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Aims Brugada syndrome (BrS) is associated with an increased risk of sudden cardiac death due to ventricular tachycardia/fibrillation (VT/VF) in young, otherwise healthy individuals. Despite SCN5A being the most commonly known mutated gene to date, the genotype–phenotype relationship is poorly understood and remains uncertain. This study aimed to elucidate the genotype–phenotype correlation in BrS. Methods and results Brugada syndrome probands deemed at high risk of future arrhythmic events underwent genetic testing and phenotype characterization by the means of epicardial arrhythmogenic substrate (AS) mapping, and were divided into two groups according to the presence or absence of SCN5A mutation. Two-hundred probands (160 males, 80%; mean age 42.6 ± 12.2 years) were included in this study. Patients harbouring SCN5A mutations exhibited a spontaneous type 1 pattern and experienced aborted cardiac arrest or spontaneous VT/VF more frequently than the other subjects. SCN5A-positive patients exhibited a larger epicardial AS area, more prolonged electrograms and more frequently observed non-invasive late potentials. The presence of an SCN5A mutation explained >26% of the variation in the epicardial AS area and was the strongest predictor of a large epicardial area. Conclusion In BrS, the genetic background is the main determinant for the extent of the electrophysiological abnormalities. SCN5A mutation carriers exhibit more pronounced epicardial electrical abnormalities and a more aggressive clinical presentation. These results contribute to the understanding of the genetic determinants of the BrS phenotypic expression and provide possible explanations for the varying degrees of disease expression.

An altered balance between metalloproteinases (MMPs) and their inhibitor tissue inhibitor of metalloproteinases (TIMPs) may influence the healing process of a minor amputation following a successful vein graft. To speed up this process, negative pressure wound therapy (NPWT) and advanced moist wound dressing have been proposed. We determined the systemic and local release of MMP‐1, ‐2, ‐3, ‐9, TIMP‐1, and TIMP‐2 by enzyme linked immunosorbent assay (ELISA) technique and their influences in the healing process in 26 patients who underwent minor amputation after a successful revascularisation procedure. Twelve patients (group 1) were medicated with NPWT and 14 (group 2) with advanced moist wound dressing. Plasma samples were collected on the morning of surgery and thereafter at 1, 3, and 5 months; exudates were collected 3 days after surgery when amputation was performed and thereafter at 1, 3, and 5 months. Fifteen age‐matched healthy male volunteers served as controls. All wounds healed in 5 ± 0.5 months. Follow‐up plasma and local release of MMP‐1, ‐2, ‐3, and ‐9 were overall significantly lower when compared with the preoperative levels, while those of TIMP‐1 and ‐2 were significantly higher with no differences among the groups. Despite no differences in the healing process being observed among the two types of medications, at 1 month the local release of MMP‐2 and ‐9 was significantly lower (P = .013 and .047, respectively) and that of TIMP‐1 was significantly higher (P = .042) in group 1 as compared to group 2. A correct and aggressive local approach to the wound is able to promote the healing of the lesion stimulating the extracellular matrix turnover with local MMP/TIMP adequate balance and favouring the creation of granulation tissue. However, a successful restoration of an adequate blood flow remains the key point of a durable and rapid wound healing.

Patients with COVID-19 frequently have non-typical ECG changes in the QRS and T-wave morphology. The novel CineECG uses using the mean temporal spatial isochrones (mTSI) to relate the activation and recovery pathway to the cardiac anatomy. The aim of this feasib ility study is to use the novel CineECG to separate normal from abnormal ECGs. The ECGs of 100 normal controls were used to obtain the normal mTSI paths values for the QRS, ST segment and T-wave. These normal CineECG values were used to classify the COVID-19 ECGs as either as normal or abnormal of 107 patients being treatedfor COVID-19 in the University Medical Center Utrecht. The CineECG was able to classify 98% of the normal ECG correctly and 94% of the abnormal ECG in comparison to expert ECG classifications. The ability of the CineECG to relate the ECG to the cardiac anatomy supports the detection of abnormal ECGs. The CineECG might be a novel ECG screening tool to detect potential cardiac involvement of the COVID-19 disease for non-ECG experts.

