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Recent studies indicate that regulatory T cells (Tregs) attenuate murine atherosclerosis. Since interleukin (IL)-2 induces Tregs proliferation, we tested the impact of L19-IL2, a fusion antibody specific to extra-domain B of fibronectin (ED-B) containing an active human IL-2 molecule, in experimental atherosclerosis.L19-IL2 or appropriate controls were given intravenously to 6 month old Western diet-fed apoE(-/-) mice on day 1, 3, and 5. Human IL-2 was detected on day 7 within atherosclerotic plaques of L19-IL2-treated mice, and magnetic resonance imaging of the plaques showed a significant adventitial gadolinium enhancement on day 7 and 13, suggesting microvascular leakage as a result of the pharmacodynamic activity of L19-IL2. Treatment with L19-IL2 significantly reduced the size of pre-established atherosclerotic plaques at the thoracic aorta (Sudan III stained area) and in the aortic root area (microscopic, morphometric analysis) on day 7 as compared to controls (L19, D1.3-IL2, NaCl) as well as compared to baseline (day 0). Tregs markers Foxp3 and CTLA4 were highly increased in plaques after L19-IL2 treatment compared to controls (p0.01), whereas the macrophage marker Mac3 was significantly reduced (p0.03). Co-treatment with IL-2-receptor blocking antibody PC61 abrogated L19-IL2-induced plaque reduction compared with IgG control (p0.03).L19-IL2 delivers functional IL-2 to pre-established atherosclerotic plaques of WD-fed apoE(-/-) mice resulting in significant plaque size reduction mediated by local Tregs.

Vascular smooth muscle cell (VSMC) invasion is a key element in atherogenesis and restenosis, requiring integrins for adhesion/de-adhesion as well as matrix metalloproteinases (MMPs) for focalized proteolysis. Among the MMP family, pro-MMP-2 is unique in its activation, depending on the formation of a multiprotein complex with MT1-MMP/TIMP-2 at the cell surface, in which integrin alphavbeta3 participates. Integrin alphav and MT1-MMP are synthesized from precursors via furin-dependent cleavage of their pro-peptide. Furin is the prototypical proprotein convertase highly expressed in VSMCs and human atherosclerotic lesions. Its precise role in the tight network involving MMPs/integrins and their coordination and cooperation required for VSMC invasion is unknown. We demonstrate that furin-inhibition with decanoyl-RVKR-chloromethylketone inhibits VSMC invasion in a comparable degree to MMP inhibitors, which reduce the MT1-MMP-MMP-2 proteolytic cascade. Furin-inhibition did not prevent MT1-MMP/MMP-2 maturation. In contrast, it strongly reduced pro-alphav cleavage, but did not lessen its cell membrane expression. However, inhibition of pro-alphav processing via furin-inhibition strongly reduced pro-MMP-2 binding to the cell surface, thereby lessening its full maturation and diminishing the cell surface in situ proteolysis required for invasion. Thus, our data demonstrate a novel mechanism of furin-dependent alphav cleavage that enhances pro-MMP-2 binding and activation at the cell membrane in cooperation with MT1-MMP in primary VSMCs. Processing of alphav by furin contributes to the recruitment of enzymatic energy to the cell surface, thereby providing focalized proteolysis associated with VSMC invasion.

It has been shown that ED-B fibronectin (ED-B) is a potential target for plaque imaging. The aim of this study was to test a novel modified single chain anti-ED-B antibody (scFv) conjugated for near infrared fluorescence imaging (NIRF) with tetrasulfonated carbocyanine-maleimide (TSC-scFv) and to examine the association of ED-B with the presence of macrophages in a murine model of atherosclerosis. Expression of ED-B was observed in plaque areas in apolipoprotein E-deficient (apoE(-/-)) mice which increased with age and plaque load. Robust imaging was possible after explantation of the aorta and demonstrated a strong NIRF signal intensity in focal aortic and brachiocephalic plaque lesions, whereas no signals were found in undiseased areas. Plaque lesion ED-B was expressed by smooth muscle cell and was closely associated to macrophage infiltrates. Although not expressed by the same cell type, there was a significant correlation (p0.01) between ED-B and macrophage immunoreactivity. In vitro human coronary and mouse smooth muscle cells significantly increased ED-B expression after angiotensin II and TNF-alpha treatment. This study demonstrates that plaque NIRF imaging is feasible with a novel single chain antibody and that ED-B expression is closely associated with inflammation in experimental atherosclerosis.

Target temperature management (TTM) after cardiac arrest (CA) improves outcome in patients with acute coronary syndrome (ACS). Previous data point to an interaction between hypothermia and drug metabolism, potentially impacting on platelet function in patients on antiplatelet therapy.To compare clopidogrel metabolism and platelet function in clopidogrel naïve ACS patients treated with TTM (33°C, n=15) and in ACS patients (troponin positive) without TTM (n=18).Platelet function was measured by multiple electrode platelet aggregometry (MEA), light transmittance aggregometry (LTA) and VASP analysis before and after administration of a 600mg clopidogrel loading dose. Plasma levels of clopidogrel and its metabolites were measured. All patients were screened for CYP2C19*2 polymorphism and scheduled for PCI. TTM was carried out for 24h at a target temperature of 33°C using a computer feedback surface cooling device in cardiac arrest patients.Plasma concentration of clopidogrel and metabolites was lower in the TTM group after 2 and 4h, respectively (all p0.005 vs. controls), and platelet function tests revealed an attenuated response to clopidogrel with respect to baseline platelet activity in the TTM group. This was significant for MEA, LTA and VASP analysis (all p0.05). Moreover, there was no significant difference in genotype and platelet function determined ex vivo at 33 or 37°C, respectively.Inhibition of platelet function is significantly lessened in TTM at 33°C, likely due to reduced clopidogrel absorption. Patients with TTM might thus have a higher risk for further cardiovascular events despite antiplatelet therapy with clopidogrel.

Serum concentrations of soluble L-selectin by far exceed those of other soluble adhesion molecules, and serum soluble L-selectin concentrations are remarkably stable upon prolonged storage. We present evidence for Ca(2+)-dependent binding interactions between human serum amyloid P (SAP), a proteolysis-resistant pentraxin glycoprotein, and L-selectin, as shown by surface plasmon resonance measurements, protein band shift assays in a native PAGE system, and after SDS-PAGE and membrane transfer. Monoclonal antibodies to L-selectin strongly reduced binding of biotinylated SAP to L-selectin-IgG chimeras immobilized on microtiter plates. As binding was reduced by prior glycopeptidase F treatment of L-selectin but not of SAP, it appears to be based on SAP lectin domain interactions with N-linked L-selectin carbohydrates. In freshly prepared human lymphocytes, SAP incubation induced expression of a beta2 integrin neoepitope associated with high-affinity binding. This was partially blocked by pre-incubation with Fab fragments of two anti-L-selectin antibodies. In flow chamber experiments, SAP inhibited the adherence of human neutrophils to activated endothelium under shear stress. Thus, SAP binds to human L-selectin and affects L-selectin-dependent leukocyte-endothelial interactions.

Objective: Angiotensin II (AII) promotes cardiac fibrosis by increased extracellular matrix production and enhanced interaction of matrix proteins with their cellular receptors, including integrins. AII and other growth factors augment h1-integrin-dependent adhesion and spreading by binside-out signalingQ without affecting the total number of integrin receptors. bInside-out signalingQ involves specific signaling pathways, including protein kinase C (PKC), leading to activation of h1-integrins. In the present study we investigated the mechanisms involved in AII-increased adhesion of adult rat cardiac fibroblasts (CFBs), obtained from Sprague–Dawley rats, to collagen I mediated by h1-integrin. Methods and results: Treatment of CFBs with AII induced a concentration-dependent increase in adhesion to collagen I (2.2-fold, pb0.01) within 3–6 h of treatment. This effect was mediated by h1-integrin via the angiotensin AT1 receptor and was significantly reduced by protein kinase C inhibition. AII significantly induced phosphorylation of PKC epsilon (PKCq), which is involved in h1-integrin-dependent adhesion and motility, and phosphorylation of the cytoplasmatic tail of h1-integrin at threonine 788/789, required for adhesion. Phosphorylation of h1integrin and an increase in adhesion was also induced by l-a-phosphatidylinositol-3,4,5-triphosphate (l-a-PIP3), an activator of endogeneous PKCq. AII failed to increase adhesion in myofibroblasts obtained from PKCq (/) mice, but not in cells obtained from control mice. Coimmunoprecipitation and double immunofluorescence demonstrated that AII induced a close association of PKCq with h1-integrin in CFBs. Conclusion: The present study demonstrates that AII increased h1-integrin-dependent adhesion to collagen I in cardiac fibroblasts by insideout signaling via PKCq and phosphorylation of the cytoplasmatic tail of the h1-integrin. D 2005 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.

