Laparoscopic repositioning of a dislocated pacemaker into the rectovesical pouch.

Dislocation of the epicardial pacemaker into the peritoneal cavity is an uncommon but potentially life-threatening complication. We report a case of a 74 year old with an abdominally implanted epicardial pacemaker that migrated through the peritoneum to the excavatio rectovesicalis. The laparoscopic approach was chosen because of the increased risks of perioperative morbidity and decreased survival. The generator was implanted into a pocket beneath the anterior rectus sheath and the lead was peritonalized with a running suture. In conclusion, a laparoscopic retrieval is feasible and safe in the treatment of a displaced pacemaker in the rectovesical pouch.

Electromagnetic interference of avalanche transceivers with cardiac pacemakers and implantable cardioverter defibrillators.

BACKGROUND: Avalanche transceivers are essentials tools in locating persons who were buried by an avalanche. In the past few years, avalanche transceivers have become widely available and affordable, but it is largely unknown whether they are a source of electromagnetic interference for implanted cardiac devices. We aimed to determine the potential interaction between avalanche transceivers and pacemakers or implantable cardioverter defibrillators (ICDs). METHODS: One hundred and one patients, 41 with pacemakers and 60 with ICDs, were enrolled (mean age 66 ± 15 years). Four avalanche transceivers (Pieps DSP [Pieps GmbH, Lebring, Austria], Ortovox x1, Ortovox m2, and Ortovox f1 [Otovox Sportartikel GmbH, Taufkirchen, Germany]) were evaluated in transmit as well as in receive mode. Surface electrocardiograms, intracardiac electrograms, and marker channels were continuously recorded and observed by an experienced physician. Electromagnetic interference events were categorized as direct interference with the function of the implanted device itself or as interference with the telemetric communication without compromising device function. RESULTS: Among all patients, there was no interference with the intrinsic function of their pacemakers or ICDs. A total of 120 episodes of telemetry interference occurred in 48% of the patients. Of those episodes, 112 of 404 (28%) were observed in transmit and eight of 404 (2%) in receive mode (P < 0.0001). The digital avalanche transceiver (Pieps DSP) was associated with significantly less telemetry interference (20/202; 10%) than the analog transceiver (Ortovox f1) (39/202; 19%) (P = 0.0108). CONCLUSIONS: Avalanche transceivers are safe for patients with pacemakers and ICDs. Despite the observed telemetry interferences, the intrinsic function of the implanted devices was never compromised.

Performance evaluation of a right atrial automatic capture verification algorithm using two different sensing configurations.

BACKGROUND: This acute data collection study evaluated the performance of a right atrial (RA) automatic capture verification (ACV) algorithm based on evoked response sensing from two electrode configurations during independent unipolar pacing. METHODS: RA automatic threshold tests were conducted. Evoked response signals were simultaneously recorded between the RA(Ring) electrode and an empty pacemaker housing electrode (RA(Ring)-->Can) and the electrically isolated Indifferent header electrode (RA(Ring)-->Ind). The atrial evoked response (AER) and the performance of the ACV algorithm were evaluated off-line using each sensing configuration. An accurate threshold measurement was defined as within 0.2 V of the unipolar threshold measured manually. Threshold tests were designed to fail for small AER (< 0.35 mV) or insufficient signal-to-artifact ratio (SAR < 2). Manual threshold measurements were obtained during RA unipolar and bipolar pacing and compared across device indications. RESULTS: Data were collected from 38 patients with RA bipolar leads from four manufacturers. AER signals were analyzed from 34 patients who were indicated for a pacemaker (five), implantable cardioverter-defibrillator (11), or cardiac resynchronization therapy pacemaker (six) or defibrillator (12). The minimum AER amplitude was larger (P < 0.0001) when recorded from RA(Ring)-->Can (1.6+/-0.9 mV) than from RA(Ring)-->Ind (1.3+/-0.8 mV). The algorithm successfully measured the pacing threshold in 96.8% and 91.0% of tests for RA(Ring)-->Can and RA(Ring)-->Ind, respectively. No statistical difference between the unipolar and bipolar pacing threshold was observed. CONCLUSIONS: The RA(Ring)-->Can AER sensing configuration may provide a means of implementing an independent pacing/sensing method for ACV in the RA. RA bipolar pacing therapy based on measured RA unipolar pacing thresholds may be feasible.

PPARalpha inhibits TGF-beta-induced beta5 integrin transcription in vascular smooth muscle cells by interacting with Smad4.