Background: In Brugada syndrome (BrS), diagnosed in presence of a spontaneous or ajmaline-induced type-1 pattern, ventricular arrhythmias originate from the right ventricle outflow tract (RVOT). We developed a novel CineECG method, obtained by inverse electrocardiogram (ECG) from standard 12-lead ECG, to localize the electrical activity pathway in patients with BrS. Methods: The CineECG enabled the temporospatial localization of the ECG waveforms, deriving the mean temporospatial isochrone from standard 12-lead ECG. The study sample included (1) 15 patients with spontaneous type-1 Brugada pattern, and (2) 18 patients with ajmaline-induced BrS (at baseline and after ajmaline), in whom epicardial potential duration maps were available; (3) 17 type-3 BrS pattern patients not showing type-1 BrS pattern after ajmaline (ajmaline-negative); (4) 47 normal subjects; (5) 18 patients with right bundle branch block (RBBB). According to CineECG algorithm, each ECG was classified as Normal, Brugada, RBBB, or Undetermined. Results: In patients with spontaneous or ajmaline-induced BrS, CineECG localized the terminal mean temporospatial isochrone forces in the RVOT, congruent with the arrhythmogenic substrate location detected by epicardial potential duration maps. The RVOT location was never observed in normal, RBBB, or ajmaline-negative patients. In most patients with ajmaline-induced BrS (78%), the RVOT location was already evident at baseline. The CineECG classified all normal subjects and ajmaline-negative patients at baseline as Normal or Undetermined, all patients with RBBB as RBBB, whereas all patients with spontaneous and ajmaline-induced BrS as Brugada. Compared with standard 12-lead ECG, CineECG at baseline had a 100% positive predictive value and 81% negative predictive value in predicting ajmaline test results. Conclusions: In patients with spontaneous and ajmaline-induced BrS, the CineECG localized the late QRS activity in the RVOT, a phenomenon never observed in normal, RBBB, or ajmaline-negative patients. The possibility to identify the RVOT as the location of the arrhythmogenic substrate by the noninvasive CineECG, based on the standard 12-lead ECG, opens new prospective for diagnosing patients with BrS.

Brugada syndrome (BrS) is diagnosed by the presence of an elevated ST-segment and can result in sudden cardiac death. The most commonly found mutated gene is SCN5A, which some argue is the only gene that has been definitively confirmed to cause BrS, while the potential causative effect of other genes is still under debate. While the issue of BrS genetics is currently a hot topic, current knowledge is not able to result in molecular confirmation of over half of BrS cases. Therefore, it is difficult to develop research models with wide potential. Instead, the clinical genetics first need to be better understood. In this study, we provide crucial human data on the novel heterozygous variant NM_198056.2:c.4285G>A (p.Val1429Met) in the SCN5A gene, and demonstrate its segregation with BrS, suggesting a pathogenic effect. These results provide the first disease association with this variant and are crucial clinical data to communicate to basic scientists, who could perform functional studies to better understand the molecular effects of this clinically-relevant variant in BrS.

Background: The relationship between the typical electrocardiographic pattern and electromechanical abnormalities has never been systematically explored in Brugada syndrome (BrS). Objectives: The aims of this study were to characterize the electromechanical substrate in patients with BrS and to evaluate the relationship between electrical and mechanical abnormalities. Methods: We enrolled 50 consecutive high-risk patients with BrS (mean age 42 ± 7.2 years), with implantable cardioverter-defibrillator implantation for primary or secondary prevention of ventricular tachyarrhythmias (ventricular tachycardia/ventricular fibrillation [VT/VF]), undergoing substrate mapping and ablation. Patients underwent 3-dimensional (3D) echocardiography with 3D wall motion/deformation quantification and electroanatomic mapping before and after ajmaline administration (1 mg/kg in 5 minutes); 3D mechanical changes were compared with 50 age- and sex-matched controls. The effect of substrate ablation on electromechanical abnormalities was also assessed. Results: In all patients, ajmaline administration induced Brugada type 1 pattern, with a significant increase in the electrical substrate (P

Aims Brugada syndrome (BrS) represents a major cause of sudden cardiac death in young individuals. The risk stratification to forecast future life-threatening events is still controversial. Non-invasive assessment of late potentials (LPs) has been proposed as a risk stratification tool. However, their nature in BrS is still undetermined. The purpose of this study is to assess the electrophysiological determinants of non-invasive LPs. Methods and Results Two hundred and fifty consecutive patients with (Group 1, n = 96) and without (Group 2, n = 154) BrS-related symptoms were prospectively enrolled in the registry. Signal-averaged electrocardiogram (SAECG) was performed in all subjects before undergoing epicardial mapping. Group 1 patients exhibited larger arrhythmogenic substrates (AS; 5.8 ± 2.8 vs. 2.6 ± 2.1 cm2, P Conclusion The results of this study support the role of the epicardial AS as an electrophysiological determinant of non-invasive LPs, which may serve as a tool in the non-invasive assessment of the BrS substrate, as SAECG-LPs could be considered an expression of the abnormal epicardial electrical activity. ClinicalTrials.gov number (NCT02641431; NCT03106701).