Integrins are heterodimeric alpha/beta receptors that link the cytoskeleton with the extracellular matrix, thereby regulating several cell functions important in atherosclerosis. In vitro, the subtilisin/kexin-like proprotein convertases (PCs), namely PC5 and furin, have been shown to be responsible for the endoproteolytic activation of the alpha(v) integrin subunit. Based on their cleavage activity, these PCs are potential targets in atherosclerosis. In the present study, we investigated the localization of furin and PC5 in different stages of human atherosclerosis. Immunohistochemical analysis of furin and PC5 revealed their presence in vascular smooth-muscle cells and endothelial cells in atherosclerotic and non-atherosclerotic lesions. However, in the more advanced lesions, furin and PC5 staining was significantly expressed in macrophages/foam cells. In vitro, THP-1 derived macrophages contained furin and PC5, and maturation of monocytes to macrophages was accompanied by enhanced alpha(v)beta3 cell-surface expression. Inhibition of furin/PC5 with the specific pharmacological furin-like PC-inhibitor dec-CMK inhibited alpha(v) endoproteolytic activation but did not abolish alpha(v)beta3 cell-surface expression. This indicates that furin/PC5 is required for alpha(v) endoproteolytic activation but not for alpha(v) routing and sorting to the cell surface. In conclusion, our study demonstrates that furin and PC5 are significantly expressed in mononuclear cells in advanced human atherosclerotic lesions, where they regulate alpha(v) endoproteolytic activation.

The IGF-1 receptor (IGF-1R) and MT1-MMP are synthesized as larger precursor proproteins, which require endoproteolytic activation by the proprotein convertases (PCs) furin/PC5 to gain full biological activity. The aim of this study was to investigate the contribution of PCs to IGF-1R and/or MT1-MMP activation in vascular smooth muscle cells (VSMCs) as well as VSMC proliferation/migration, which are key elements in vascular remodeling. Furin and PC5 mRNAs and proteins were found in VSMCs. Inhibition of furin-like PCs with the specific pharmacological inhibitor dec-CMK inhibited IGF-1R endoproteolytic activation. Inhibition of IGF-1R maturation abrogated IGF-induced IGF-1R autophosphorylation, PI3-kinase and MAPK induction, as well as VSMC proliferation (p

Background— Integrins play an important role for vascular smooth muscle cell (VSMC) migration during the development of atherosclerosis and restenosis. Integrin α v -subunit consists of disulphide-bound 125-kDa heavy and 25-kDa light chains, which are generated by endoproteolytic cleavage. This type of activation requires the presence of suitable proprotein convertases (PCs). Based on ex vivo and in vitro data, the PC5 isozyme has been suggested to be the major integrin convertase. We have recently demonstrated that PC5 is upregulated during vascular remodeling in rodents, colocalizing with α v in VSMCs. The aim of this study was to investigate the activation of α v by PCs in VSMCs and its consequences for α v -dependent cell functions. Methods and Results— Immunoblotting demonstrated that inhibition of PC activity by the specific pharmacological inhibitor dec-CMK inhibits α v cleavage in VSMCs. These results were confirmed using PC5-specific antisense oligonucleotides. PC5-antisense oligonucleotides and dec-CMK inhibited VSMC adhesion to the α v β 3 /β 5 ligand vitronectin (both P P v cleavage inhibited the adhesion-dependent focal adhesion kinase Y397 -autophosphorylation and subsequent Akt activation, whereas phosphorylation of extracellular signal-regulated kinase 1/2 was not affected. In human endarterectomy lesions, PC5 colocalized with α v integrin in VSMCs in the atherosclerotic plaques. Conclusions— The present study demonstrates that α v endoproteolytic activation is necessary for integrin-mediated adhesion and migration as well as signaling and requires PC5 in VSMCs. The colocalization of PC5 and α v in human carotid plaques indicates that PC5 might play a key role for α v activation in vivo.

Activation of MAPK pathways by angiotensin II (Ang II) is important for cardiac fibroblast (CFB) proliferation and migration. Activity of MAP-kinases is closely controlled by a group of dual-specific MAP kinase phosphatases (MKPs). Lipopolysaccharides (LPS) and cytokines are elevated in patients with heart failure and may contribute to disease progression. In this study, we investigate the effect of LPS on Ang II-induced CFB function. Pretreatment of CFBs with LPS (1 microg/mL; 30 min) almost completely inhibited Ang II-induced DNA-synthesis and inhibited Ang II directed chemotaxis by more than 80%. Compared to controls, LPS pretreatment significantly reduced phosphorylation levels of ERK1/2- and p38 MAPK and induced MKP-1 levels. Silencing MKP-1 with antisense oligodesoxynucleotides reversed the antimitogenic effect of LPS on Ang II-induced CFB DNA-synthesis and migration. Induction of MKP-1 by LPS was inhibited by the protein kinase C (PKC)-inhibitor calphostin C, but not by the ERK1/2-pathway inhibitor PD98059, suggesting that PKC but not ERK1/2 is required for LPS-mediated MKP-1 induction in CFBs. Our data demonstrate that LPS have direct cellular effects in CFBs through an inhibition of Ang II-induced MAPK activity via PKC-mediated induction of MKP-1. This might be relevant with regard to the decreased MAPK activity and increased levels in MKPs reported during chronic heart failure in humans.

The present study examined non-insulin-treated streptozotocin (STZ)-induced diabetic rats to determine the role of kinins in diabetic nephropathy. Their involvement in the renoprotective effect of the angiotensin-converting enzyme inhibitor (ACEI) ramipril was investigated using the bradykinin (BK) B 2 -receptor antagonist, icatibant (HOE 140), or a combination of the two drugs. Although, none of the treatments prevented the decline of the glomerular filtration rate (GFR) in diabetic rats, ramipril (3 mg/kg/day), but not icatibant (HOE 140; 500 μg/kg/day), prevented proteinuria in these animals. However, the antiproteinuric effect of ramipril was reduced by 45% when combined with icatibant. To explore whether the renal kallikrein–kinin system (KKS) belongs to the underlying mechanisms of these findings, we also determined urinary BK levels, renal kallikrein (KLK) and angiotensin-converting enzyme (ACE) activity as well as renal cortical mRNA levels of neutral endopeptidase 24.11 (NEP) and low-molecular weight (LMW) kininogen. STZ led to a reduction of renal KLK and ACE activity and NEP expression and to a three-fold increase of urinary BK excretion and renal kininogen expression. Icatibant given alone had no effect on these parameters. In contrast, ramipril treatment normalized urinary protein and BK excretion as well as kininogen mRNA expression without affecting NEP mRNA expression or KLK and ACE activity. Our data demonstrate that renal BK is increased in severe STZ-induced diabetes mellitus, but may affect glomerular regulation only to a minor degree under this condition. However, kinins are partly involved in the antiproteinuric action of ACEI at this stage of diabetic nephropathy.

Integrin alphav is involved in intracellular-extracellular signaling important for cytoskeleton alterations and control of cell movement. In vitro experiments indicate that the integrin alphav-subunit undergoes post-translational endoproteolytic cleavage. This type of activation requires the presence of suitable kexin/subtilisin-like proprotein convertases. In vitro experiments have demonstrated that, among several proprotein convertases, PC5A, and to a threefold lesser extent furin, can activate alphav integrin. The biological significance of these in vitro data would be further supported by a coexpression and coordinated regulation of the gene expression of alphav integrin and its activating enzyme PC5 in vivo. In the present study we investigated the regulation of alphav integrin and PC5 following balloon injury in vivo. Comparative immunocytochemistry revealed a coordinated regulation of alphav integrin and PC5 during vascular remodeling in rodents. Integrin alphav was found to be upregulated in PCNA-positive, proliferating vascular smooth muscle cells. Northern blots revealed no significant regulation of furin mRNA, whereas PC5A mRNA increased during vascular remodeling, suggesting that PC5 is the major convertase during neointima formation in vivo. Incubation of vascular smooth muscle cells with the Golgi-disturbing agent brefeldin A inhibited alphav integrin maturation, indicating that endoproteolytic cleavage occurs in the trans-Golgi network, were PC5 is localized. Thus, the present study further supports the concept that activation of alphav integrin occurs in the trans-Golgi network in vascular smooth muscle cells and involves PC5.

We have recently demonstrated that furin, PC5, and PC7, members of the subtilisin/kexin-like mammalian proprotein convertases (PCs), are found in rodent aorta. These PCs have been identified to activate several growth factors, adhesion molecules and extracellular matrix compounds by endoproteolytic cleavage. In the present study, we investigated the regulation of PC5 in vascular smooth muscle cells (VSMCs) in vitro and in vivo. Stimulation of rat aortic VSMCs with platelet-derived growth factor (PDGF)-BB (20 ng/mL), angiotensin II (Ang II, 1 μmol/L), or 10% fetal calf serum (FCS) for 48 hours increased DNA synthesis, as assessed by proliferating cell nuclear antigen (PCNA) immunoblotting. PC5 was strongly upregulated by PDGF-BB and 10% FCS (both 8-fold, P s6 -kinase (both P s6 -kinase dependent. Thus, growth factors regulate the proprotein convertase PC5, which may play an important role during VSMC growth.

