Monocytes prevent apoptosis of iPSCs and promote differentiation of kidney organoids.

BACKGROUND: Induced pluripotent stem cells (iPSCs)-derived kidney organoids are a promising model for studying disease mechanisms and renal development. Despite several protocols having been developed, further improvements are needed to overcome existing limitations and enable a wider application of this model. One of the approaches to improve the differentiation of renal organoids in vitro is to include in the system cell types important for kidney organogenesis in vivo, such as macrophages. Another approach could be to improve cell survival. Mesodermal lineage differentiation is the common initial step of the reported protocols. The glycogen synthase kinase-3 (GSK-3) activity inhibitor, CHIR99021 (CHIR), is applied to induce mesodermal differentiation. It has been reported that CHIR simultaneously induces iPSCs apoptosis that can compromise cell differentiation. We thought to interfere with CHIR-induced apoptosis of iPSCs using rapamycin. METHODS: Differentiation of kidney organoids from human iPSCs was performed. Cell survival and autophagy were analyzed using Cell counting kit 8 (CCK8) kit and Autophagy detection kit. Cells were treated with rapamycin or co-cultured with human monocytes isolated from peripheral blood or iPSCs-macrophages using a transwell co-culture system. Monocyte-derived extracellular vesicles (EVs) were isolated using polyethylene glycol precipitation. Expression of apoptotic markers cleaved Caspase 3, Poly [ADP-ribose] polymerase 1 (PARP-1) and markers of differentiation T-Box Transcription Factor 6 (TBX6), odd-skipped related 1 (OSR1), Nephrin, E-Cadherin, Paired box gene 2 (Pax2) and GATA Binding Protein 3 (Gata3) was assessed by RT-PCR and western blotting. Organoids were imaged by 3D-confocal microscopy. RESULTS: We observed that CHIR induced apoptosis of iPSCs during the initial stage of renal organoid differentiation. Underlying mechanisms implied the accumulation of reactive oxygen species and decreased autophagy. Activation of autophagy by rapamacin and by an indirect co-culture of differentiating iPSCs with iPSCs-macrophages and human peripheral blood monocytes prevented apoptosis induced by CHIR. Furthermore, monocytes (but not rapamycin) strongly promoted expression of renal differentiation markers and organoids development via released extracellular vesicles. CONCLUSION: Our data suggest that co-culturing of iPSCs with human monocytes strongly improves differentiation of kidney organoids. An underlying mechanism of monocytic action implies, but not limited to, an increased autophagy in CHIR-treated iPSCs. Our findings enhance the utility of kidney organoid models.

Calcium dobesilate reduces SARS-CoV-2 entry into endothelial cells by inhibiting virus binding to heparan sulfate.

Recent reports demonstrate that SARS-CoV-2 utilizes cell surface heparan sulfate as an attachment factor to facilitate the initial interaction with host cells. Heparan sulfate interacts with the receptor binding domain of SARS-CoV-2 spike glycoprotein, and blocking this interaction can decrease cell infection. We and others reported recently that the family of compounds of 2,5-dihydroxyphenylic acid interferes with the binding of the positively charged groove in growth factor molecules to negatively charged cell surface heparan sulfate. We hypothesized that Calcium Dobesilate (CaD)-calcium salt of 2,5-dihydroxyphenylic acid-may also interfere with the binding of SARS-CoV-2 spike protein to heparan sulfate. Using lentiviral SARS-CoV-2 spike protein pseudotyped particles we show that CaD could significantly reduce pseudovirus uptake into endothelial cells. On the contrary, CaD did not affect cell infection with VSVG-expressing lentivirus. CaD could also prevent retention of SARS-CoV-2 spike protein in ex vivo perfused mouse kidney. Using microfluidic culture of endothelial cells under flow, we show that CaD prevents spike protein interaction with heparan sulfate glycocalyx. Since CaD has no adverse side effects and is approved in humans for other medical indications, our findings can rapidly translate into clinical studies.