Prodromal signs of a non-healing wound after revascularisation, which might be strictly linked with impending failure of vascular reconstructions, are associated with an inflammatory response mediated by several circulating adhesion molecules, extracellular endopeptidases, and cytokines. The aim of our study was to investigate the role of selected plasma biomarkers in the prediction of both wound healing and failure of infrapopliteal vein graft or percutaneous trans-luminal angioplasty (PTA) with selective stent positioning of the superficial femoral artery (SFA) in a population affected with critical limb ischaemia. A total of 68 patients who underwent either surgical or endovascular revascularisation of the inferior limb with autologous saphenous vein infrapopliteal bypass or PTA and selective stenting of the SFA were enrolled in our study. Patients were divided into two groups according to treatment: 41 patients were included in Group 1 (open surgery) and 27 in Group 2 (endovascular procedure). Plasma and blood samples were collected on the morning of surgery and every 6 months thereafter for up to 2 years of follow-up or until an occlusion occurred of either the vein bypass graft or the vessel treated endovascularly. Fifteen age-matched healthy male volunteers were considered a reference for biological parameters. Vascular cell adhesion molecule 1 [VCAM-1]/CD106, inter-cellular adhesion molecule-1 [ICAM-1]/CD54), interleukin-1 (IL-1), interleukin-6 (IL-6), tumour necrosis factor alpha (TNF-α), and metalloproteinases (MMP)-2 and -9 plasma levels were measured with enzyme-linked immunosorbent assay (ELISA) kits. The mean observed time to heal of 54 wounds was 13 ± 4 months, with no statistically significant differences among the groups. The healing failure of the remaining wounds was strictly related to an unsuccessful open (n = 12) or endovascular (n = 8) treatment. The 2-year primary patency rate was 65% (SE = .09) in Group 1 and 52% (SE = .1) in Group 2. When compared with mean concentration values of Group 1, VCAM-1 and ICAM-1 were always significantly higher during follow-up in patients of Group 2 (P < .05). Furthermore, in the same group, IL-6 and tumour necrosis factor alpha (TNF-α) were found to be significantly higher at 6- and 12-month (P < .05) when compared with surgically treated patients. Cox regression analysis showed that elevated plasma levels of VCAM-1, ICAM-1, IL-6, and TNF-α during follow up were strongly related to impaired wound healing and/or revascularisation failure (P < .05). Elevated plasma levels of inflammatory markers VCAM-1, ICAM-1, IL-6, and TNF-α may be related to the failure of wound healing and revascularisation procedures. Interestingly, we have observed that endovascular treatments cause a higher level of these inflammation biomarkers when compared with a vein graft, although wound-healing and patency and limb salvage rates are not influenced.

Abnormal, persistent inflammation after bypass surgery could prevent healing of an ischaemic foot lesion. In 37 patients with peripheral arterial disease (PAD) (Rutherford Grade III Category 5) who underwent infrapopliteal vein graft and midfoot amputation, plasma levels of fibrinogen, C-reactive protein (CRP), interleukin-1 (IL-1), interleukin-6 (IL-6), tumour necrosis factor-α (TNF-α), and matrix metalloproteinase-2 and -9 (MMP-2 and MMP-9) were determined preoperatively and during the follow up. Nine patients without clinical and Doppler evidence of arterial disease, who underwent post-traumatic midfoot primary amputation, were included in the experiment group, and 15 age-matched healthy volunteers served as control. In patients who had midfoot amputation for trauma, all wounds healed. Seven (19%) wounds in patients with an occluded graft healed, and five (13%) required major amputation because of a non-healing wound. Time required for complete healing of the lesion was similar between trauma and PAD patients (8 ± 2 months vs 11 ± 6, respectively, P = NS). Univariate analysis demonstrated that, in PAD patients, the postoperative high levels of TNF-α, IL-6, and MMP-2 and -9 were predictive for wound healing failure at 3, 6, and 9 months (P < 0.05), respectively. Furthermore, the subgroup of patients who experienced occlusion of the vein graft during follow up had a significant increase of MMP-2, -9, IL-6, and TNF-α at 3, 6, and 9 months (P < 0.05), respectively. Monitoring inflammatory markers allows the determination of patients at risk of healing failure of midfoot amputation after distal revascularisation and might predict the fate of the vein graft.