The aim of this study was to investigate whether transformation of quiescent vascular smooth muscle cells (VSMCs) into proliferating secretory cells is accompanied by an expression of processing enzymes that activate de novo-synthesized growth factors. Three enzymes belonging to the family of the kexin/subtilisin-like mammalian proprotein convertases (PCs), furin, PC5, and PC7, were found to be upregulated after balloon denudation in vivo. To determine their importance in these cell processes, we investigated their gene regulation using a short-term organ culture system. After incubation of rat aorta for 4 and 24 hr in serum-free medium, we demonstrated a significant induction of VSMC proliferation. The affected subset of VSMCs, positive for α-smooth muscle actin, also expressed proliferating cell nuclear antigen (PCNA). Our results revealed a parallel upregulation of furin, PC5, and PC7 in PCNA-immunolabeled cells. As a substrate model for comparison with PCs we used nerve growth factor (NGF). NGF is known to be activated by PCs. As shown by Northern blotting analysis, NGF mRNA concentration was significantly increased in cultured explants. NGF was released into the culture medium. In conclusion, both PCs and NGF are coordinately modulated on induction of VSMC proliferation.

Neointima formation involves tissue expression of matrix proteins and growth factors. The role of αvβ3, but not αvβ5 integrin in vascular cells has been sufficiently investigated. The aim of the present study was to determine and compare the function of αvβ3 and αvβ5 integrins in rat aortic (RASMC) and human coronary vascular smooth muscle cells (HCSMC) and to characterize their expression accompanying neointima formation in vivo. RASMC and HCSMC express αvβ3 and αvβ5 integrin subunits. The αvβ5 integrin predominantly mediated adhesion of RASMCs to vitronectin and spreading on vitronectin via RGD-binding sequences. In contrast, the αvβ3 integrin did not contribute to the adhesion and spreading on fibronectin, vitronectin, gelatin or collagen I coated layers. PDGF-directed migration through gelatin coated membranes involved both αvβ3 and αvβ5 integrins. Selective blocking antibodies for αvβ3 and αvβ5 inhibited migration of RASMC and HCSMC by more than 60% (p < 0.01). Integrin expression was studied in vivo in thoracic aorta of Sprague Dawley rats before and after balloon injury. In situ hybridization demonstrated low signals for αvβ3 and β5 mRNA in uninjuried aorta, which increased significantly at 14 days, localized predominantly in the neointima. Northern analysis of aorta after 14 days of injury also demonstrated an upregulation of αvβ3 and β5 mRNA compared to uninjured aorta. Consistent with the increase in message levels, increased inegrin protein expression was seen in the neointima after 7 and 14 days. This study provides evidence that αvβ3 and αvβ5 are elevated during neointima formation in the rat and indicates a novel role for αvβ5 participating in mechanisms regulating smooth muscle cell migration.

CD40-CD154-mediated cell signalling is impor tant for T-cell function under various physiological and pathophysiological conditions. It has been re cently described to play a role in cellular functions of macrophages (MACs) and vascular smooth-muscle cells (VSMCs) in cardiovascular diseases. In the present study, the expression of CD40 receptor protein and its function was studied in VSMCs ob tained from human coronary arteries. In addition, we studied the expression and distribution of CD40 in atherosclerotic lesions of 43 patients. VSMCs isolated from human coronary arteries and aorta treated with interferon-g (INF-γ) demon strated a upregulation of CD40 compared to low basal expression. CD40 expressing VSMCs treated with CD154 (CD40 ligand) resulted in an increas ed expression of inflammatory proteins, such as vascular-cell adhesion molecule-1 (VCAM-1) and Interleukin-8 (Il-8). Serial immunohistological stainings of human iliac arteries from 43 patients were performed (from lesions according to WHO criteria) and CD40 expression and distribution was analyzed. Human atherosclerotic lesions revealed a significant increase of intimal thickness, number of inflammatory infiltrates, and CD40-positive MACs and VSMCs with progression of the lesions. A significant correlation between intimal thickness and CD40-positive MACs and CD40-positive VS-MCs was observed. Ligation of the cellular CD40 receptors on VSMCs contributes to inflammatory cellular events in the vascular wall. The histological data suggest a direct association of CD40 expres sion in atherosclerotic lesions with plaque develop ment and progression of atherosclerosis.

Troglitazone (TRO) is an oral insulin-sensitizer that has direct effects on the vasculature to inhibit cell growth and migration. In vascular smooth muscle cells (VSMCs), insulin transduces a mitogenic signal that is dependent on the ERK1/2 MAP kinases. We examined the effects of TRO on this pathway and found that it inhibits mitogenic signaling. In quiescent VSMCs, insulin (1 microM) induced a 3.2-fold increase in DNA synthesis. TRO (1-20 microM) inhibited insulin-stimulated DNA synthesis by 72.8% at the maximal concentration. TRO at I and 10 microM had no significant effect on insulin-stimulated ERK1/2 activity. At 20 microM, however, TRO modestly enhanced insulin-stimulated ERK1/2 activity by 1.5-fold. ERKs transduce a mitogenic signal by phosphorylating transcription factors such as Elk-1. which regulate critical growth-response genes. We used GAL-Elk-1 expression plasmids to detect ERK-dependent activation of Elk-1. TRO at 1-20 microM potently inhibited insulin-stimulated, ERK1/2-dependent Elk-1 transcription factor activity. Neither early steps in insulin signaling nor the phosphatidylinositol 3-kinase (PI3K) branch of this pathway were affected by TRO, because it had no effect on IRS-1 phosphorylation, PI3K/IRS-1 association, or Akt phosphorylation. Because TRO is a known ligand for the nuclear transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma), we tested two other ligands for this receptor, rosiglitazone (RSG) and 15-deoxy-delta12,14 prostaglandin J2 (15d-PGJ2). Both also inhibited insulin-induced DNA synthesis. In summary, these data show that TRO inhibits mitogenic signaling by insulin at a point distal of ERK1/2 activation, potentially by a PPARgamma-mediated inhibition of ERK-dependent phosphorylation and activation of nuclear transcription factors that regulate cell growth.

Abstract —We recently demonstrated that α v β 3 integrins are involved in the mechanisms of angiotensin II (Ang II)–induced DNA synthesis and collagen gel contractions in rat cardiac fibroblasts (CFBs), cellular mechanisms that are relevant for cardiac remodeling. The aim of the present study was to elucidate the effect of Ang II and other growth factors on the regulation of the α v β 3 integrins in fibroblasts from neonatal rat hearts. The α v β 3 integrin receptor expression was significantly increased ( P 1 (TGF-β 1 ), and platelet-derived growth factor (PDGF) for 8 and 16 hours. The surface expression of the α v and β 3 integrin subunits was elevated after 32 and 48 hours ( P v β 3 integrin (both P v β 3 integrin but was mainly mediated by an α v β 5 integrin. Relevant in vivo expression of α v β 3 integrin by CFBs was confirmed with in situ hybridization with probes for α v and β 3 mRNA in rat hearts. The present study demonstrates that the expression of α v β 3 integrin is augmented by Ang II, PDGF, and TGF-β 1 in neonatal CFBs. Furthermore, this integrin is involved in the chemotaxis, motility, and adhesion of CFBs. The present findings support the current concept that integrins participate in the control of fibroblast behavior during cardiac remodeling mechanisms.

Glycoprotein IIb/IIIa blockade by abciximab and tirofiban, non-peptidergic inhibitors, leads to sustained clinical benefits in the treatment of acute coronary syndromes. The purpose of this study was to clarify the functional effects of abciximab and tirofiban on vascular vitronectin receptors, αvβ3- and αvβ5-integrins. Integrin expression and 7E3 binding in human umbilical venous endothelial cells, human umbilical venous smooth muscle cells, and human iliac arterial smooth muscle cells were observed in the following intensity: αvβ3 — human umbilical venous endothelial cells>human umbilical venous smooth muscle cells>human iliac arterial smooth muscle cells/αvβ5 — human iliac arterial smooth muscle cells>human umbilical venous smooth muscle cells>human umbilical venous endothelial cells. 7E3 binding correlated with αvβ3-expression in all cell types. Integrin-mediated cell functions were analysed with adhesion and spreading assays on vitronectin. In human umbilical venous endothelial cells, these functions were mediated by αvβ3 and in human iliac arterial smooth muscle cells by αvβ5. In human umbilical venous smooth muscle cells, both vitronectin receptors were involved. Abciximab potently inhibited αvβ3-mediated cell adhesion and spreading. With tirofiban, no significant inhibition of vascular cell functions was observed. The present data demonstrate that vitronectin–cell interactions in vascular cells are mediated via two distinct integrin-receptors, αvβ3 and αvβ5. Abciximab, which solely inhibits αvβ3-mediated cell functions, may be particularly effective in human endothelium and in β3-integrin expressing vascular smooth muscle cells.

Insulin-stimulated DNA synthesis, MAP kinase (MAPK) activity and c-fos expression in vascular smooth muscle cells (VSMCs) was blocked by the MAPK inhibitor PD 98059. Regulation of c-fos expression by the transcription factor Elk-1 at the serum response element (SRE) is dependent on its phosphorylation by MAPK. PD 98059 also suppressed insulin-induced Elk-1 transcriptional activity through the SRE. These data show that MAPK plays a critical role in both insulin-mediated growth and Elk-1-dependent induction of c-fos in VSMCs.