TLR4 Response to LPS Is Reinforced by Urokinase Receptor.

GPI-anchored uPAR is the receptor for the extracellular serine protease urokinase-type plasminogen activator (uPA). Though uPAR role in inflammatory processes is documented, underlying mechanisms are not fully understood. In this study we demonstrate that uPAR is a part of Toll-like receptor 4 (TLR4) interactome. Downregulation of uPAR expression resulted in diminished LPS-induced TLR4 signaling, less activation of NFκB, and decreased secretion of inflammatory mediators in myeloid and non-myeloid cells in vitro. In vivo uPAR-/- mice demonstrated better survival, strongly diminished inflammatory response and better organ functions in cecal ligation and puncture mouse polymicrobial sepsis model. Mechanistically, GPI-uPAR and soluble uPAR colocalized with TLR4 on the cell membrane and interacted with scavenger receptor CD36. Our data show that uPAR can interfere with innate immunity response via TLR4 and this mechanism represents a potentially important target in inflammation and sepsis therapy.

Heparanase-2 protects from LPS-mediated endothelial injury by inhibiting TLR4 signalling.

The endothelial glycocalyx and its regulated shedding are important to vascular health. Endo-ß-D-glucuronidase heparanase-1 (HPSE1) is the only enzyme that can shed heparan sulfate. However, the mechanisms are not well understood. We show that HPSE1 activity aggravated Toll-like receptor 4 (TLR4)-mediated response of endothelial cells to LPS. On the contrary, overexpression of its endogenous inhibitor, heparanase-2 (HPSE2) was protective. The microfluidic chip flow model confirmed that HPSE2 prevented heparan sulfate shedding by HPSE1. Furthermore, heparan sulfate did not interfere with cluster of differentiation-14 (CD14)-dependent LPS binding, but instead reduced the presentation of the LPS to TLR4. HPSE2 reduced LPS-mediated TLR4 activation, subsequent cell signalling, and cytokine expression. HPSE2-overexpressing endothelial cells remained protected against LPS-mediated loss of cell-cell contacts. In vivo, expression of HPSE2 in plasma and kidney medullary capillaries was decreased in mouse sepsis model. We next applied purified HPSE2 in mice and observed decreases in TNFα and IL-6 plasma concentrations after intravenous LPS injections. Our data demonstrate the important role of heparan sulfate and the glycocalyx in endothelial cell activation and suggest a protective role of HPSE2 in microvascular inflammation. HPSE2 offers new options for protection against HPSE1-mediated endothelial damage and preventing microvascular disease.

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.

Protein kinase C α inhibition prevents peritoneal damage in a mouse model of chronic peritoneal exposure to high-glucose dialysate.

Chronic exposure to commercial glucose-based peritoneal dialysis fluids during peritoneal dialysis induces peritoneal membrane damage leading to ultrafiltration failure. In this study the role of protein kinase C (PKC) α in peritoneal membrane damage was investigated in a mouse model of peritoneal dialysis. We used 2 different approaches: blockade of biological activity of PKCα by intraperitoneal application of the conventional PKC inhibitor Go6976 in C57BL/6 wild-type mice and PKCα-deficient mice on a 129/Sv genetic background. Daily administration of peritoneal dialysis fluid for 5 weeks induced peritoneal upregulation and activation of PKCα accompanied by epithelial-to-mesenchymal transition of peritoneal mesothelial cells, peritoneal membrane fibrosis, neoangiogenesis, and macrophage and T cell infiltration, paralleled by reduced ultrafiltration capacity. All pathological changes were prevented by PKCα blockade or deficiency. Moreover, treatment with Go6976 and PKCα deficiency resulted in strong reduction of proinflammatory, profibrotic, and proangiogenic mediators. In cell culture experiments, both treatment with Go6976 and PKCα deficiency prevented peritoneal dialysis fluid-induced release of MCP-1 from mouse peritoneal mesothelial cells and ameliorated transforming growth factor-ß1-induced epithelial-to-mesenchymal transition and peritoneal dialysis fluid-induced MCP-1 release in human peritoneal mesothelial cells. Thus, PKCα plays a crucial role in the pathophysiology of peritoneal membrane dysfunction induced by peritoneal dialysis fluids, and we suggest that its therapeutic inhibition might be a valuable treatment option for peritoneal dialysis patients.