Background: Guidelines recommend the use of implanted cardioverter-defibrillators in patients with Brugada syndrome and induced ventricular tachyarrhythmias, but there is no evidence supporting it. Objectives: This prospective registry study was designed to explore clinical and electrophysiological predictors of malignant ventricular tachyarrhythmia inducibility in Brugada syndrome. Methods: A total of 191 consecutive selected patients with (group 1; n = 88) and without (group 2; n = 103) Brugada syndrome–related symptoms were prospectively enrolled in the registry. Patients underwent electrophysiological study and substrate mapping or ablation before and after ajmaline testing (1 mg/kg/5 min). Results: Overall, before ajmaline testing, 53.4% of patients had ventricular tachyarrhythmia inducibility, which was more frequent in group 1 (65.9%) than in group 2 (42.7%; p < 0.001). Regardless of clinical presentation, larger substrates with more fragmented long-duration ventricular potentials were found in patients with inducible arrhythmias than in patients without inducible arrhythmias (p < 0.001). One extrastimulus was used in more extensive substrates (median 13 cm2; p < 0.001), and ventricular fibrillation was the more frequently induced rhythm (p < 0.001). After ajmaline, patients without arrhythmia inducibility had arrhythmia inducibility without a difference in substrate characteristics between the 2 groups. The substrate size was the only independent predictor of inducibility (odds ratio: 4.51; 95% confidence interval: 2.51 to 8.09; p < 0.001). A substrate size of 4 cm2 best identified patients with inducible arrhythmias (area under the curve: 0.98; p < 0.001). Substrate ablation prevented ventricular tachyarrhythmia reinducibility. Conclusions: In Brugada syndrome dynamic substrate variability represents the pathophysiological basis of lethal ventricular tachyarrhythmias. Substrate size is independently associated with arrhythmia inducibility, and its determination after ajmaline identifies high-risk patients missed by clinical criteria. Substrate ablation is associated with electrocardiogram normalization and not arrhythmia reinducibility. (Epicardial Ablation in Brugada Syndrome [BRUGADA_I]; NCT02641431; Epicardial Ablation in Brugada Syndrome: An Extension Study of 200 BrS Patients; NCT03106701)

Objectives The aim of our study was to correlate flow dynamics and the release of inflammatory cytokines Interleukin 1, 2, 6, TNF (Tumour Necrosis Factor) alfa, both in vitro and in vivo. Materials and methods Endothelial cells were exposed to laminar flow (6 dyne/cm 2 ) in an in vitro circulatory system and the release of Interleukin 1, 2, 6 and TNF alfa was quantified by ELISA. Interleukin 1, 2, 6 and TNF alfa release was also assessed in vein grafts implanted in the arterial circulation of Lewis rats. Arterial vein grafts were explanted at different time intervals from 3 days to 12 weeks after surgery. Vein grafts implanted in the arterial circulation for 4 weeks, were re implanted in the venous circulation of syngenic Lewis rats, and the release of Interleukin 1, 2, 6 and TNF alfa, was assessed in an organ culture. Six vein grafts (4 occluded, 2 patent) implanted in humans as femorodistal bypass were examined for the presence of myointimal hyperplasia and perigraft inflammatory cells. Results In vitro, endothelial cells exposed to laminar flow released an increased amount of Interleukin 1, 2, 6 and TNF alfa in comparison to endothelial cells not exposed to flow. In experimental vein grafts implanted in the arterial circulation there was an increased release of inflammatory cytokines associated to inflammatory changes in the adventitia. Once the vein grafts were re implanted in the venous circulation, the release of these cytokines diminished, while the inflammatory changes in the adventitia regressed. Conclusions Increased shear stress induces release of cytokines and inflammatory changes in the adventitia. These inflammatory changes can contribute to plaque progression and to un stable plaque. These findings support the use of anti-inflammatory therapy in patients prone to develop atherosclerosis and in those who had arterial reconstructive surgery.

Transplant vasculopathy limits the clinical results of solid organ transplantation.Thirty-three arterial grafts were implanted in the abdominal aorta of Lewis rats. The animals were humanely sacrificed 4 wk after surgery. The study groups had 15 arterial isografts and 18 arterial allografts. Growth factors and inflammatory cytokines, released by the removed grafts, were studied in organ culture. The released growth factors were analyzed in vitro to assess their effect on the proliferation of endothelial, smooth muscle cells and fibroblasts.In arterial isogenic and allogenic grafts, platelet-derived growth factor and basic fibroblastic growth factor release was minimal (P 0.01). There was a significant release of granulocyte-macrophage colony-stimulating factor and tumor necrosis factor-α (TNF-α; P 0.001) in allografts. GM-CSF and TNF-α, at concentrations in the allograft organ cultures, stimulated significantly the growth of smooth muscle cells. The simultaneous action of TNF-α and GM-CSF had an exponential growth effect on endothelial cells and smooth muscle cells. Interleukin (IL)-1, IL-2, and IL-9 were released in high quantities by allografts. In vitro, IL-1, IL-2, and IL-9 facilitated the growth effect of GM-CSF and TNF-α.Transplant vasculopathy depends on the simultaneous and complementary additive effects of several growth factors and cytokines, which have a continuous "cross talk."

Background The production of growth factors from several experimental arterial conduits was determined. Methods We implanted 105 experimental arterial grafts that were 1 cm long in the abdominal aorta of Lewis rats (average weight, 250 g). Five different types of grafts were analyzed: arterial isografts, vein grafts, arterial allografts, and polytetrafluoroethylene (PTFE) grafts with normal or decreased compliance. Animals were killed humanely 4 weeks after surgery and the production of platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), transforming growth factor-β, tumor necrosis factor-α, and interleukin-1 was analyzed. Results Myointimal hyperplasia (MH) was evident in vein grafts, arterial allografts, and PTFE grafts, but not in arterial isografts. Growth factor production was increased for grafts prone to develop MH like vein, PTFE grafts, and arterial allografts. PDGF and bFGF were increased significantly for PTFE and vein grafts, but not for arterial allografts. The importance of bFGF and PGDF was confirmed by the capability of antibody to PDGF and to bFGF to reduce the mitogenic activity of smooth muscle cells, in vivo and in vitro, for PTFE and vein grafts, but not for arterial allografts, in which a predominant role was played by interleukin-1 and tumor necrosis factor-α. Conclusions Agents able to neutralize this increased production of growth factors, either directly or by competition with their receptors, can prevent MH formation.

