Non-HLA antibodies targeting angiotensin II Type 1 receptor and endothelin-1 Type A receptors induce endothelial injury via ß2-arrestin link to mTOR pathway.

Functional non-HLA antibodies (antibodies to non-human leukocyte antigens) targeting the G protein-coupled receptors angiotensin II type 1 receptor (AT1R) and endothelin-1 type A receptor (ETAR) are implicated in the pathogenesis of transplant vasculopathy. While ERK signaling (a regulator of cell growth) may represent a general cellular response to agonist stimulation, the molecular link between receptor stimulation and development of vascular obliteration has not been fully established. Here we hypothesize involvement of the versatile adaptor proteins, ß-arrestins, and the major regulator of cell growth, PI3K/mTOR signaling, in impaired endothelial repair. To test this, human microvascular endothelial cells were treated with AT1R/ETAR antibodies isolated from patients with kidney transplant vasculopathy. These antibodies activated both mTOR complexes via AT1R and ETAR in a PI3K-dependent and ERK-independent manner. The mTOR inhibitor, rapamycin, completely abolished activation of mTORC1 and mTORC2 after long-term treatment with receptor antibodies. Imaging studies revealed that ß2- but not ß1-arrestin was recruited to ETAR in response to ET-1 and patient antibodies but not with antibodies isolated from healthy individuals. Silencing of ß2-arrestin by siRNA transfection significantly reduced ERK1/2 and mTORC2 activation. Non-HLA antibodies impaired endothelial repair by AT1R- and ETAR-induced mTORC2 signaling. Thus, we provide evidence that functional AT1R/ETAR antibodies induce ERK1/2 and mTOR signaling involving ß2-arrestin in human microvascular endothelium. Hence, our data may provide a translational rationale for mTOR inhibitors in combination with receptor blockers in patients with non-HLA receptor recognizing antibodies.

Autoantibodies Targeting AT1- and ETA-Receptors Link Endothelial Proliferation and Coagulation via Ets-1 Transcription Factor.

Scleroderma renal crisis (SRC) is an acute life-threatening manifestation of systemic sclerosis (SSc) caused by obliterative vasculopathy and thrombotic microangiopathy. Evidence suggests a pathogenic role of immunoglobulin G (IgG) targeting G-protein coupled receptors (GPCR). We therefore dissected SRC-associated vascular obliteration and investigated the specific effects of patient-derived IgG directed against angiotensin II type 1 (AT1R) and endothelin-1 type A receptors (ETAR) on downstream signaling events and endothelial cell proliferation. SRC-IgG triggered endothelial cell proliferation via activation of the mitogen-activated protein kinase (MAPK) pathway and subsequent activation of the E26 transformation-specific-1 transcription factor (Ets-1). Either AT1R or ETAR receptor inhibitors/shRNA abrogated endothelial proliferation, confirming receptor activation and Ets-1 signaling involvement. Binding of Ets-1 to the tissue factor (TF) promoter exclusively induced TF. In addition, TF inhibition prevented endothelial cell proliferation. Thus, our data revealed a thus far unknown link between SRC-IgG-induced intracellular signaling, endothelial cell proliferation and active coagulation in the context of obliterative vasculopathy and SRC. Patients' autoantibodies and their molecular effectors represent new therapeutic targets to address severe vascular complications in SSc.

Real-time monitoring of hypertrophy in HL-1 cardiomyocytes by impedance measurements reveals different modes of growth.

Hypertrophic growth is a response of the heart to increased mechanical load or physiological stress. Thereby, cardiomyocytes grow in length and/or width to maintain cardiac pump function. Major signaling pathways involved in cardiomyocyte growth and remodeling have been identified during recent years including calcineurin-NFAT and PI3K-Akt signaling. Modulation of these pathways is of certain interest for therapeutic treatment of cardiac hypertrophy. However, quantification and characterization of hypertrophy in response to different stimuli or modulators is difficult. This study aims to test different read-out systems for detection and quantification of differences in hypertrophic growth in response to prohypertrophic stimuli. Real-time impedance measurements allowed the detection of distinct differences in hypertrophic growth in response to endothelin, norepinephrine, phenylephrine or BIO, which were not observable by other methods such as flow cytometry. Endothelin treatment induced a rapid and strong peak in the impedance signal concomitant with a massive reorientation of the actin cytoskeleton. Changes in expression of hypertrophy-associated genes were detected and stabilization of ß-catenin was identified as a common response to all hypertrophic stimuli used in this study. Hypertrophic growth was blocked by the PI3K/mTOR inhibitor PI-103.