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 osteogenic differentiation of mesenchymal stem cells and vascular calcification via the complement C5a receptor.

Vascular calcification is a severe consequence of several pathological processes with a lack of effective therapy. Recent studies suggest that circulating and resident mesenchymal stem cells (MSC) contribute to the osteogenic program of vascular calcification. Molecular mechanisms underlying MSC osteogenic potential and differentiation remain, however, sparsely explored. We investigated a role for the complement receptor C5aR in these processes. We found that expression of C5aR was upregulated upon differentiation of human MSC to osteoblasts. C5aR inhibition by silencing and specific antagonist impaired osteogenic differentiation. We demonstrate that C5aR expression upon MSC differentiation was regulated by the multifunctional urokinase receptor (uPAR). uPAR targeting by siRNA resulted in complete abrogation of C5aR expression and consequently in the inhibition of MSC-osteoblast differentiation. We elucidated the NFκB pathway as the mechanism utilized by the uPAR-C5aR axis. MSC treatment with the NFκB inhibitor completely blocked the differentiation process. Nuclear translocation of the p65 RelA component of the NFκB complex was induced under osteogenic conditions and impaired by the inhibition of uPAR or C5aR. Dual-luciferase reporter assays demonstrated enhanced NFκB signaling upon MSC differentiation, whereas uPAR and C5aR downregulation lead to inhibition of the NFκB activity. We show involvement of the Erk1/2 kinase in this cascade. In vivo studies in a uPAR/LDLR double knockout mouse model of diet-induced atherosclerosis revealed impaired C5aR expression and calcification in aortic sinus plaques in uPAR(-/-)/LDLR(-/-) versus uPAR(+/+)/LDLR(-/-) control animals. These results suggest that uPAR-C5aR axis via the underlying NFκB transcriptional program controls osteogenic differentiation with functional impact on vascular calcification in vivo.

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.

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 ß 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. CONCLUSIONS: 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-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-γ (PPARγ) 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-PPARγ interaction, resulting in PPARγ nuclear export to the cytosol. CONCLUSIONS: This study provides the first evidence that uPA interferes with PPARγ 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.

The tight junction protein ZO-2 and Janus kinase 1 mediate intercellular communications in vascular smooth muscle cells.

Recent evidence points to a multifunctional role of ZO-2, the tight junction protein of the MAGUK (membrane-associated guanylate kinase-like) family. Though ZO-2 has been found in cell types lacking tight junction structures, such as vascular smooth muscle cells (VSMC), little is known about ZO-2 function in these cells. We provide evidence that ZO-2 mediates specific homotypic cell-to-cell contacts between VSMC. Using mass spectrometry we found that ZO-2 is associated with the non-receptor tyrosine kinase Jak1. By generating specific ZO-2 constructs we further found that the N-terminal fragment of ZO-2 molecule is responsible for this interaction. Adenovirus-based expression of Jak1 inactive mutant demonstrated that Jak1 mediates ZO-2 tyrosine phosphorylation. By means of RNA silencing, expression of Jak1 mutant form and fluorescently labeled ZO-2 fusion protein we further specified that active Jak1, but not Jak1 inactive mutant, mediates ZO-2 localization to the sites of intercellular contacts. We identified the urokinase receptor uPAR as a pre-requisite for these cellular events. Functional requirement of the revealed signaling complex for VSMC network formation was confirmed in experiments using Matrigel and in contraction assay. Our findings imply involvement of the ZO-2 tight junction independent signaling complex containing Jak1 and uPAR in VSMC intercellular communications. This mechanism may contribute to vascular remodeling in occlusive cardiovascular diseases and in arteriogenesis.