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-beta (TGF-beta) is highly expressed in restenotic and atherosclerotic lesions, and known to induce integrin expression. Peroxisome proliferator-activated receptor alpha (PPARalpha), a member of the nuclear receptor superfamily, regulates gene expression in a variety of vascular cells. We investigated the effects of PPARalpha ligands on TGF-beta-induced beta3 and beta5 integrin expression and potential interaction between PPARalpha and TGF-beta signaling. PPARalpha ligands WY-14643 (100 micromol/L) and 5,8,11,14-eicosatetranoic acid (ETYA, 50 micromol/L) inhibited TGF-beta-induced beta5 integrin protein expression by 72+/-6.8% and 73+/-7.1%, respectively (both P<0.05). TGF-beta-stimulated beta3 integrin expression was not affected by PPARalpha ligands. Both PPARalpha ligands also suppressed TGF-beta-induced beta5 integrin mRNA levels. PPARalpha ligands inhibited TGF-beta-inducible transcription of beta5 integrin by an interaction with a TGF-beta response element between nucleotides -63 and -44, which contains a Sp1/Sp3 transcription factor binding site. Nuclear complexes binding to the TGF-beta response region contained Sp1/Sp3 and TGF-beta-regulated Smad 2, 3, and 4 transcription factors. TGF-beta-stimulated Sp1/Smad4 nuclear complex formation was inhibited by WY-14643 and ETYA with a parallel induction of PPARalpha/Smad4 interactions. However, in vitro pull-down experiments failed to demonstrate direct binding between PPARalpha/Smad4. Both PPARalpha ligands blocked PDGF-directed migration of TGF-beta-pretreated VSMCs, a process mediated, in part, by beta5 integrins. The present study demonstrates that PPARalpha activators inhibit TGF-beta-induced beta5 integrin transcription in VSMCs through a novel indirect interaction between ligand-activated PPARalpha and the TGF-beta-regulated Smad4 transcription factors. The full text of this article is available at http://www.circresaha.org.

Endoproteolytic activation of alpha(v) integrin by proprotein convertase PC5 is required for vascular smooth muscle cell adhesion to vitronectin and integrin-dependent signaling.

BACKGROUND: Integrins play an important role for vascular smooth muscle cell (VSMC) migration during the development of atherosclerosis and restenosis. Integrin alpha(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 alpha(v) in VSMCs. The aim of this study was to investigate the activation of alpha(v) by PCs in VSMCs and its consequences for alpha(v)-dependent cell functions. METHODS AND RESULTS: Immunoblotting demonstrated that inhibition of PC activity by the specific pharmacological inhibitor dec-CMK inhibits alpha(v) cleavage in VSMCs. These results were confirmed using PC5-specific antisense oligonucleotides. PC5-antisense oligonucleotides and dec-CMK inhibited VSMC adhesion to the alpha(v)beta3/beta5 ligand vitronectin (both P<0.05). Furthermore, PC5-asODNs inhibited VSMC migration on vitronectin-coated wells (P<0.05). Inhibition of PC activity and consequently alpha(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 alpha(v) integrin in VSMCs in the atherosclerotic plaques. CONCLUSIONS: The present study demonstrates that alpha(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 alpha(v) in human carotid plaques indicates that PC5 might play a key role for alpha(v) activation in vivo.

Three-dimensional rotational venography of the coronary sinus tree facilitates left ventricular lead implantation for CRT.

BACKGROUND: Precise knowledge of the coronary sinus (CS) tree anatomy facilitates catheter-based intubation of the CS, target vein and lead selection and reduces the need for fluoroscopy, contrast medium and overall procedure time in cardiac resynchronization therapy (CRT). Three-dimensional rotational angiography (3DRA) provides a new means of multiangle imaging of the CS tree that can be applied preoperatively. PURPOSE AND METHODS: Our study aims to investigate the feasibility of preoperative rotational CS venography and its implications for CRT device implantation procedures. For this purpose, CS visualization was done either conventionally with retrograde CS venography and standard fluoroscopic views (SFV, n = 30), or with antegrade CS imaging via contrast medium injection into the left coronary artery using 3DRA in the venous phase of contrast flow (3DRA, n = 30). RESULTS: 3DRA successfully identified the posterior and left marginal veins in 92% of patients and allowed target vein visualization in 86%. Additional retrograde venography was necessary in four patients (14%). Fluoroscopy time and contrast medium administration for stable CS intubation were lower in the group with 3DRA than in those with SFV (all p < 0.05). The time for CS lead placement after guiding catheter intubation was 8.9 ± 5.5 min in the 3DRA group versus 14.7 ± 7.4 min in SFV patients (p < 0.05). Consequently, total fluoroscopy time (-12.1 min), volume of contrast medium (-29.5 ml) and overall procedure time (-32.8 min) were significantly lower in 3DRA than in SFV patients (p < 0.05). CONCLUSION: 3DRA offers reliable multiangle visualization of the CS anatomy and facilitates successful CRT lead implantation.