We investigated the role and influence of platelet derived growth factor (PDGF) and transforming growth factor beta1 (TGF) in the pathologic mechanism at the basis of plaque instability regulating the expression of matrix metalloproteinases (MMPs).Plaques obtained from 70 patients who underwent carotid endarterectomy were classified histologically as stable or unstable. Serum levels of PDGF and TGF were measured pre- and postoperatively. The serum activities of MMP-2 and MMP-9 were also analyzed. Human umbilical artery smooth muscle cells (HUASMCs) were stimulated in vitro with PDGF at various concentrations (20 and 50 ng/mL) and TGF (2 and 5 ng/mL) in a serum-free medium. The release of MMPs in the conditioned medium was assessed by enzyme-linked immunosorbent assay. Release of the MMPs was confirmed by Western blot analysis; their activity and expression were determined by zymography and reverse transcription-polymerase chain reaction. Specific inhibition tests were performed on HUASMCs to evaluate the role of these growth factors.Forty-two (60%) patients had an unstable carotid plaque and 28 (40%) a stable plaque. Preoperatively, patients affected with unstable carotid plaques had higher PDGF and lower TGF plasma levels than patients with stable carotid plaques (P.001); the levels returned to normal at 1 and 30 days postoperatively, compared with 20 non-operated healthy volunteers. Release, activity, protein level, and expression of MMPs in PDGF-stimulated HUASMCs were greater than in the controls (P.001), whereas these values in the TGF-stimulated HUASMCs were lower (P.001). The addition of monoclonal anti-PDGF antibodies decreased the release, activity, protein level, and expression of MMPs, whereas the addition of monoclonal anti-TGF antibodies increased the release, activity, protein level and expression of MMPs (P.001).TGF seems to be an important stabilizing factor and prevents plaque rupture through the decrease of MMPs.

Background. The role of thrombin in the stimulation of endothelial cell (EC) proliferation is controversial. The aim of this study was to investigate if thrombin regulates cell proliferation and production of platelet-derived growth factor (PDGF), bovine fibroblast growth factor (bFGF), and transforming growth factor β1 (TGF-β1) by bovine aortic ECs. Methods. ECs, obtained from thoracic aortas of calves, were stimulated with thrombin at various concentrations (from 0.05 to 1.0 IU/ml) in serum free culture. Mitogenic activity of thrombin on ECs was determined by tritiated thymidine uptake. The release of PDGF, bFGF, and TGF-β1 was assessed by ELISA. PDGF release was confirmed by Western blot and bFGF and TGF-β1 mRNA expression was determined by polymerase chain reaction (PCR). Results. Thrombin at high concentrations did not cause any increase in EC proliferation after 72 h of culture and induced inhibition of EC proliferation after 96 h and 8 days of culture. It induced a decrease in PDGF release and an increase in TGF-β1 release. Thrombin at low concentrations induced a significant increase in EC proliferation at 72 h, 96 h, and 8 days of culture. It induced an increase in PDGF release and a decrease in TGF-β1 release. bFGF release was higher than control at all thrombin concentrations. These data were confirmed by Western blot and PCR studies. Conclusions. Thrombin regulates EC growth through the inhibition of EC proliferation at high concentrations and through the stimulation of EC proliferation at low physiological concentrations. EC proliferation is partially mediated by autocrine production of PDGF, bFGF, and TGF-β1.

Background: Cigarette smoking influences and enhances the development of atherosclerosis. We investigated if nicotine, an important constituent of cigarette smoking, has a stimulatory effect on bovine smooth muscle cell proliferation in vitro through the mediation of bFGF and TGF-β1. Methods: Bovine aortic smooth muscle cells (SMC) were stimulated with (-)-nicotine at various concentrations ranging from 6 × 10-4 mol/L to 6 × 10-8 mol/L. SMC viability and count were assessed. The presence of bFGF and TGF-β1 in serum-free conditioned media was determined by the inhibition antibody-binding assay, and the mitogenic activity of (-)-nicotine on SMC was analyzed by the 3H-thymidine uptake. Polymerase chain reaction was used to study the expression of bFGF and TGF-β1. Results: The bFGF release after (-)-nicotine stimulation was greater than in the controls, whereas TGF-β1 release was lower. The greatest mitogenic activity was found at a (-)-nicotine concentration of 6 × 10-6 mol/L. The addition of monoclonal antibody anti-bFGF decreased the 3H-thymidine uptake of SMC exposed to (-)-nicotine, whereas the addition of monoclonal antibody anti-TGF-β1 increased the 3H-thymidine uptake of stimulated SMC. bFGF mRNA expression was significantly higher in SMC exposed to (-)-nicotine than in the controls, but TGF-β1 mRNA expression was significantly lower in SMC exposed to 6 × 10-6 mol/L (-)-nicotine than in SMC treated with the other concentrations of (-)-nicotine and in controls. Conclusions: Nicotine is a potent regulator of bFGF and TGF-β1 production and release by aortic SMC, and it seems to play an important role in the development and progression of atherosclerosis and neointimal fibrous hyperplasia. (Surgery 2000:127:316-22.)