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.

Stat1 nuclear translocation by nucleolin upon monocyte differentiation.

BACKGROUND: Members of the signal transducer and activator of transcription (Stat) family of transcription factors traverse the nuclear membrane through a specialized structure, called the nuclear pore complex (NPC), which represents a selective filter for the import of proteins. Karyophilic molecules can bind directly to a subset of proteins of the NPC, collectively called nucleoporins. Alternatively, the transport is mediated via a carrier molecule belonging to the importin/karyopherin superfamily, which transmits the import into the nucleus through the NPC. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we provide evidence for an alternative Stat1 nuclear import mechanism, which is mediated by the shuttle protein nucleolin. We observed Stat1-nucleolin association, nuclear translocation and specific binding to the regulatory DNA element GAS. Using expression of nucleolin transgenes, we found that the nuclear localization signal (NLS) of nucleolin is responsible for Stat1 nuclear translocation. We show that this mechanism is utilized upon differentiation of myeloid cells and is specific for the differentiation step from monocytes to macrophages. CONCLUSIONS/SIGNIFICANCE: Our data add the nucleolin-Stat1 complex as a novel functional partner for the cell differentiation program, which is uniquely poised to regulate the transcription machinery via Stat1 and nuclear metabolism via nucleolin.

The tight junction protein ZO-2 mediates proliferation of vascular smooth muscle cells via regulation of Stat1.

AIMS: Recent evidence suggests that the zonula occludens protein 2 (ZO-2) might have additional cellular functions, beyond regulation of paracellular permeability of epithelial and endothelial cells. Deregulation of ZO-2 in response to ischaemia, hypertensive stress, and vascular injury implies its involvement in cardiovascular disorders, most likely via regulating the functional behaviour of vascular smooth muscle cells (VSMC). However, a role of ZO-2 in VSMC biology has yet to be established. Our study was designed to understand the specific functions of ZO-2 in human VSMC. METHODS AND RESULTS: The expression of ZO-2 and Stat1 upon vascular injury was studied using ex vivo organ culture of coronary arteries combined with immunohistochemistry. ZO-2 silencing in human primary VSMC was achieved by means of lentiviral gene transfer. Cell proliferation was assessed by analysing DNA synthesis and by cell counting. Stat1 expression was examined using immunoblotting, immunocytochemistry, TaqMan, and fluorescence activated cell sorting (FACS) analysis. Functional relevance of Stat1 up-regulation was studied using a Stat1 promoter-luciferase reporter assay and intracellular microinjections of a Stat1 specific antibody. ZO-2 was highly expressed in the media and neointima of dilated but not of control arteries, whereas expression of the transcription factor Stat1 was inversely regulated upon injury. Analysis of VSMC with down-regulated ZO-2 revealed increased expression of Stat1 in these cells, whereas Stat1 phosphorylation was not affected. Stat1 up-regulation in VSMC with ZO-2 silencing resulted in a coordinate activation of Stat1-specific genes and consequently led to inhibition of cell proliferation. This effect was restored by microinjection of a Stat1 neutralising antibody. CONCLUSION: Our data suggest that the tight junction protein ZO-2 is involved in regulation of VSMC growth control upon vascular injury that is mediated by the transcription factor Stat1. Our findings point to a novel function of ZO-2 in VSMC and implicate ZO-2 as a novel important molecular target in pathological states of vascular remodelling in cardiovascular diseases.

Urokinase induces survival or pro-apoptotic signals in human mesangial cells depending on the apoptotic stimulus.