Ambulatory respiratory rate trends identify patients at higher risk of worsening heart failure in implantable cardioverter defibrillator and biventricular device recipients: a novel ambulatory parameter to optimize heart failure management.

PURPOSE: Respiratory distress is the primary driver for heart failure (HF) hospitalization. Implantable pacemakers and defibrillators are capable of monitoring respiratory rate (RR) in ambulatory HF patients. We investigated changes in RR prior to HF hospitalizations and its near-term risk stratification power. METHODS: NOTICE-HF was an international multi-center study. Patients were implanted with an implantable cardioverter defibrillator or cardiac resynchronization therapy defibrillator, capable of trending daily maximum, median, and minimum RR (maxRR, medRR, minRR). RR from 120 patients with 9 months of follow-up was analyzed. One-tailed Student's t test was used to compare RR values prior to HF events to baseline defined as 4 weeks prior to the events. A Cox regression model was used to calculate the hazard ratios (HR) for the 30-day HF hospitalization risk based on RR values in the preceding month. RESULTS: Daily maxRR, medRR, and minRR were significantly elevated prior to HF events compared to baseline (ΔmaxRR 1.8 ± 3.0; p = 0.02; ΔmedRR, 2.1 ± 2.8; p = 0.007; ΔminRR, 1.5 ± 2.1, p = 0.008). Risk of experiencing HF events within 30-days was increased if the standard deviation of medRR over the preceding month was above 1.0 br/min (HR = 12.3, 95 % confidence interval (CI) 2.57-59, p = 0.002). The risk remained high after adjusting for clinical variables that differed at enrollment. CONCLUSION: Ambulatory daily respiratory rate trends may be a valuable addition to standard management for HF patients.

Increased myocardial expression of osteopontin in patients with advanced heart failure.

The expression of the adhesion protein osteopontin (OP) is associated with cardiac hypertrophy and is significantly increased after transition to heart failure in experimental animal models. We, therefore, hypothesized that OP could be upregulated in heart failure in humans. In the present study, we investigated the expression of OP in myocardial biopsies obtained from patients with heart failure due to dilated cardiomyopathy (mean LVEF=30.3+/-4.4%, mean+/-S.D., n=10, group A) compared to patients with a normal left-ventricular ejection fraction (mean LVEF=61+/-11.2%, n=9; group B). Myocardial immunoreactivity for OP was examined using two different antibodies against OP. The expression of cardiac myocyte OP was significantly upregulated in group A in comparison to group B (P<0.0001). Both groups also displayed OP immunoreactivity in non-myocytes, including vascular smooth muscle cells and cardiac fibroblasts (P=not significant). Statistical analysis revealed a significant correlation of increased OP immunoreactivity in cardiac myocytes of patients with impaired left ventricular function, assessed by hemodynamic data (LVEF, RVEF, LVESVI, LVEDVI and LVEDP, R=-0.828, -0.671, 0.751, 0.685 and 0.461, respectively; all P<0.05). Furthermore, OP expression correlated with cardiac myocyte hypertrophy (mean diameter 21.0+/-1.8 microm in group A and 16.6+/-2.1 microm in group B; P<0.0001). In conclusion, the present study indicates, that factors and/or mechanisms involved in heart failure in patients with dilated cardiomyopathy, lead to induction of OP expression in humans.

Phrenic nerve stimulation in CRT patients and benefits of electronic lead repositioning: the ERACE trial.