Background: Myointimal hyperplasia is a common complication after vascular reconstruction. Increasing shear stress has been shown to reduce formation of myointimal hyperplasia. The aims of our study were (1) to analyze the correlation between shear stress and release of transforming growth factor (TGF)–β1 by endothelial cells and (2) to determine the effect of TGF-β1 on smooth muscle cell proliferation. Methods: Bovine arterial endothelial cells were subjected to increasing shear stress in an in vitro serum-free system. The release of TGF-β1 by endothelial cells was assessed by enzyme-linked immunosorbent assay and Western blot analysis. The effect of TGF-β1 on the proliferation of the subconfluent monolayer of bovine smooth muscle cells was determined by tritiated thymidine uptake. Results: Shear stress induced a significant increase of the release of TGF-β1 by endothelial cells (p < 0.001). This phenomenon was proportional to the level of shear stress. The amount of TGF-β1 released by endothelial cells subjected to shear stress had a significant inhibitory effect on growth rate and tritiated thymidine uptake of smooth muscle cells. Conclusions: On the basis of the results of our study, we conclude that increasing shear stress induces release of TGF-β1 by arterial endothelial cells in a concentration that has a clear inhibitory effect on smooth muscle cell proliferation. This phenomenon could explain the inhibitory effect of increasing shear stress on the formation of myointimal hyperplasia. (Surgery 1998;123:212-7.)

The importance of rosuvastatin at therapeutic dosage in regulating the release, activity, protein level, and expression of matrix metalloproteinases (MMP)-2 and MMP-9 was investigated.Human umbilical artery smooth muscle cells were stimulated, in vitro, in a serum-free medium with rosuvastatin at various concentrations (2, 4, 7, and 10 ng/mL, which correspond to the maximal plasma concentration observed in healthy men after a daily oral intake of 5, 10, 20, and 40 mg, respectively). The release of MMP-2 and MMP-9 in the conditioned medium was assessed by enzyme-linked immunosorbent assay and confirmed by Western blot, the activity and expression were determined by zymography and polymerase chain reaction, respectively.Human umbilical artery smooth muscle cells stimulated with rosuvastatin at 7 and 10 ng/mL had a significant lower release, activity, protein level, and expression of MMP-2 and MMP-9, when compared with those stimulated at 2 and 4 ng/mL (MMP-2 =p0.0001 and p 0.0001, respectively; MMP-9 =p0.0001 and p0.0001, respectively).The effects of rosuvastatin in reducing MMP-2 and MMP-9, which might stabilize the atherosclerotic plaques, are dose-dependent.

ObjectivesThis study aimed to determine whether the plasma levels of matrix metalloproteinases (MMPs)-2 and -9 and their specific inhibitors (tissue inhibitors of metalloproteinases (TIMPs-1 and -2)) were altered in patients with symptomatic and asymptomatic, severe, recurrent carotid artery stenosis.PatientsFifty-two patients (out of a total of 621) who had undergone successful carotid artery endarterectomy (CEA) between 1999 and 2003 and developed recurrent carotid artery stenosis (≥70%) were included in the study. Restenosis was symptomatic in 23 patients and asymptomatic in 29 patients.MethodsRecurrent carotid artery stenosis was classified based on presentation, and as early–intermediate (6 months to 3 years) or late (>3 years). A detailed clinical history was taken and two blood samples were drawn from each patient to determine plasma levels of MMPs and TIMPs along with other biological parameters. Recurrent stenosis was confirmed with computed tomographic angiography.ResultsPatients with symptomatic restenosis had significantly (p