Abstract Clinical data suggest a link between the activation of the renin-angiotensin system and cardiovascular ischemic events. Leukocyte accumulation in the vessel wall is a hallmark of early atherosclerosis and plaque progression. E-Selectin, vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) are adhesion molecules participating in mediating interactions between leukocytes and endothelial cells and have been found to be expressed in atherosclerotic plaques. We investigated whether angiotensin II, the effector of the renin-angiotensin system, influences the endothelial expression of E-selectin, VCAM-1, and ICAM-1. In coronary endothelial cells derived from explanted human hearts, angiotensin II (10 −11 to 10 −5 mol/L) induced a concentration-dependent increase in E-selectin expression. The effect was measured by cell ELISA and duplex reverse-transcription polymerase chain reaction (RT-PCR) and reached its maximum at 10 −7 mol/L. Angiotensin II induced only a small increase in E-selectin expression in cardiac microvascular endothelial cells. VCAM-1 and ICAM-1 were not affected by angiotensin II stimulation. In addition, the effect of angiotensin II–induced E-selectin expression on leukocyte adhesion was quantified under flow conditions. Angiotensin II (10 −7 mol/L) increased leukocyte adhesion significantly to 67% of the maximal effect by tumor necrosis factor-α at a wall shear stress of 2 dyne/cm 2 . This adhesion was found to be E-selectin dependent, as demonstrated by blocking antibodies. The AT 1 -receptor antagonist DUP 753 significantly reduced E-selectin–dependent adhesion, whereas the AT 2 -receptor antagonist PD 123177 had no inhibitory effect. In addition, only AT 1 -receptor, but not AT 2 -receptor, mRNA could be detected by RT-PCR in coronary endothelial cells. Therefore, it is suggested that AT 1 receptors mediate the effects of angiotensin II on E-selectin expression and leukocyte adhesion on coronary endothelial cells.

The expression level of angiotensin II (ANG II) type 1 receptors (AT1) determines the magnitude of ANG II signaling in vascular smooth muscle cells (VSMC). AT1 mRNA expression in cultured bovine VSMC increased twofold after 8 h of protein kinase C (PKC) activation with phorbol 12-myristate 13-acetate (PMA), whereas stimulation with forskolin did not alter the AT1 mRNA level. The expression of AT1 promoter/exon 1 [-513/+92 base pairs (bp)] luciferase constructs transfected into VSMC increased 2.4-fold with PMA stimulation. In-gel kinase assays demonstrated rapid phosphorylation of mitogen-activating protein kinases (MAPK) ERK1 and ERK2 by PMA. Electrophoretic gel mobility shift assays showed sequence-specific binding of nuclear proteins from PMA-activated VSMC, identified as activator protein 1 (AP-1) complex in competition assays, to a radiolabeled AT1-promoter fragment (-368/-399 bp). Recombinant AP-1 binds in a sequence-specific manner to the -386/-399-bp region. Site-specific mutagenesis destroying the AP-1 site, the adjacent polyoma enhancer activator 3 element, or both sites simultaneously indicated that both sites together are necessary and sufficient to control basal and PMA-induced activation of the human AT1 promoter in transfected VSMC. The capability of the phorbol ester PMA to activate the human AT1 promoter in VSMC via an AP-1 element suggests a prominent role for PKC/MAPK and Ets proteins in AT1 regulation.

Vascular smooth muscle cell (VSMC) proliferation and mi- gration are responses to arterial injury that are highly im- portant to the processes of restenosis and atherosclerosis. In the arterial balloon injury model in the rat, platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) are induced in the vessel wall and regulate these VSMC activities. Novel insulin sensitizing agents, thiazo- lidinediones, have been demonstrated to inhibit insulin and epidermal growth factor-induced growth of VSMCs. We hypothesized that these agents might also inhibit the effect of PDGF and bFGF on cultured VSMCs and intimal hyper- plasia in vivo. Troglitazone (1 m M), a member of the thiazo- lidinedione class, produced a near complete inhibition of both bFGF-induced DNA synthesis as measured by bro- modeoxyuridine incorporation (6.5 6 3.9 vs. 17.6 6 4.3% cells labeled, P , 0.05) and c-fos induction. This effect was asso- ciated with an inhibition (by 73 6 4%, P , 0.01) by troglita- zone of the transactivation of the serum response element, which regulates c-fos expression. Inhibition of c-fos induc- tion by troglitazone appeared to occur via a blockade of the MAP kinase pathway at a point downstream of MAP kinase activation by MAP kinase kinase. At this dose, troglitazone also inhibited PDGF-BB-directed migration of VSMC (by 70 6 6%, P , 0.01). These in vitro effects were operative in vivo. Quantitative image analysis revealed that troglita- zone-treated rats had 62% ( P , 0.001) less neointima/media area ratio 14 d after balloon injury of the aorta compared with injured rats that received no troglitazone. These results suggest troglitazone is a potent inhibitor of VSMC prolifera- tion and migration and, thus, may be a useful agent to pre- vent restenosis and possibly atherosclerosis. ( J. Clin. Invest. 1996. 98:1897-1905.) Key words: restenosisplatelet- derived growth factorbasic fibroblast growth factorc-fos • MAP kinase

The degradation of bradykinin by angiotensin-converting-enzyme (ACE) activity in cultured human endothelial cells was studied by direct measurement of bradykinin and by its effect on the release of endothelium-derived relaxing factors. The half-life of exogenous bradykinin (10,000 pg/ml) was calculated from the decay of the bradykinin concentration as 46 +/- 2 min in cell monolayers, 133 +/- 15 min in conditioned medium, and 24 +/- 2 min in homogenates. Most of the bradykinin-degrading activity in cell monolayers could be inhibited in a concentration-dependent manner by the ACE inhibitors lisinopril, ramiprilat, and captopril. Bradykinin-degrading activity was released into the culture medium containing one-fourth of the bradykinin-degrading activity found in the presence of cell monolayers. In cell homogenates higher unspecific bradykinin-degrading activities were present. The functional consequence of bradykinin degradation was demonstrated by the potentiating effect of ramiprilat on the generation of endothelium-derived relaxing factors nitric oxide and prostacyclin from endothelial cells. The study supports the concept of increased vasodilatory effects of bradykinin during ACE inhibition.

The study was designed to evaluate the effects of chronic inhibition of angiotensin-converting enzyme (ACE) on the reactivity of the endothelium and the smooth muscle to vasoconstrictor and vasodilator stimuli in normal rats. Male rats were treated orally for 6 weeks with enalapril (10 mg/kg/day, n = 10) or with placebo (n = 10). Endothelium-dependent relaxations to acetylcholine and adenosine diphosphate were augmented in aortic rings from rats treated with enalapril compared with controls, whereas the response to the endothelium-independent vasodilator SIN-1 were similar. Contractions induced by phenylephrine and angiotensin II remained unchanged. Thus, the results suggest that chronic ACE inhibition enhances the release of relaxing factors from the endothelium in normotensive rats.

The interaction of angiotensin-converting enzyme (ACE) inhibitors and bradykinin was investigated in isolated bovine and human coronary arteries. Rings with and without endothelium were mounted in organ chambers for measurement of isometric force. The effects of the ACE inhibitors lisinopril, enalaprilat, fosinoprilat, ramiprilat, and captopril were determined during submaximal stimulation with bradykinin or other vasodilators. Lisinopril and captopril alone did not affect vascular tone; however, in rings with endothelium partially relaxed with bradykinin (or = 10(-10) M), all ACE inhibitors caused further relaxations. Lisinopril did not affect bradykinin concentrations in the incubation medium. Mechanical removal of the endothelium or incubation with nitro-L-arginine or the bradykinin2-receptor antagonist Hoe 140 prevented the relaxations to bradykinin and lisinopril. Other vasodilators including acetylcholine, adenosine diphosphate, substance P, or SIN-1 did not prime the rings to respond to ACE inhibitors. Endothelium-dependent relaxations to lisinopril were also observed in human coronary arteries treated with bradykinin (or = 10(-7) M). Thus, ACE inhibitors potentiate endothelium-dependent relaxations to submaximal concentrations of bradykinin in bovine and human coronary arteries. This local mechanism occurs regardless of elevated bradykinin concentrations in the blood and reduced angiotensin II generation.

