repair in cancer cells. On the contrary, downregulation of PLAUR expression results in delayed DNA repair. We found PLAUR to be essential for activation of Checkpoint kinase 1 (CHK1); maintenance of cell cycle arrest after DNA damage in a TP53-dependent manner; expression, nuclear import and recruitment to DNA-damage foci of RAD51 recombinase, the principal protein involved in the homologous recombination repair pathway. Underlying mechanism implies auto-/paracrine signaling of PLAUR/TLR4 receptor complex leading to activation of CHK1 and DNA repair. The signaling is induced by a danger molecule released by DNA-damaged cells and mediates, at least partially, activation of DNA-damage response. This study describes a new mechanism of DNA repair activation initiated by auto-/paracrine signaling of membrane receptors PLAUR/TLR4. It adds to the understanding of role of PLAUR in cancer and provides a rationale for therapeutic targeting of PLAUR/TLR4 interaction in TP53-positive cancers.