Our aim was to investigate the release, activity, and expression of matrix metalloproteinases (MMPs)-1, -2, -3 and -9, and tissue inhibitors of metalloproteinases (TIMPs)-1 and -2 in patients undergoing carotid endarterectomy and to determine whether altered plasma levels of MMPs and TIMPs may be correlated with carotid instability.The carotid plaques of 53 consecutive patients who underwent carotid endarterectomy were classified histologically as stable or unstable. The release of MMPs and TIMPs was analyzed in the serum of patients with stable and unstable carotid plaques, and in 15 age-matched healthy volunteers. The production, activity, and expression of MMPs and TIMPs were determined by Western blotting, zymography, and reverse transcriptase polymerase chain reaction in the carotid specimens.Twenty-nine (55%) patients had an unstable carotid plaque and 24 (45%) a stable plaque. Plasma levels of MMPs were higher in patients with unstable plaques compared to patients with stable plaques and healthy volunteers ( P.001), whereas plasma levels of TIMPs were lower in patients with unstable plaques compared to patients with stable plaques and healthy volunteers ( P.001). In the carotid specimens, we found increased activity, production, and expression of MMPs, and decreased activity, production and expression of TIMPs in unstable plaques compared to stable plaques ( P.001). After endarterectomy, plasma levels of MMPs and TIMPs in patients with unstable and stable plaques returned to the values found in healthy volunteers.Our study demonstrated that an imbalance exists between MMPs and TIMPs in unstable carotid plaques, which is reflected in the plasma levels of these markers. These data may help in selecting patients at high risk for cerebral events.

Background. Vascular endothelial growth factor (VEGF), a highly specific chemotactic and mitogenic factor for vascular endothelial cells (EC), appears to be involved in the development of atherosclerosis. The purpose of our study was to assess if VEGF might indirectly stimulate SMC migration and proliferation in a EC-SMC coculture system, through the mediation of growth factors released by EC. Methods. Bovine aortic SMC were cocultured with bovine aortic EC treated with hrVEGF, to assess SMC proliferation and migration. The release and mRNA expression of basic fibroblast growth factor (bFGF) and transforming growth factor β1 (TGFβ1) were assessed by ELISA and PCR analysis. Results. hrVEGF (10 ng/ml), added to EC cocultured with SMC, induced a significant increase in tritiated thymidine uptake by SMC as compared to controls (P < 0.01) and a significant increase in SMC migration in respect to control (27%; P < 0.01). EC stimulated with hrVEGF increased the release and the expression of bFGF and decreased the release and the expression of TGFβ1 with a statistically significant difference in respect to controls (P < 0.001). Conclusions. VEGF indirectly stimulates SMC proliferation and migration through the modulation of bFGF and TGFβ1 released by EC.

Background: Cigarette smoking is implicated in atherosclerotic plaque formation, but the role of nicotine in this process is not completely understood. The release of platelet-derived growth factor (PDGF) by the bovine aortic smooth muscle cell (SMC) after nicotine administration at a concentration similar to that ingested by active and passive smokers and the role of PDGF in SMC cytoskeletal modification were studied. Methods: SMC, harvested with enzymatic digestion from calf aorta, were stimulated in a serum-free medium for 72 hours with (-)-nicotine (from 6 × 10-4 mol/L to 6 × 10-8 mol/L). The release of PDGF was assessed by inhibition antibody-binding assay and confirmed by Western blotting. Mitogenic activity of nicotine on SMCs was also determined. The SMC cytoskeleton was studied with specific antibodies anti-α-actin fibers, anti-vimentin, and anti-β-tubulin, and the modification induced by PDGF was assessed by blocking PDGF activity with specific antibodies. Results: The greatest PDGF release (1.24 ± 0.14 ng/104 cells vs control 0.43 ± 0.07 ng/104 cells) was noted at a (-)-nicotine concentration of 6 × 10-7 mol/L (P

Background. Cigarette smoking has been directly linked to atherosclerosis formation and vascular graft failures but the role of nicotine in these processes is not yet completely understood. We investigated the release of platelet-derived growth factor BB (PDGF BB) by the bovine aortic endothelial cell (EC) after nicotine administration at concentrations similar to those found in plasma of active and passive smokers and the role of PDGF BB, autocrinally released, in EC cytoskeletal modification. Methods. EC were stimulated in a serum-free medium for 72 h with (−)-nicotine (from 6 × 10−4 to 6 × 10−8 M). The release of PDGF BB was assessed by inhibition antibody-binding assay and confirmed by Western blotting. Mitogenic activity of nicotine on EC was also determined. The EC cytoskeleton was studied with specific antibodies anti-α-actin fibers and anti-vimentin and the modification induced by PDGF BB was assessed by blocking PDGF BB activity with specific antibodies. Results. The greatest PDGF BB release was noted at a (−)-nicotine concentration of 6 × 10−6 M (P < 0.001). The addition of antibody anti-PDGF BB to EC exposed to (−)-nicotine decreased tritiated thymidine uptake by 20% (P < 0.001). EC exposed to (−)-nicotine concentrations of 6 × 10−6 and 6 × 10−8 M had a significant alteration in the expression of α-actin fibers and vimentin as compared with control. Administration of the antibody anti-PDGF BB in the culture medium reversed cytoskeletal alteration. Conclusions. Nicotine enhanced the release of PDGF BB by EC which in turn caused an alteration in cytoskeletal organization.