High field magnetic resonance imaging (MRI) was performed to investigate the long-term effect of ezetimibe (eze), a cholesterol resorption blocker, on atherosclerotic lesion formation in the thoracic aorta of apolipoprotein E-deficient mice (apoE ( -/- )) in comparison to wild type mice (WT). Fifteen-month-old apoE ( -/- ) (Western type diet), apoE ( -/-eze ) (Western type diet with eze) which received eze (5 mc/kg/day) continuously, and age-matched WT (normal chow) were studied using contrast-enhanced 3D turbo-spin-echo sequences (RARE factor 2) on a 7 Tesla scanner. Vessel parameters were analyzed in the aortic root (AR) and aortic arch (AA) and compared to those found in histology. Plasma cholesterol levels were reduced at 15 months by 71% (P0.01) in apoE ( -/-eze ) compared to apoE ( -/- ). Vessel wall thickness was increased in the AR and AA in apoE ( -/- ) by 189.1 and 147.2%, respectively compared to WT. ApoE ( -/-eze ) showed reduced wall thickness in the AR (127.4%) and AA (102.8%, both P0.05 vs. apoE ( -/- )). A significant increase in total aortic vessel area was determined in the AR and AA in apoE ( -/- ) by 134.7 and 118.3%, respectively, compared to WT. This effect was inhibited in apoE ( -/-eze ) (AR: 126.7%, AA: 86.4%, both P0.05). Histological analysis confirmed the effect of eze observed by MRI and demonstrated a significant correlation between the two techniques (P0.001). MRI demonstrates that ezetimibe significantly reduces atherosclerotic disease in apoE ( -/- ). MRI is therefore a useful technique to perform in vivo interventional studies in experimental atherosclerosis.

Ezetimibe (EZE), an inhibitor of cholesterol absorption, reduces atherosclerosis in apolipoprotein E–deficient (apo –/– ) mice. The matrix protein ED-B fibronectin (ED-B) is upregulated in atherosclerotic lesions. Using a novel conjugate for near-infrared fluorescence (NIRF) imaging targeting ED-B, we studied the effect of EZE on plaque lesion formation in apoE –/– mice. ApoE –/– mice received EZE (5 μg/kg/d) or chow up to the age of 4, 6, and 8 months. NIRF imaging of aortic lesions was performed 24 hours after intravenous application ex vivo and in vivo. Plaque lesion formation was analyzed by histology and immunohistochemistry. Aortic lesion formation detected by Sudan staining and NIRF imaging was significantly reduced at 6 and 8 months ( p < .001). Plaque areas determined by NIRF imaging significantly correlated with Sudan staining ( p < .001). EZE treatment resulted in a significant reduction in plaque macrophage and ED-B immunoreactivity (both p < .05) in brachiocephalic lesions. There was a significant reduction in plaque size in brachiocephalic arteries in 8-month-old mice treated with EZE compared with mice during short-term treatment ( p < .05), indicating EZE plaque regression. Targeted NIRF imaging showed a correlation to histologic lesion extension during therapeutical intervention in experimental atherosclerosis.

Objective: Insulin resistance and hyperinsulinemia are major causes of cardiovascular morbidity and mortality. Matrix metalloproteinases (MMPs), highly expressed in activated mononuclear cells in vulnerable atherosclerotic lesions, are the main proteolytic enzymes controlling plaque stability. The aim of this study was to investigate the regulation of monocyte MMP-9 by insulin. Methods and results: Stimulation of MMP-9 expression by insulin was time- and concentration-dependent in human monocytic THP-1 cells. Inhibition of insulin receptor (IR) maturation via inhibition of its activating convertase furin with the pharmacological furin-inhibitor decanoyl-RVKR-chloromethylketone, as well as blocking of IGF-1R function with a IGF-1R blocking antibody, demonstrated that insulin mediates increases in MMP-9 via IR activation. Inhibition of insulin's “metabolic” phosphatidylinositol 3-kinase signaling with wortmannin (50 nmol/L) or LY294002 (2.5 μmol/L) did not prevent insulin-dependent MMP-9 induction. In contrast inhibition of insulin's “mitogenic” Ras-Raf-mitogen-activated protein kinase–kinase pathways with PD98059 (15 μmol/L) or U0126 (2 μmol/L) inhibited insulin-induced MMP-9 gelatinolytic activity in THP-1 cells. Likewise, PD98059 inhibited insulin augmented MMP-9 levels in primary human monocytes, whereas wortmannin had no effect. Conclusion: This study demonstrates that insulin can induce MMP-9 via mitogenic signaling pathways in monocytes, whereas phosphatidylinositol 3-kinase-dependent signaling, typically altered in insulin resistance, is not required. Induction of MMP-9 by insulin may potentially contribute to a pro-inflammatory state and the increased cardiovascular morbidity and mortality in type 2 diabetics.

Angiotensin II (AII) plays a crucial role in controlling the proliferation and migration of vascular smooth muscle cells (VSMCs). The present study was undertaken to determine if troglitazone (Tro) has an effect on the G-protein coupled signaling through AII type I (AT-1) receptors in cultured rat aortic VSMCs. AII-induced MAP kinase activation was inhibited 67.9% by Tro. AII-induced DNA synthesis and migration was completely inhibited by Tro or by the AT-1 receptor blocker irbesartan. The present study demonstrates that troglitazone inhibits AII-induced DNA synthesis, migration and MAP kinase activation in VSMCs which are important molecular events for the development of neointimal hyperplasia and atherosclerosis.

Background— Accumulation of macrophages and their in situ expression of matrix metalloproteinases (MMPs) are important determinants of plaque stability. Activation of membrane-bound MT1-MMP, the major activator of pro-MMP-2, requires intracellular endoproteolytic cleavage of its precursor protein. This type of activation typically requires suitable furin-like proprotein convertases (PCs), specifically furin and PC5. The present study was done to investigate the function of MT1-MMP as well as furin-like PCs in mononuclear inflammatory cells. Methods and Results— Macrophage differentiation of human monocytic THP-1 cells was accompanied by increased expression of furin, PC5, and MT1-MMP. Some pro-MMP-2 activation was found in macrophages, but pro-MMP-2 level or activation was not enhanced after stimulation with the proinflammatory mediators tumor necrosis factor-α or lipopolysaccharide. However, culturing of macrophages in conditioned medium from serum-starved vascular smooth muscle cells, which constitutively secrete pro-MMP-2, resulted in a strong pro-MMP-2 activation. Inhibition of furin-like PCs with the specific pharmacological inhibitor decanoyl-RVKR-chloromethylketone (dec-CMK) inhibited MT1-MMP activation in macrophages. Dec-CMK or furin-specific small interfering RNA significantly inhibited macrophage MT1-MMP–dependent activation of vascular smooth muscle cell–derived pro-MMP-2. Flow cytometry demonstrated that human circulating monocytes express furin and PC5, and MT1-MMP and immunohistochemistry revealed their colocalization in macrophages in advanced human atherosclerotic lesions. Conclusions— Furin-like PCs (furin and PC5) play a central role in a MT-MMP–MMP-2 proteolytic cascade, involving provision of macrophage MT1-MMP for the activation of pro-MMP-2 synthesized by other cells. Furin and PC5 are expressed in human peripheral blood mononuclear cells and colocalize with MT1-MMP in macrophages in the atherosclerotic plaque, supporting the hypothesis that they are potential targets in atherosclerosis.

Background—Accumulating evidence suggests that C-reactive protein (CRP), in addition to being a predictor of coronary events, may have direct actions on the vessel wall in the evolution of atherosclerosis. Although accumulation of vascular smooth muscle cells (VSMCs) in the intima is a key event in the development of arterial lesions, apoptosis of VSMCs also plays an important role in progression of atherosclerotic lesions and contributes to increased plaque vulnerability.Methods and Results—In the present study we demonstrate that CRP induces caspase-mediated apoptosis of human coronary VSMCs. DNA microarray analysis was used to identify CRP-regulated genes. The growth arrest– and DNA damage–inducible gene 153 (GADD153) mRNA expression was prominently upregulated by CRP. As confirmed by Northern blot analysis, CRP induced a time- and dose-dependent increase of GADD153 mRNA expression. GADD153, a gene involved in growth arrest and apoptosis in vascular and nonvascular cells, is regulated at both transcriptional and posttranscriptional levels. CRP regulation of GADD153 mRNA expression in VSMCs occurs primarily at the posttranscriptional level by mRNA stabilization. Small interfering RNA (siRNA) specifically targeted to GADD153 reduced CRP-induced apoptosis. GADD153 also specifically colocalized to apoptotic VSMCs in human coronary lesions, further supporting a functional role for GADD153 in CRP-induced cell death.Conclusions—These results demonstrate that GADD153 is a CRP-regulated gene in human VSMCs and plays a causal role in CRP-induced apoptosis. Pharmacological targeting of CRP expression or action may provide a novel therapy for atherosclerosis.