PURPOSE: Despite novel left ventricular (LV) lead technologies, phrenic nerve stimulation (PNS) remains an adverse effect observed in many patients with cardiac resynchronization therapy (CRT). Beyond anatomic repositioning, modern CRT devices allow avoidance of PNS also by software-based adaption of the pacing configuration. The Electronic Repositioning With Acuity and Easytrak Leads study evaluated the incidence of PNS in a CRT population and examined how often LV lead relocation can be avoided by "electronic repositioning" (ER). METHODS: Patients who had an indication for implantation of a first CRT defibrillator with the option of ER were enrolled. Primary endpoint was the efficiency of ER determined by the frequency of PNS with the standard pacing configuration (LV tip to RV coil) avoidable by ER. PNS and pacing parameters were evaluated during implant, predischarge, and first routine follow-up (FU) using four different pacing configurations available by ER. RESULTS: In total, 292 patients were enrolled and provided with a transvenous LV lead (82.2 % male, 65.5 ± 9.2 years old). The majority of the population was in NYHA III (84.2 %) with a LV ejection fraction of 25.3 ± 6.8 % and mean QRS width of 155 ± 27 ms, ischemic cardiomyopathy was present in 43.6 %. Median FU was 116 days. In the standard pacing configuration, PNS was inducible in 19.0/25.6/24.6 % at implant/predischarge/FU, respectively, resulting in 32.2 % of the patients presenting at least once with PNS. The safety margin for the standard pacing configuration between LV and PNS threshold was <1.0 V at 0.5 ms in 5.6/7.0/5.0 % of the patients, corresponding with a total rate of 11.6 % during the FU. In the finally chosen configuration, clinically relevant PNS occurred in 1.0/2.2/1.3 %. The four vector configurations allowed all but 6 of 292 (2 %) patients to be reprogrammed using ER without reoperation. CONCLUSIONS: The incidence of inducible PNS in CRT patients is considerable. In this study, PNS could be avoided in the majority of the patients by means of electronic repositioning. Thus, the use of ER should be considered for CRT patients.

Combined use of three-dimensional rotational angiogram of left atrium and circular multi-electrode ablation catheter for pulmonary vein isolation.

BACKGROUND: A novel circular pulmonary vein ablation catheter (PVAC) has been introduced for pulmonary vein isolation (PVI). Accurate delineation of left atrium-pulmonary vein (LA-PV) anatomy is important for this technique. The aim of this study was to test whether the 3-dimensional rotational angiography (3D RTA) of the left atrium can facilitate PVI using PVAC technique. METHODS: Twenty patients with paroxysmal atrial fibrillation (AF) were enrolled in this study. The 3D RTA was reconstructed and registered with live fluoroscopy in all the patients. AF ablation was performed with a PVAC catheter in the navigation of registered 3D RTA. RESULTS: The 3DRTA image was successfully reconstructed and registered with live fluoroscopy in all patients (100%). The LA-PV anatomy was delineated clearly in all patients. Navigation of the PVAC inside the registered 3D RTA, ensured accurate placement within the atrium to perform ablation, and the PVAC was correctly placed inside the PV ostium to verify the PVI. All the PVs were isolated. Total procedural time was (87.5 ± 12.1) minutes, and fluoroscopy time was (20.1 ± 6.3) minutes. Follow-up after (7.1 ± 1.5) months showed freedom from AF in 70% (14/20) patients. No PV stenosis was observed. CONCLUSIONS: Intraprocedure reconstructed and registered 3D RTA can clearly delineate the LA-PV anatomy in real-time. The results demonstrate the feasibility and reliability of combining use of 3DRA and PVAC in AF ablation procedures.

Chronic CRP-exposure inhibits VEGF-induced endothelial cell migration.

UNLABELLED: C-reactive protein (CRP) is a pluripotent mediator of inflammation and is present at sites of vascular injury and in atherosclerotic lesions. CRP stimulates endothelial cell adhesion molecule expression and monocyte migration, thereby contributing to the development and progression of vascular lesion formation. In addition, chronic exposure to CRP is known to inhibit angiogenesis and endothelial cell (EC) proliferation. AIM: Whether CRP also affects EC migration, however, has yet to be determined. The present study investigates how long-term exposure to CRP interacts with vascular endothelial growth factor (VEGF) -induced EC migration. METHODS AND RESULTS: Using a Transwell chamber migration assay, VEGF (20 ng/mL, 5 h incubation)-induced migration of human umbilical vein EC was significantly inhibited in cells pretreated with CRP (10 microg/mL) for 24 h by more than 75%. EC migration in response to VEGF is known to require activation of the protein kinase B (Akt)/endothelial NO synthase (eNOS)- and the extracellular signal-regulated protein kinase 1/2 (ERK1/2) pathway. We therefore investigated the long-term effects of CRP on these signalling events. Immunoblotting with phosphospecific antibodies revealed rapid and transient activation/phosphorylation of the protein kinase Akt within 20 minutes after stimulation with VEGF, which was inhibited by 86% in EC pretreated with CRP (10 microg/mL, 24 h, p<0.05). Subsequent VEGF-induced phosphorylation of eNOS downstream of Akt was completely inhibited in CRP-treated EC. In contrast, CRP-pretreatment did not affect VEGF-induced phosphorylation of ERK1/2. Interestingly, stimulation of EC with CRP for 16-24 h induced marked expression of the phosphatase and tensin homolog (PTEN), which functions as a negative regulator of phosphatidylinositol 3 kinase (PI3K) -->Akt signalling. CONCLUSION: The observed time course for CRP-mediated PTEN upregulation corresponds to the exposure time needed for inhibition of Akt phosphorylation and migration and may therefore constitute a potential mechanism by which CRP inhibits inducible Akt phosphorylation and EC migration.