Deregulated apoptosis of MCs (mesangial cells) is associated with a number of kidney diseases including end-stage diabetic nephropathy. Cell death by apoptosis is a tightly orchestrated event, whose mechanisms are not completely defined. In the present study we show that the uPA (urokinase-type plasminogen activator)/uPAR (uPA receptor) system can initiate both cell survival and pro-apoptotic signals in human MCs in response to different apoptotic stimuli. uPA abrogated MC apoptosis induced by serum withdrawal conditions and enhanced apoptosis initiated in MCs by high glucose. Effects of uPA were independent of its proteolytic activity and required uPAR for both pro- and anti-apoptotic effects. Studies on the uPAR interactome provide evidence that the opposing effects of uPA were directed via different uPAR-interacting transmembrane partners. Exposure of MCs to RGD (Arg-Gly-Asp) peptide led to abrogation of the anti-apoptotic effect of uPA, which implies involvement of integrins in this process. A pro-apoptotic effect of uPA under high-glucose conditions was mediated via association of uPAR and the cation-independent M6P (mannose-6-phosphate)/IGF2R (insulin-like growth factor 2 receptor). Both receptors were co-precipitated and co-localized in MCs. Studies on the underlying signalling indicate that the ERK1/2 (extracellular-signal-regulated kinase 1/2), Akt and BAD (Bcl-2/Bcl-X(L)-antagonist, causing cell death) protein were involved in regulation of apoptosis by uPA in MCs. M6P/IGF2R mediated BAD perinuclear localization during apoptosis initiated by uPA and high glucose. In conclusion, we provide evidence that, in MCs, the uPA/uPAR system regulates survival/apoptosis processes in a stimulus-specific fashion via a mitochondria-dependent mechanism and that BAD protein serves as a downstream molecule.

Tyk2 mediates effects of urokinase on human vascular smooth muscle cell growth.

The urokinase (uPA)/uPA receptor (uPAR) system plays a role in the response of the vessel wall to injury, presumably by modulating vascular smooth muscle cell (VSMC) functional behaviour. The Jak/Stat signaling pathway has been implicated to mediate the uPA/uPAR-directed cell migration and proliferation in VSMC. We have therefore investigated the underlying molecular mechanisms, which remained not completely understood. In particular, we aimed at identification of the kinase involved in the signaling cascade leading to Stat1 phosphorylation by uPA and its impact on VSMC growth. We performed expression in VSMC of kinase-deficient mutant forms of the Janus kinases Jak1 and Tyk2 and used different cell culture models imitating the response to vascular injury. We provide evidence that Tyk2, but not Jak1, mediates uPA-induced Stat1 phosphorylation and VSMC growth inhibition and suggest a novel function for Tyk2 as an important modulator of the uPA-directed VSMC functional behaviour at the place of injury.

Rosuvastatin regulates vascular smooth muscle cell phenotypic modulation in vascular remodeling: role for the urokinase receptor.

The urokinase (uPA)/urokinase receptor (uPAR) multifunctional system is an important mediator of migration and proliferation of vascular smooth muscle cells (VSMC). However, whether uPA/uPAR-directed mechanisms are involved in the beneficial effects of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors on vascular remodeling remains unexplored. In this study, we have investigated the effect of the hydrophilic statin rosuvastatin on neointimal remodeling, and the role of uPAR. Using an ex vivo organ and in vitro cell culture models we demonstrate that rosuvastatin decreases injury-induced neointima formation and proliferation of medial VSMC in porcine coronary arteries, as well as migration and proliferation of human coronary VSMC. Studies on the underlying mechanisms show that rosuvastatin impairs VSMC transition from their physiological contractile to the pathophysiological synthetic phenotype. These effects are mediated, at least in part, via uPAR, as confirmed by means of rosuvastatin-directed uPAR expression and uPAR silencing in both models. Our findings provide evidence that rosuvastatin modulates VSMC phenotypic changes and subsequently their proliferation and migration, and indicate the important role for uPAR in these processes. This mechanism contributes to the beneficial non-lipid lowering effect of rosuvastatin on negative vascular remodeling.