Objective : Heart failure is characterized by an imbalance of matrix synthesis/turnover, finally resulting in fibrosis. Cardiac myocytes and fibroblasts play a pivotal role in the remodeling process. Cardiac remodeling involves the expression of TGF-β1 and matrix metalloproteinases (MMPs) in cardiac fibroblasts (CFBs). Furin, a subtilisin/kexin-like proprotein convertase (PC), activates TGF-β1 and membrane-bound MT1-MMP, which facilitates pro-gelatinase A (MMP-2) activation. Even though several reports identified TGF-β1 as a pro-fibrotic cytokine in the heart, it increases MMP-activity and cell migration/invasion in several cell types. The present study was done to investigate the contribution of TGF-β1 and furin to CFBs MMP-activity and motility. Methods and results : Stimulation of CFBs from adult Sprague–Dawley rats with TGF-β1 (20 ng/ml) induced furin, but had no effect on the closely related PC5. Inhibition of furin inhibited angiotensin II-induced TGF-β1 activation, indicating that TGF-β1 amplifies its activating convertase in CFBs. Pretreatment of CFBs with TGF-β1 (20 ng/ml, 24 h) increased their migration by about two-fold ( p

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors have been reported to inhibit vascular smooth muscle cell growth, a key event in the pathogenesis of proliferative vascular diseases. The mechanism by which HMG-CoA reductase inhibitors exert their antiproliferative activity is not fully understood, especially their effect on DNA replication. We therefore investigated the effects of atorvastatin on minichromosome maintenance (MCM) protein 6 and 7 expression in vascular smooth muscle cells, two proteins essential for initiation of DNA replication. Stimulation of quiescent rat aortic vascular smooth muscle cells with fetal bovine serum induced MCM6 and MCM7 protein and mRNA expression, which was potently attenuated by atorvastatin in a dose-dependent fashion. Mevalonate completely abrogated the inhibitory effect on serum-induced MCM6 and MCM7 expression, demonstrating that biosynthesis of isoprenoids was likely the specific pathway blocked by atorvastatin. Transient transfection experiments revealed that atorvastatin inhibited MCM6 and MCM7 promoter activity, implicating a transcriptional mechanism. The MCM6 and MCM7 promoters contain several E2F sites critical for their transcriptional activation. Activity of a luciferase reporter plasmid containing four E2F elements was also strongly inhibited by atorvastatin. The inhibitory effect of atorvastatin on MCM6 and MCM7 was reversed by adenoviral-mediated overexpression of E2F, indicating that their downregulation by atorvastatin involves an E2F-dependent mechanism. These findings demonstrate that MCM proteins play an essential role during the proliferation of vascular smooth muscle cells and may provide a novel therapeutic target for proliferative vascular diseases. Inhibition of MCM6 and MCM7 expression by blocking E2F function may contribute importantly to the inhibition of vascular smooth muscle cell DNA synthesis by atorvastatin.

Integrins play an important role in vascular smooth muscle cell (VSMC) migration, a crucial event in the development of restenosis and atherosclerosis. Transforming growth factor-β (TGF-β) is highly expressed in restenotic and atherosclerotic lesions, and known to induce integrin expression. Peroxisome proliferator-activated receptor α (PPARα), a member of the nuclear receptor superfamily, regulates gene expression in a variety of vascular cells. We investigated the effects of PPARα ligands on TGF-β–induced β 3 and β 5 integrin expression and potential interaction between PPARα and TGF-β signaling. PPARα ligands WY-14643 (100 μmol/L) and 5,8,11,14-eicosatetranoic acid (ETYA, 50 μmol/L) inhibited TGF-β–induced β 5 integrin protein expression by 72±6.8% and 73±7.1%, respectively (both P 3 integrin expression was not affected by PPARα ligands. Both PPARα ligands also suppressed TGF-β–induced β 5 integrin mRNA levels. PPARα ligands inhibited TGF-β–inducible transcription of β 5 integrin by an interaction with a TGF-β response element between nucleotides −63 and −44, which contains a Sp1/Sp3 transcription factor binding site. Nuclear complexes binding to the TGF-β response region contained Sp1/Sp3 and TGF-β–regulated Smad 2, 3, and 4 transcription factors. TGF-β–stimulated Sp1/Smad4 nuclear complex formation was inhibited by WY-14643 and ETYA with a parallel induction of PPARα/Smad4 interactions. However, in vitro pull-down experiments failed to demonstrate direct binding between PPARα/Smad4. Both PPARα ligands blocked PDGF-directed migration of TGF-β–pretreated VSMCs, a process mediated, in part, by β 5 integrins. The present study demonstrates that PPARα activators inhibit TGF-β–induced β 5 integrin transcription in VSMCs through a novel indirect interaction between ligand-activated PPARα and the TGF-β–regulated Smad4 transcription factors. The full text of this article is available at http://www.circresaha.org.

Activation of the local and systemic renin-angiotensin system is directly and indirectly involved in mechanisms of vascular remodeling during chronic hypertension. This study investigated the effect of angiotensin II (AII) on rat vascular smooth muscle cell (VSMC) migration towards platelet-derived growth factor-BB (PDGF-BB) in vitro. Pre-treatment with AII (1 microM) for 48 or 72 h induced a significant increase in PDGF-BB-directed migration by 77 +/- 21 % and 58 +/- 24 %, respectively (both p0.01). This effect was concentration dependent and inhibited by the selective angiotensin receptor type I (AT(1)) blocker DUP 753. PDGF-directed migration of VSMCs was significantly inhibited by antibodies against beta(3)-and beta(5)-integrins, indicating an important role of these integrins in VSMC migration. However, AII augmented migration was not accompanied by an increased expression of beta(3)- and beta(5)-integrin mRNA and protein levels in VSMCs. Inhibition of the mitogen-activated protein kinase ERK 1/2 with PD 98059 (30 microM) completely abolished the effect of AII on PDGF-BB-directed VSMC migration (p0.01). The proline-rich tyrosine kinase 2 (Pyk2) and focal adhesion kinase (FAK) are cytoskeleton-associated protein kinases participating in integrin-dependent signaling. Therefore, expression and phosphorylation of these kinases was determined 48 h after AII treatment, revealing a significant increase in Pyk2 and FAK protein levels (up to 2-fold, both p0.05) and increased phosphorylation of Pyk2 (2-fold, p0.05) and ERK 1/2 (4-fold, p0.05) as compared to controls. Furthermore, immunofluorescence and Western blot analysis demonstrated a translocation of Pyk2 from the plasma membrane to the cytosol, as well as a perinuclear enrichment of ERK 1/2 protein 48 h after AII treatment. In conclusion, our data suggest that changes in the levels of Pyk2 and ERK 1/2 phosphorylation, responsible for integrin-dependent signaling, as well as their subcellular translocation are important for the enhanced chemotactic response of VSMCs after AII pre-treatment.

Tumor necrosis factor alpha (TNFalpha) interferes with insulin signaling in adipose tissue and may promote insulin resistance. Insulin resistance is associated with vascular injury, but little is known about the interaction of TNFalpha and insulin in the vasculature. By activating the Insulin receptor (IR) --IRS-1 --phosphatidylinositol-3-kinase (PI3K) --Akt-pathway, insulin protects vascular smooth muscle cells (VSMC) from undergoing apoptosis. We therefore investigated the effect of TNFalpha on insulin's antiapoptotic signaling in rat aortic VSMC. Insulin induced rapid tyrosine-phosphorylation of the IR and IRS-1 and caused a 2.8-fold increase of IRS-1-bound PI3K. TNFalpha had no effect on insulin-induced tyrosine-phosphorylation of IR or IRS-1, but inhibited insulin-stimulated IRS-1/PI3K-association by 84%. Insulin-induced phosphorylation of Akt downstream of PI3K was inhibited by TNFalpha in a similar pattern. We next examined the effect of TNFalpha on insulin's protective actions on H(2)O(2)-induced apoptosis. Insulin alone prevented 72.8% of H(2)O(2)-induced apoptosis, which was significantly inhibited by TNFalpha. TNFalpha alone did not induce apoptosis. In contrast, TNFalpha had no effect on PDGF-induced antiapoptotic signal transduction via Akt. Thus, TNFalpha selectively interferes with insulin's antiapoptotic signaling in VSMC by inhibiting the association of IRS-1/PI3K and the downstream activation of Akt.

CD40-CD154-mediated signaling has recently been described as playing a role in cellular functions involved in atherosclerotic processes. CD40 is expressed in macrophages, lymphocytes, endothelial cells, and vascular smooth muscle cells. However, cross-sectional studies investigating the expression of CD40 in atherosclerotic lesions are lacking. In the present study the expression of CD40 was studied in atherosclerotic lesions from 43 patients classified according to the World Health Organization criteria. Serial immunohistologic stainings of human iliac arteries from 43 patients were performed using monoclonal antibodies. Lesions were classified according to World Health Organization criteria, and CD40 expression was analyzed with regard to cell morphology and cellular markers by 2 independent observers. Human atherosclerotic lesions revealed a significant increase in intimal thickness, number of inflammatory infiltrates, and CD40-positive macrophages and vascular smooth muscle cells with progression of the lesions. This increase was most prominent from stage 0 to stage I. A significant correlation between intimal thickness and CD40-positive macrophages (r = 0.75, p

Abstract —β 1 -Integrins play an important role for adhesion and spreading of human smooth muscle cells. In the present study we examined the influence of angiotensin II and platelet-derived growth factor (PDGF)-BB on β 1 -integrin–dependent functions of human smooth muscle cells obtained from iliac arteries. Treatment of these cells with PDGF-BB (20 ng/mL) and Angiotensin II (1 μmol/L) did not change β 1 -integrin expression up to 48 hours as analyzed by flow cytometry and reverse transcription polymerase chain reaction. β 1 -integrins predominantly mediated adhesion of human smooth muscle cells to collagen I (79.7±4.4%, P P P 1 receptor. The PDGF-BB mediated increase of adhesion was inhibited in the presence of genestein, a tyrosine-kinase inhibitor and by protein kinase C downregulation with phorbol 12-myristate 13-acetate. Spreading of smooth muscle cells also was β 1 -integrin dependent on collagen I and α 5 β 1 -integrin dependent on fibronectin. Angiotensin II and PDGF-BB increased cell spreading on fibronectin up to 276% and 318%, respectively, and on collagen I up to 133% and 138% (all P 1 -integrin, α 5 -integrin on fibronectin, the AT 1 receptor blocker irbesartan, and genestein. The present data demonstrate that angiotensin II and as well PDGF-BB enhance β 1 -integrin–dependent adhesion and spreading of human vascular smooth muscle cells. Furthermore, the experiments with PDGF suggest an involvement of protein kinase C activation leading to these enhanced effects.