Rapid ventricular pacing to optimize rotational angiography in atrial fibrillation ablation.

BACKGROUND: Conventional pulmonary vein (PV) angiography cannot precisely delineate the left atrium (LA)-PV anatomy, which is essential for the ablation of atrial fibrillation (AF). The aim of the study was to test the feasibility of a novel method of rotational angiography for the AF ablation. METHODS AND RESULTS: Forty-one patients were enrolled in this study. CT scanning was performed in all patients before the procedure. Rotational angiography (rotating from right anterior oblique 55 degrees to left anterior oblique 55 degrees ) was performed before AF ablation. Rapid ventricular pacing (RVP, 300 ms) was carried out to reduce cardiac output while contrast medium was injected into the LA via a pigtail catheter. RVP was successfully performed in 36 (87.8%) patients. The ostia of all PVs and the LA appendage were visible in all these 36 cases. There was a good correlation in the PV ostial diameters as assessed by rotational angiography via RVP as compared to CT imaging (r (2) > 0.85). CONCLUSIONS: Rotational angiography by RVP is able to delineate the LA-PV anatomy. There is a good correlation in the PV ostial diameters as assessed by rotational angiography via RVP and CT imaging. Rotational angiography by RVP is feasible during AF ablation.

Clinical evaluation of two different evoked response sensing methods for automatic capture detection in the left ventricle.

BACKGROUND: This acute feasibility study compared two different automatic capture detection methodologies, the reduced coupling capacitor (RCC) and the independent pace/sense (IPS) methods, for the left ventricle (LV). METHODS: LV threshold tests were performed in DDD mode, with LV-only and bi-ventricular (BiV) pacing using an external cardiac resynchronization therapy (CRT) defibrillator. Evoked response (ER) signals from LV leads were recorded using the LV(Tip) (LV(Tip)-->Can) and LV(Ring) (LV(Ring)-->Can) to empty pulse generator (Can) housing sensing vectors to evaluate the two methodologies. Pacing vector, pulse duration, atrioventricular delay, and interventricular delay were varied to assess their effects on ER. The minimum ER amplitude (ER(min)), signal-to-artifact ratio (SAR), and ER amplitude voltage dependence were evaluated. ER(min)>2 mV and SAR(min)>2 define potential automatic LV capture detection for the two methodologies. RESULTS: Data collected from 43 patients (63.7 +/- 11.0 years) were analyzed, including unipolar and bipolar (14/29) LV leads. Neither ER sensing method was affected by changing the pacing vector. The LV(Tip)-->Can ER(min) was significantly decreased at the 1.0-ms pulse duration when compared to 0.4-ms (p < 0.05). During BiV pacing, LV(Tip)-->Can ER(min) increased at negative interventricular delays and decreased at positive interventricular delays relative to simultaneous pacing. LV(Tip)-->Can resulted in fewer patients with sufficient ER characteristics for capture detection, albeit only significantly at the extended pulse duration (79% vs 97%, p < 0.05) and at simultaneous and positive interventricular delays (81% vs 97%, p < 0.05). CONCLUSIONS: Though LV capture detection was feasible using both investigated methods, the RCC method (LV(Tip)-->Can) sensitivity to the evaluated pacing parameters suggests the IPS method (LV(Ring)-->Can) provides a more robust performance.

LPS regulate ERK1/2-dependent signaling in cardiac fibroblasts via PKC-mediated MKP-1 induction.

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.

Proprotein convertase PC5 regulation by PDGF-BB involves PI3-kinase/p70(s6)-kinase activation in vascular smooth muscle cells.