Intrinsic Deregulation of Vascular Smooth Muscle and Myofibroblast Differentiation in Mesenchymal Stromal Cells from Patients with Systemic Sclerosis.

INTRODUCTION: Obliterative vasculopathy and fibrosis are hallmarks of systemic sclerosis (SSc), a severe systemic autoimmune disease. Bone marrow-derived mesenchymal stromal cells (MSCs) from SSc patients may harbor disease-specific abnormalities. We hypothesized disturbed vascular smooth muscle cell (VSMC) differentiation with increased propensity towards myofibroblast differentiation in response to SSc-microenvironment defining growth factors and determined responsible mechanisms. METHODS: We studied responses of multipotent MSCs from SSc-patients (SSc-MSCs) and healthy controls (H-MSCs) to long-term exposure to CTGF, b-FGF, PDGF-BB or TGF-ß1. Differentiation towards VSMC and myofibroblast lineages was analyzed on phenotypic, biochemical, and functional levels. Intracellular signaling studies included analysis of TGF-ß receptor regulation, SMAD, AKT, ERK1/2 and autocrine loops. RESULTS: VSMC differentiation towards both, contractile and synthetic VSMC phenotypes in response to CTGF and b-FGF was disturbed in SSc-MSCs. H-MSCs and SSc-MSCs responded equally to PDGF-BB with prototypic fibroblastic differentiation. TGF-ß1 initiated myofibroblast differentiation in both cell types, yet with striking phenotypic and functional differences: In relation to H-MSC-derived myofibroblasts induced by TGF-ß1, those obtained from SSc-MSCs expressed more contractile proteins, migrated towards TGF-ß1, had low proliferative capacity, and secreted higher amounts of collagen paralleled by reduced MMP expression. Higher levels of TGF-ß receptor 1 and enhanced canonical and noncanonical TGF-ß signaling in SSc-MSCs accompanied aberrant differentiation response of SSc-MSCs in comparison to H-MSCs. CONCLUSIONS: Deregulated VSMC differentiation with a shift towards myofibroblast differentiation expands the concept of disturbed endogenous regenerative capacity of MSCs from SSc patients. Disease related intrinsic hyperresponsiveness to TGF-ß1 with increased collagen production may represent one responsible mechanism. Better understanding of repair barriers and harnessing beneficial differentiation processes in MSCs could widen options of autologous MSC application in SSc patients.

17ß-Estradiol regulates mTORC2 sensitivity to rapamycin in adaptive cardiac remodeling.

Adaptive cardiac remodeling is characterized by enhanced signaling of mTORC2 downstream kinase Akt. In females, 17ß-estradiol (E2), as well as Akt contribute essentially to sex-related premenopausal cardioprotection. Pharmacologic mTOR targeting with rapamycin is increasingly used for various clinical indications, yet burdened with clinical heterogeneity in therapy responses. The drug inhibits mTORC1 and less-so mTORC2. In male rodents, rapamycin decreases maladaptive cardiac hypertrophy whereas it leads to detrimental dilative cardiomyopathy in females. We hypothesized that mTOR inhibition could interfere with 17ß-estradiol (E2)-mediated sexual dimorphism and adaptive cell growth and tested responses in murine female hearts and cultured female cardiomyocytes. Under physiological in vivo conditions, rapamycin compromised mTORC2 function only in female, but not in male murine hearts. In cultured female cardiomyocytes, rapamycin impaired simultaneously IGF-1 induced activation of both mTOR signaling branches, mTORC1 and mTORC2 only in presence of E2. Use of specific estrogen receptor (ER)α- and ERß-agonists indicated involvement of both estrogen receptors (ER) in rapamycin effects on mTORC1 and mTORC2. Classical feedback mechanisms common in tumour cells with upregulation of PI3K signaling were not involved. E2 effect on Akt-pS473 downregulation by rapamycin was independent of ERK as shown by sequential mTOR and MEK-inhibition. Furthermore, regulatory mTORC2 complex defining component rictor phosphorylation at Ser1235, known to interfere with Akt-substrate binding to mTORC2, was not altered. Functionally, rapamycin significantly reduced trophic effect of E2 on cell size. In addition, cardiomyocytes with reduced Akt-pS473 under rapamycin treatment displayed decreased SERCA2A mRNA and protein expression suggesting negative functional consequences on cardiomyocyte contractility. Rictor silencing confirmed regulation of SERCA2A expression by mTORC2 in E2-cultured female cardiomyocytes. These data highlight a novel modulatory function of E2 on rapamycin effect on mTORC2 in female cardiomyocytes and regulation of SERCA2A expression by mTORC2. Conceivably, rapamycin abrogates the premenopausal "female advantage".