Abstract —Angiotensin II (Ang II) plays an important role in cardiac remodeling through stimulation of proliferation and extracellular matrix (ECM) production in cardiac fibroblasts. Integrins are a family of transmembrane receptors that mediate the attachment of cells to ECM. We hypothesized that Ang II regulation of integrins further contributes to its role in cardiac remodeling. We cultured adult rat cardiac fibroblasts with and without Ang II (100 nmol/L) to determine the effects on mRNA and protein levels of integrins, as well as α-actinin and other cytoskeletal proteins that link to integrins at the site of focal adhesions. Ang II was also added in the presence of irbesartan (10 μmol/L), a specific Ang II type 1 (AT 1 ) receptor antagonist, or PD 123319 (10 μmol/L), a specific Ang II type 2 receptor antagonist. To investigate the function of these integrins, we determined the effects of blocking antibodies on Ang II–induced adhesion to ECM. We also treated spontaneously hypertensive rats (SHR) with an AT 1 receptor blocker, losartan, or with hydralazine to investigate integrin and α-actinin expression in treated and untreated SHR. Ang II enhanced α v , β 1 , β 3 , and β 5 integrins; osteopontin; and α-actinin mRNA and protein levels in cardiac fibroblasts. All of these effects were inhibited by irbesartan but not by PD 123319. Pretreatment of cardiac fibroblasts with Ang II enhanced cell attachment to ECM proteins and induced focal adhesion kinase phosphorylation. Blocking antibodies to β 3 and α v β 5 attenuated Ang II–induced adhesion. In SHR, ventricular α v and β 5 integrin expression and α-actinin were increased compared with those in Wistar-Kyoto rats. Although both losartan and hydralazine lowered mean arterial pressure and decreased peripheral vascular resistance, only losartan attenuated the increased integrin, α-actinin, fibronectin laminin, and osteopontin expression and the increased left ventricular mass (as determined with echocardiography). Hydralzine had none of these effects. Although both agents attenuated β-myosin heavy chain expression, a marker of hypertrophy, losartan had a greater effect. These results suggest that integrins and α-actinin are upregulated by Ang II and in left ventricular hypertrophy and that the block of expression of these proteins through inhibition of the AT 1 receptor is associated with attenuation of the hypertrophic response. Ang II induces integrin and α-actinin expression in cardiac fibroblasts that is associated with adhesion and left ventricular hypertrophy and blocked through inhibition of the AT 1 receptor.

Parallel zu den Arbeiten mit kultivierten Fibroblasten stellt sich die Frage, ob Osteopontin auch von anderen kardialen Zellen exprimiert wird. Immunhistochemie in Rattenherzen mit nekrotischen Arealen (Murry et al., 1994) und in syrischen Hamstern mit dilatativer Kardiomyopathie zeigten eine myokardiale Osteopontin-Expression in Assoziation mit Entzundungszellen, am ehesten Makrophagen (Williams et al., 1995). Hinweise, das Kardiomyozyten Osteopontin produzieren, kamen von einer Arbeitsgruppe, die Osteopontin in mikrovaskularen Endothelzellen und Kardiomyozyten fand (Singh et al., 1995). Sie konnten zeigen, das Behandlung mit Glukokortikoiden die Transkription von Osteopontin steigert. Wir konnten zeigen, das die Expression von Osteopontin in Fibroblasten durch Wachstumsfaktoren wie Angiotensin II, TGF-s, die in der Pathophysiologic der kardialen Hypertrophie relevant sind, deutlich gesteigert wurde. Inwieweit Osteopontin auch von Kardiomyozyten exprimiert und reguliert wird, war zu Beginn der folgenden Untersuchungen nicht bekannt. Unser besonderes Interesse galt hierbei Wachstumsfaktoren, die Myozytenhypertrophie von Kardiomyozyten induzieren, wie Noradrenalin (NA), Endothelin-1(ET-l) und Angiotensin II (A II). Diese Faktoren induzieren ANP mRNA Expression, Reexpression von fetalen kontraktilen Proteinen und andere Veranderungen, die mit der Hypertrophie der Kardiomyozyten einhergehen (Sadoshima and Izumo, 1993, Shubeita et al., 1990, Simpson et al., 1982). Im folgenden Abschnitt sind die Ergebnisse zur Untersuchung der Expression und Regulation von Osteopontin in isolierten Kardiomyozyten zusammengefast.

The thiazolidinedione troglitazone inhibits angiotensin II-induced extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase activity in vascular smooth muscle cells. Activation of extracellular signal-regulated kinase 1/2 by angiotensin II is a multistep process involving both its phosphorylation by mitogen-activated protein kinase extracellular signal-regulated kinase kinase in the cytoplasm and a subsequent translocation to the nucleus. The cytoplasmic activation of extracellular signal-regulated kinase 1/2 in vascular smooth muscle cells proceeds through the protein kinase Czeta --> mitogen-activated protein kinase extracellular signal-regulated kinase kinase --> extracellular signal-regulated kinase pathway. Troglitazone did not affect the angiotensin II-induced activation of protein kinase Czeta or its downstream signaling kinases extracellular signal-regulated kinase 1/2 in the cytosol. In contrast, angiotensin II-induced activation of protein kinase Czeta and extracellular signal-regulated kinase 1/2 in the nucleus were both inhibited by troglitazone. Nuclear translocation of extracellular signal-regulated kinase 1/2 induced by angiotensin II was completely blocked by troglitazone. Protein kinase Czeta, however, did not translocate upon angiotensin II stimulation. Troglitazone, therefore, inhibits both angiotensin II-induced nuclear translocation of extracellular signal-regulated kinase 1/2 and the nuclear activity of its upstream signaling kinase protein kinase Czeta. Since extracellular signal-regulated kinase 1/2 nuclear translocation may be a critical signaling step for multiple growth factors that stimulate vascular smooth muscle cells proliferation and migration, troglitazone may provide a new therapeutical approach for the prevention and treatment of atherosclerosis and restenosis.

Large clinical trials have demonstrated profound benefits of aldosterone inhibition by spironolactone in patients with heart failure (impaired left ventricle function

Growth factor-directed migration is a critical component of the wound healing response although little is known about the signaling pathways involved. We examined the effect of inhibiting the mitogen-activated protein kinase (MAPK) pathway on platelet-derived growth factor (PDGF) and fibronectin-induced cell migration of human retinal pigment epithelial (RPE) cells. Using transwell cell-culture chambers, the effect of PDGF-BB (10–50 ng/ml) and fibronectin on components of migration was measured with or without the MAPK pathway inhibitor PD98059 (10–30 μM). MAPK activation of serum-starved cells by PDGF-BB was demonstrated by an immunoprecipitation/kinase assay and by immunohistochemistry using antibody specific for phosphorylated MAPK. PDGF-BB (10 ng/ml) stimulated MAPK activity in RPE (10 min) and its nuclear localization (1 h). PD98059 inhibited the activation of MAPK by PDGF-BB or serum. PDGF-BB stimulated RPE chemokinesis, chemotaxis, and haptotaxis; chemokinesis was additively increased and chemotaxis synergistically increased by the presence of a fibronectin substratum. PD98059 potently inhibited fibronectin-induced haptotaxis and PDGF-BB-induced chemotaxis but inhibited chemokinesis only at higher PDGF-BB (50 ng/ml) concentrations in the presence of fibronectin substratum. These results demonstrate that MAPK is critically involved in multiple components of RPE migrationin vitroand suggest the potential of targeting MAPK to inhibit RPE migrationin vivo.

Background Osteopontin (OP) has been identified in cultured rat cardiac fibroblasts, where it contributes to angiotensin II (AII)-induced remodeling processes; in cultured cardiomyocytes; and in macrophages in cardiac tissues with inflammation. However, the presence of OP has not been reported in histological sections of myocardial tissue. In the present study, we investigated (1) the regulation of OP mRNA expression in cultured rat cardiomyocytes; (2) the localization of OP mRNA in neonatal and adult normal and hypertrophied rat hearts; and (3) the histology of OP expression in myocardial specimens from humans either with myocyte hypertrophy or with no pathological changes. Methods and Results Cultured neonatal cardiomyocytes expressed OP mRNA and were immunoreactive for OP. Endothelin-1 (ET-1) and norepinephrine (NE) increased both OP and atrial natriuretic peptide (ANP) mRNA levels twofold to threefold ( P r 2 =.87, P Conclusions The present study provides the first evidence that cardiomyocytes are a prominent source of OP in vivo and suggests that induction of OP expression is strongly associated with ventricular hypertrophy.