Nicotine, a constituent of cigarette smoking, may induce atherosclerosis through the production of growth factors. The pattern of bFGF and TGF beta1 production and release by bovine aortic endothelial cells (EC) stimulated with nicotine (from 6 x 10(-4) to 6 x 10(-8) M) was studied. EC viability and count were assessed. The presence of bFGF and TGF beta1 in serum-free conditioned media was determined by the inhibition antibody-binding assay and Western blot analysis. Mitogenic activity of nicotine on EC was also determined. Polymerase chain reaction (PCR) was used to study the expression of bFGF and TGF beta1. The bFGF release after nicotine stimulation was greater than controls, whereas TGF beta1 release was lower. At a nicotine concentration of 6 x 10(-6) M we noted the greatest mitogenic activity. The addition of monoclonal antibody anti-bFGF decreased the tritiated thymidine uptake of EC exposed to nicotine but the addition of monoclonal antibody anti-TGF beta1 had no significant effect. bFGF mRNA expression was significantly higher in EC exposed to nicotine than in controls, whereas TGF beta1 mRNA expression was not modified. From these data we concluded that nicotine regulates bFGF production and release and TGF beta1 release and may have a key role in the development and progression of atherosclerosis.

Myointimal hyperplasia is a common complication of arterial recontructive surgery. The serine protease thrombin has a major role in vessel wall healing and eventual myointimal hyperplasia formation. The aim of this study was to determine the effect of thrombin on the production of PDGF AA and bFGF by arterial smooth muscle cells.Bovine smooth muscle cells were stimulated with thrombin in a serum-free culture. The release of PDGF AA and bFGF was assessed by ELISA. The effect of thrombin on the proliferation of confluent monolayers of bovine smooth muscle cells was determined by tritiated thymidine uptake.Smooth muscle cells stimulated with thrombin released more PDGF AA (P0.001) and bFGF (P0.001) than the control. Addition of anti-PDGF AA and anti-bFGF antibodies to the medium of smooth muscle cell cultures neutralized the mitogenic effect of thrombin (P0.001).The findings of our study suggest that thrombin may lead to myointimal hyperplasia formation through induction of PDGF and bFGF production by smooth muscle cells.

Objectives:Elevated concentrations of oxidised low density lipoproteins (OxLDL) are associated with accelerated atherogenesis. The aim of our study was to determine the effect of OxLDL on the proliferation rate and platelet derived growth factor (PDGF) AA production on aortic smooth muscle cells. High density lipoproteins (HDL), which are known to have a protective effect against atherosclerosis, were used as control.Materials and methods:Bovine aortic smooth muscle cells were grown in presence of increased concentrations of OxLDL and HDL and in presence of control medium culture (DMEM). Proliferation rate was assessed by 3H-thymidine uptake. PDGF AA production was determined by ELISA and Western Blot Analysis.Results:OxLDL increased the proliferation rate of aortic smooth muscle cells as compared to DMEM and HDL (p

Objective(s). We hypothesized that basic fibroblast growth factor (bFGF) may exert a role in carotid plaque instability by regulating the expression of matrix metalloproteinases (MMP).Methods. Plaques obtained from 40 consecutive patients undergoing carotid endarterectomy were preoperatively classified as soft or hard. Serum bFGF was pre- and postoperatively measured. The release of MMP-2 and MMP-9 in the blood serum, and the activity, production and expression in the carotid specimens was analyzed. Specific anti-bFGF inhibition tests were performed in vitro on human umbilical artery smooth muscle cells (HUASMC) to evaluate the role of bFGF in the activity, production and expression of MMP-2 and -9.Results. Twenty-one (53%) patients had a soft carotid plaque and 19 (48%) a hard plaque. Preoperative bFGF serum levels were higher in patients with soft plaques [soft=34 (28–39) pg/mL and hard=20 (17–22) pg/mL—p

Background The phenomena involved in regression of arterial myointimal hyperplasia have not been analyzed in detail. Materials and methods In 24 Lewis rats, a 1-cm-long venous graft, obtained from syngenic Lewis rats, was implanted in the infrarenal aorta. After 4 wk, the grafts were removed and analyzed using scanning electron microscopy and histochemistry. The grafts showed evidence of myointimal hyperplasia; 16 of these explanted grafts were reimplanted in the vein circulation of syngenic Lewis rats. These grafts were harvested 2 wk (8 animals) and 8 wk (8 animals) later, showing complete regression of myointimal hyperplasia. Results Regression of experimental myointimal hyperplasia was correlated with the simultaneous and complementary action of Transforming Growth Factor beta and Tumor Necrosis Factor alfa. Inflammatory cytokines (IL1, IL2, and IL6) inhibit Tumor Necrosis Factor alfa-induced apoptosis. Conclusions Regression of myointimal hyperplasia is an active process, which implies the action of several inhibitory factors. The analysis of these phenomena can lead to new therapeutic approaches to prevent myointimal hyperplasia progression.