Sex differences in exercise-induced physiological myocardial hypertrophy are modulated by oestrogen receptor beta.

AIMS: Oestrogen receptor alpha (ERα) and beta (ERß) are involved in the regulation of pathological myocardial hypertrophy (MH). We hypothesize that both ER are also involved in physiological MH. Therefore, we investigated the role of ER in exercise-induced physiological MH in loss-of-function models and studied potential mechanisms of action. METHODS AND RESULTS: We performed 1 and 8 weeks of voluntary cage wheel running (VCR) with male and female C57BL/6J wild-type (WT), ERα- and ERß-deleted mice. In line with other studies, female WT mice ran more than males (P ≤ 0.001). After 8 weeks of VCR, both sexes showed an increase in left ventricular mass (females: P ≤ 0.01 and males: P ≤ 0.05) with more pronounced MH in females (P < 0.05). As previously shown, female ERα-deleted mice run less than female WT mice (P ≤ 0.001). ERß-deleted mice showed similar running performance as WT mice (females vs. male: P ≤ 0.001), but did not develop MH. Only female WT mice showed an increase in phosphorylation of serine/threonine kinase (AKT), ERK1/2, p38-mitogen-activated protein kinase (MAPK), and ribosomal protein s6, as well as an increase in the expression of key regulators of mitochondrial function and mitochondrial respiratory chain proteins (complexes I, III, and V) after VCR. However, ERß deletion abolished all observed sex differences. Mitochondrial remodelling occurred in female WT-VCR mice, but not in female ERß-deleted mice. CONCLUSION: The sex-specific response of the heart to exercise is modulated by ERß. The greater increase in physiological MH in females is mediated by induction of AKT signalling, MAPK pathways, protein synthesis, and mitochondrial adaptation via ERß.

Sex-specific mTOR signaling determines sexual dimorphism in myocardial adaptation in normotensive DOCA-salt model.

The deoxycorticosterone acetate (DOCA)-salt mouse model exhibits adverse cardiac remodeling in male mice and cardiac protection in female mice, even when blood pressure is normalized. We hypothesized that intact mammalian target of rapamycin (mTOR) signaling is necessary for cardiac protection in females. We first tested sex differences and intracellular signaling after mTOR targeting with rapamycin in wild-type mice. Radio-telemetric blood pressure was maintained at normal for 6 weeks. Rapamycin significantly reduced left ventricular hypertrophy, preserved ejection fraction, inhibited fibrosis, and maintained capillary structure in male mice. Decreased mTORC1 and increased mTORC2 activity were detected in rapamycin-treated male mice compared with vehicle controls. In contrast, female mice developed dilative left ventricular hypertrophy, cardiac fibrosis, and capillary loss similar to DOCA-salt females lacking the estrogen receptor ß (ERß(-/-)) that we described earlier. Because rapamycin downregulated ERß in female mice, we next studied ERß(-/-) normotensive DOCA-salt females. Vehicle-treated wild-type females maintained their high constitutive mTORC1 and mTORC2 in response to DOCA-salt. In contrast to males, both mTORCs were decreased by rapamycin, in particular mTORC2 by 60%. ERß(-/-) DOCA-salt females showed similar mTORC1 and mTORC2 response patterns. We suggest that ERß-dependent regulation involves sex-specific use of mTOR signaling branches. Maintenance of both mTORC1 and mTORC2 signaling seems to be essential for adaptive cardiac remodeling in females and supports a rationale for sex-specific therapeutic strategies in left ventricular hypertrophy.

Recovery of senescent endothelial cells from injury.

Percutaneous coronary intervention is increasingly performed in elderly patients. Because the procedure is associated with endothelial cell (EC) denudation, we compared recovery of young and old ECs from scratch injuries inflicted in culture. Although senescent ECs displayed markedly reduced potential to proliferate and migrate, they repopulated the wounds as fast as young cells. Morphometric analysis revealed that senescent cells were significantly larger and as a result far fewer senescent cells managed to cover the lesion. Compared with young EC, senescent cells displayed increased expression of senescence-associated ß-galactosidase, nitric oxide synthase (eNOS), and AKT kinase, and secreted increased amounts of growth factors (VEGF, TGF-ß), cytokines (IL-6, IL-8, MCP-1), adhesion molecules (sICAM-1), and matrix proteins (fibronectin). This secretory phenotype rather than the rate of wound closure per se may contribute to unfavorable vascular remodeling in the elderly undergoing coronary catheterization.