Angiotensin II (AII) is a critical factor in cardiac remodeling which involves hypertrophy, fibroblast proliferation, and extracellular matrix production. However, little is known about the mechanism by which AII accelerates these responses. Osteopontin is an acidic phosphoprotein with RGD (arginine-glycine-aspartate) sequences that are involved in the vascular smooth muscle cell remodeling process. We identified the presence of osteopontin mRNA and protein in cultured rat cardiac fibroblasts and its prominent regulation by AII (10(-11) M). Osteopontin message levels were increased fourfold (P < 0.01) and protein fivefold (P < 0.05) at 24 h after addition of AII (10(-7) M). This response was inhibited by the AT1 receptor blocker, losartan. Osteopontin mRNA levels were increased in hypertrophied ventricles from animals with renovascular hypertension (1.6-fold, P < 0.05) and aortic banding (2.9-fold, P < 0.05). To examine the function of osteopontin, we determined its effects on (a) the ability of cardiac fibroblasts to contract three-dimensional collagen gels and (b) cardiac fibroblast growth. A monoclonal antibody against osteopontin partially blocked AII-induced three-dimensional collagen gel contraction by cardiac fibroblasts (64+/-4 vs. 86+/-5% in the presence of antibody, P < 0.05), while osteopontin itself promoted contraction of the gels by fibroblasts (71+/-5%, P < 0.05 compared with control). Either a monoclonal antibody against beta3 integrin which is a ligand for osteopontin or the RGD peptide blocked both AII and osteopontin-induced collagen gel contraction. Thus, the osteopontin RGD sequence binds to beta3 integrins on the fibroblast to promote fibroblast binding to collagen. All induced a threefold increase in DNA synthesis of cardiac fibroblasts, which was completely blocked by antibodies against osteopontin and beta3 integrin, or by RGD peptide, but not by controls. Thus, All-induced growth of cardiac fibroblasts also requires osteopontin engagement of the beta3 integrin. Taken together, these results provide the first evidence that osteopontin is a potentially important mediator of AII regulation of cardiac fibroblast behavior in the cardiac remodeling process.

Insulin and angiotensin II are additive in stimulating TGF-β1 and matrix mRNAs in mesangial cells. Angiotensin II (Ang II) and insulin are implicated in the mesangial cell hypertrophy and excessive accumulation of mesangial matrix seen in glomerulosclerosis. Therefore, the effects of Ang II with and without insulin on mRNA levels of several important extracellular matrix genes and transforming growth factor beta-1 (TGF-β1) were examined. Ang II alone (1 µM) added to quiescent, murine mesangial cells in serum-free, insulin-free media slightly but not significantly increased TGF-β1, fibronectin, collagen I, collagen IV and laminin message levels. The slight elevations in message expression were reversed by losartan, suggesting that these modest effects are mediated by the AT-1 receptor. Ang II alone also had no significant effects on TGF-β1 and extracellular matrix message levels in quiescent rat mesangial cells. In contrast, significant increases in mRNA for collagen I (6-fold), collagen IV (4-fold), fibronectin 1 (4-fold) and TGF-β1 (2-fold) were seen with insulin alone (10−6 M) in rat mesangial cells, and a dose-response effect could be demonstrated for insulin (10−9 to 10−6 M). Ang II plus insulin further significantly increased collagen I (9-fold), collagen IV (9-fold), fibronectin 1 (5-fold) and TGF-β1 (3-fold) message expression. These effects were partially reversed in the presence of losartan. The Northern analyses were supported by measurements of active and total TGF-β1 activity (pg/ml/5 × 106 cells): 1145 ± 76 and 1960 ± 199, serum free control; 1121 ± 92 and 1932 ± 214, Ang II (10−6 M); 4589 ± 103 (P < 0.001 vs. control) and 11071 ± 1952 (P < 0.01 vs. control), insulin (10−6 M); and 6881 ± 183 (P < 0.001 vs. control) and 16626 ± 1435 (P < 0.01 vs. control), insulin plus Ang II. These results suggest that insulin, itself, significantly increases TGF-β1 and extracellular matrix gene expression in rat mesangial cells. Ang II alone has modest effects, while Ang II and insulin have additive effects. To explain the mechanism of these additive effects, we investigated the action of Ang II on insulin signaling and the effect of insulin on Ang II AT1 receptor mRNA expression. Ang II did not enhance insulin-induced insulin receptor substrate-1 (IRS-1) phosporylation or phosphatidylinositol3 (PI-3) kinase activity, but did enhance insulin-induced mitogen activated protein (MAP) kinase activity. Insulin increased message levels of AT1 receptor by twofold. These results suggest that enhancement of MAP kinase activity and AT1 receptor regulation by insulin may contribute to the additive effects of insulin and Ang II in mesangial cells.

Osteopontin (OP) is a multifunctional cytokine and adhesion protein that contains an RGD (arginin-glycin-aspartate) binding sequence that enables it to interact with several integrins, CD44 variants, and other adhesion receptors. OP receptor binding then directly or indirectly activates intracellular signaling pathways, mediating its effects on cell–matrix and cell–cell interactions. OP is increased in response to pro-inflammatory cytokines and mechanical strain in various cell types,1 and the function of its secreted protein can be altered by proteases, including thrombin.2 Thus, OP can exists as an immobilized matrix molecule (eg, in bone, atherosclerotic plaques, or calcified heart valves) or as soluble cytokine. Cell signaling by OP is predominantly mediated through integrin engagement. Cleavage of OP by thrombin exposes integrin binding sites2 (eg, for α9β1), which are important for OP-mediated adhesion/migration. OP is chemotactic for various cell types, most notably monocytes/macrophages, which are attracted to sites of injury and inflammation. The best-characterized OP-induced signal pathway is the integrin-stimulated FAK-Src-Rho pathway in osteoclasts.1 However, identification and dissection of signal transduction pathways are complicated by the fact that OP potentially binds to several cell surface receptors. Proper organization of the …

An assay system for the determination of the membrane bound IgD (mIgD) and IgM (mIgM) on B lymphocytes was developed by the combination of two new ELISA methods with the results of flow cytometry after labeling with specific antibodies. The mIgD and mIgM of B lymphocytes were prepared by incubating mononuclear cells (MNCs) in Tween 20 containing buffer and repeated freeze/thaw cycles. Optimal results were achieved with 0.2–0.4% Tween 20 and two freezing cycles. With biotinstreptavidin amplification the sensitivity of the ELISA was 30 μU/ml for IgD and 0.5 ng/ml for IgM. In healthy persons 3.5±0.5 mU mIgD were detected on 10 6 IgD + cells and 57.1±5.9 ng mIgM on 10 6 I IgM + cells. The mIgD/mIgM ratio was 0.065±0.005 mU/ng. The developed ELISA systems utilize only commercially available reagents and therefore provide a convenient reproducible tool for determining membrane bound IgD and IgM on B lymphocytes.

Objective: The effect of angiotensin converting enzyme (ACE) inhibitors on vascular tone of isolated coronary arteries was determined in the presence of bradykinin and other vasodilators to elucidate the mechanisms leading to augmented bradykinin effects during ACE inhibition. Methods: Rings of isolated bovine and human coronary arteries were mounted in organ chambers for measurement of isometric force. The effects of lisinopril, enalaprilat, and captopril were investigated in the presence of submaximal concentrations of bradykinin or other vasodilators. Results: ACE inhibitors alone did not affect vascular tone. Threshold concentrations of bradykinin (10−10 M), kallidin (10−9.5 M), and the slowly degraded bradykinin agonists D-Arg(Hyp3)-bradykinin (10−9.5 M) and [Hyp3-Tyr(Me)8]-bradykinin (10−10.5 M) caused partial relaxation of bovine rings with endothelium. Subsequent addition of ACE inhibitors markedly potentiated the relaxations to the kinins. Bradykinin concentrations in the organ bath measured by a specific bradykinin radioimmunoassay remained stable during the addition of lisinopril. Variation of the exposure time to bradykinin (10 to 60 min) did not affect the relaxations to the ACE inhibitor. The relaxations to lisinopril were not observed after either removal of the endothelium or incubation with nitro-1-arginine or the bradykinin-2 receptor antagonist Hoe 140. Other vasodilators including acetylcholine, adenosine diphosphate, substance P, or SIN-1 did not prime the rings to respond to ACE inhibitors. Endothelium dependent relaxation to lisinopril and captopril was also observed in human coronary arteries treated with bradykinin ≥10−7 M), but not in those treated with substance P (10−8 M). Conclusions: ACE inhibitors selectively potentiate endothelium dependent relaxations to submaximal concentrations of bradykinin in bovine and human coronary arteries by a local mechanism. This effect on endothelial cells might occur in addition to augmented bradykinin concentrations in the blood and reduced angiotensin II generation. Cardiovascular Research 1993; 27 :312-317