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 micromol/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<0.05), whereas Ang II had no effect on PC5 protein levels compared with controls. The PCs furin and PC7, which display a comparable subcellular localization and cleavage activity, were found in VSMCs, but their levels did not increase following PDGF-BB, Ang II, or FCS stimulation. Time-course analysis revealed a rapid increase in PC5 levels after 30 minutes of PDGF-stimulation of VSMCs. PDGF-stimulated PC5 induction was inhibited by the PI3-kinase inhibitor wortmannin, and by rapamycin, an inhibitor of mTOR/p70(s6)-kinase (both P<0.05). In contrast, the mitogen-activated protein kinase (MAPK)-pathway inhibitor PD98059 did not inhibit PDGF-stimulated PC5 induction. Immunocytochemistry and in situ hybridization revealed low PC5 protein and mRNA levels in intact rat aorta in vivo. After balloon injury, PC5 protein and mRNA levels were strongly increased in proliferating PCNA-positive VSMCs. The present data demonstrate that PC5 is upregulated during proliferation of VSMCs in vivo and in vitro. We show that PDGF-induced PC5 expression is PI3-kinase/p70(s6)-kinase dependent. Thus, growth factors regulate the proprotein convertase PC5, which may play an important role during VSMC growth.

Coordinated regulation and colocalization of alphav integrin and its activating enzyme proprotein convertase PC5 in vivo.

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.

TGF-beta(1) induces peroxisome proliferator-activated receptor gamma1 and gamma2 expression in human THP-1 monocytes.

The nuclear hormone receptor peroxisome proliferator-activated gamma (PPARgamma) is expressed as two isoforms (PPARgamma1 and gamma2), and is an important modulator of monocyte gene regulation and function. TGF-beta(1) is an essential and potent immune modulator and we therefore examined its effect on PPARgamma expression in human THP-1 monocytes. TGF-beta(1) strongly induced PPARgamma2 mRNA and protein expression with a lesser effect on PPARgamma1. Transcription from a PPARgamma2 promoter/luciferase reporter vector was activated approximately 3-fold by TGF-beta(1). Mutation of two C/EBP elements within the PPARgamma2 promoter reduced TGF-beta(1)-induced transcription by approximately 65%. TGF-beta(1) also induced the expression of three C/EBPisoforms (alpha, beta, and delta). Induction of PPARgamma1 and gamma2 in monocytes by TGF-beta(1) may contribute to the anti-inflammatory effects of this growth factor.

Hypoxia activates beta(1)-integrin via ERK 1/2 and p38 MAP kinase in human vascular smooth muscle cells.

Hypoxia plays an important role in vascular remodeling and directly affects vascular smooth muscle cell (VSMC) functions. VSMC adhesion participates in changes of vascular structure; however, little is known about VSMC adhesion under hypoxic conditions. It was the aim of the present study to investigate the effects of hypoxia on adhesion mechanisms in human VSMCs. Compared to normoxic cells, hypoxia (1% O(2), 24h) significantly increased adhesion of VSMCs to collagen I by 30.2% and fibronectin by 58.0%. This effect was completely inhibited in the presence of the pharmacological ERK 1/2 mitogen-activated protein kinase (MAPK) pathway inhibitor PD98059 (30 microM) or the p38 MAPK inhibitor SB203580 (1 microM). Basal adhesion of normoxic cells was not affected by pretreatment with PD98059 and SB203580. Hypoxia induced a time-dependent activation of ERK 1/2 and p38 MAPK activation in human VSMCs, which were completely abolished by PD98059 or SB203580, respectively. Since adhesion of VSMCs to fibronectin and collagen I involves beta(1)-integrin receptors, we used a blocking antibody against beta(1)-integrin (P5D2) to examine potential effects of hypoxia on beta(1)-integrins. P5D2 significantly reduced VSMC adhesion to fibronectin and collagen I in normoxia and hypoxia in a comparable manner; however, beta(1)-integrin protein or mRNA levels were not affected by hypoxia. As evidenced by flow cytometry, hypoxia induced a activation of beta(1)-integrins by exposing an conformationally sensitive epitope on the beta(1)-subunit. These results demonstrate that hypoxia enhances adhesion of VSMC on extracellular matrix proteins by activating beta(1)-integrin.

Angiotensin II-augmented migration of VSMCs towards PDGF-BB involves Pyk2 and ERK 1/2 activation.

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 p < 0.01). 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 (p < 0.01). 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 p < 0.05) and increased phosphorylation of Pyk2 (2-fold, p < 0.05) and ERK 1/2 (4-fold, p < 0.05) 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.