Protein kinase C inhibition ameliorates posttransplantation preservation injury in rat renal transplants.

BACKGROUND: Prolonged cold preservation frequently causes delayed renal graft function resulting from tubular epithelial injury. Inhibition of signal transduction downstream from protein kinase C (PKC) may reduce renal ischemia-reperfusion injury and confer renal graft protection. We therefore evaluated the effect of sotrastaurin, a small-molecule inhibitor of Ca²âº-dependent and Ca²âº-independent PKC isoforms, in comparison with mycophenolic acid (MPA) on rat renal transplants with prolonged cold preservation. METHODS: Donor kidneys from male Lewis rats were cold stored in University of Wisconsin solution for 24 hr before syngeneic grafting. Recipients received sotrastaurin (30 mg/kg twice daily), MPA (20 mg/kg/day), or vehicle through gavage starting 1 hr after surgery. Renal function was evaluated by serum creatinine and histology on day 2 (acute injury) and day 7 (repair phase) after transplantation. Postreperfusion inflammation was determined by real-time polymerase chain reaction of proinflammatory genes and histology. Signaling mechanisms were studied by Western blotting and immunohistochemistry. RESULTS: Sotrastaurin enhanced immediate transplant function, attenuated epithelial injury, and accelerated renal function recovery compared with MPA. Despite the stronger anti-inflammatory capacity of MPA, only sotrastaurin treatment achieved significant cellular protection with persisting reduced apoptosis of tubular epithelial cells. Decreased phosphorylation of extracellular signal-regulated protein kinase and p66Shc adaptor protein, both involved in cellular stress and apoptosis, were likely the responsible mechanism of action. CONCLUSIONS: The PKC inhibitor sotrastaurin effectively ameliorated ischemia-reperfusion organ damage and promoted cytoprotection in a clinically relevant model of extended renal cold preservation followed by transplantation. Pharmacologic targeting of PKC may be beneficial for recipients receiving renal transplants at risk for delayed graft function.

Non-HLA-antibodies targeting Angiotensin type 1 receptor and antibody mediated rejection.

Antibody-mediated mechanisms directed against non-HLA related targets may exert negative impact on allograft function and survival. Angiotensin type 1 receptor (AT(1)R) emerges as a functional target for non-HLA allo- and autoantibodies (AT(1)R-Abs) comprising of IgG1 and IgG3 subclasses. Proof of concept for pathophysiologic relevance of AT(1)R-Abs in antibody mediated rejection (AMR) in renal transplants was provided by passive transfer studies in animal model and therapeutic rescue of patients. Although AT(1)R-Abs may belong to complement fixing IgG subclasses, C4d positivity in renal transplant biopsies was not frequently detected implicating complement independent mechanisms of injury. AT(1)R-Abs exert direct effects on endothelial and vascular smooth muscle cells by induction of Erk1/2 signaling and increased DNA binding of transcription factors associated with pro-inflammatory and pro-coagulatory responses. Establishment of enzyme-linked immunosorbent assay employing extracts of cells overexpressing AT(1)R in its native conformation was instrumental for recent studies in independent cohorts. Assessing the AT(1)R-Ab-status along with the HLA-antibodies may help to identify patients at particular risk for irreversible acute or chronic allograft injuries and improve overall outcomes. This review summarizes the current state of research in AT(1)R biology, development in diagnostic strategies, discusses recent clinical studies, and provides perspectives on further refinements in understanding AT(1)R-Ab-actions.

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.

Estrogen receptor-beta signals left ventricular hypertrophy sex differences in normotensive deoxycorticosterone acetate-salt mice.

We found earlier that deoxycorticosterone acetate-salt treatment causes blood pressure-independent left ventricular hypertrophy, but only in male mice. To test the hypothesis that the estrogen receptor-ß (ERß) protects the females from left ventricular hypertrophy, we treated male and female ERß-deficient (ERß(-/-)) mice and their male and female littermates (wild-type [WT]) with deoxycorticosterone acetate-salt and made them telemetrically normotensive with hydralazine. WT males had increased (+16%) heart weight/tibia length ratios compared with WT females (+7%) at 6 weeks. In ERß(-/-) mice, this situation was reversed. Female WT mice had the greatest heart weight/tibia length ratio increases of all of the groups (+23%), even greater than ERß(-/-) males (+10%). Echocardiography revealed concentric left ventricular hypertrophy in male WT mice, whereas ERß(-/-) females developed dilative left ventricular hypertrophy. The hypertrophic response in female ERß(-/-) mice was accompanied by the highest degree of collagen deposition, indicating maladaptive remodeling. ERß(+/+) females showed robust protective p38 and extracellular signal-regulated kinase 1/2 signaling relationships compared with other groups. Calcineurin Aß expression and its positive regulator myocyte-enriched calcineurin-interacting protein 1 were increased in deoxycorticosterone acetate-salt female ERß(-/-) mice, yet lower than in WT males. Endothelin increased murine cardiomyocyte hypertrophy in vitro, which could be blocked by estradiol and an ERß agonist. We conclude that a functional ERß is essential for inducing adaptive p38 and extracellular signal-regulated kinase signaling, while reducing maladaptive calcineurin signaling in normotensive deoxycorticosterone acetate female mice. Our findings address the possibility of sex-specific cardiovascular therapies.

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.

mTOR regulates vascular smooth muscle cell differentiation from human bone marrow-derived mesenchymal progenitors.

OBJECTIVE: Vascular smooth muscle cells (VSMCs) and circulating mesenchymal progenitor cells (MSCs) with a VSMC phenotype contribute to neointima formation and lumen loss after angioplasty and during allograft arteriosclerosis. We hypothesized that phosphoinositol-Akt-mammalian target of rapamycin-p70S6 kinase (PI3K/Akt/mTOR/p70S6K) pathway activation regulates VSMC differentiation from MSCs. METHODS AND RESULTS: We studied effects of PI3K/Akt/mTOR signaling on phenotypic modulation of MSC and VSMC marker expression, including L-type Ca(2+) channels. Phosphorylation of Akt and p70S6K featured downregulation of VSMC markers in dedifferentiated MSCs. mTOR inhibition with rapamycin at below pharmacological concentrations blocked p70S6K phosphorylation and induced a differentiated contractile phenotype with smooth muscle (sm)-calponin, sm-alpha-actin, and SM protein 22-alpha (SM22alpha) expression. The PI3K inhibitor Ly294002 abolished Akt and p70S6K phosphorylation and reversed the dedifferentiated phenotype via induction of sm-calponin, sm-alpha-actin, SM22alpha, and myosin light chain kinase. Rapamycin acted antiproliferative without impairing MSC viability. In VSMCs, rapamycin increased a homing chemokine for MSCs, stromal cell-derived factor-1-alpha, at mRNA and protein levels. The CXCR4-mediated MSC migration toward conditioned medium of rapamycin-treated VSMCs was enhanced. CONCLUSIONS: We describe novel pleiotropic effects of rapamycin at very low concentrations that stabilized differentiated contractile VSMCs from MSCs in addition to exerting antiproliferative and enhanced homing effects.

The CAIRP (CAndesartan for In-stent Restenosis Prevention) Trial--a multicenter study of AT1-receptor blocker therapy in coronary stenting.

Angiotensin II (Ang II) is implicated in the development of in-stent restenosis (ISR). Ang II- and AT1-receptor blockade could possibly reduce ISR. We enrolled 206 patients into a prospective double-blind, placebo-controlled, multicenter randomized trial of candesartan cilexitil 16 mg to test this notion. Mean lumen diameter (MLD) was the primary objective measured by quantitative coronary angiography and intravascular ultrasound. The Candesartan Group showed a trend towards a larger MLD at follow up without significant differences in the binary ISR rate. In vessels < 2.75 mm, we found a larger MLD in the treatment group after 6 months. This might indicate the potential benefit of AT1-receptor blocker therapy for certain subgroups when percutaneous coronary intervention is performed with bare-metal stent implantation.

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.

Monocyte-expressed urokinase inhibits vascular smooth muscle cell growth by activating Stat1.

After vascular injury, a remodeling process occurs that features leukocyte migration and infiltration. Loss of endothelial integrity allows the leukocytes to interact with vascular smooth muscle cells (VSMCs) and to elicit "marching orders"; however, the signaling processes are poorly understood. We found that human monocytes inhibit VSMC proliferation and induce a migratory potential. The monocytes signal the VSMCs through the urokinase-type plasminogen activator (uPA). The VSMC uPA receptor (uPAR) receives the signal and activates the transcription factor Stat1 that, in turn, mediates the antiproliferative effects. These results provide the first evidence that monocytes signal VSMCs by mechanisms involving the fibrinolytic system, and they imply an important link between the uPA/uPAR-related signaling machinery and human vascular disease.