Differentiation patterning of vascular smooth muscle cells (VSMC) in atherosclerosis.

To investigate the involvement of transdifferentiation and dedifferentiation phenomena inside atherosclerotic plaques, we analyzed the differentiation status of vascular smooth muscle cells (VSMC) in vitro and in vivo. Forty normal autoptic and 20 atherosclerotic carotid endarterectomy specimens as well as 20 specimens of infrarenal and suprarenal aortae were analyzed for the expression of cytokeratins 7 and 18 and beta-catenin as markers (epithelial transdifferentiation) as well as CD31 and CD34 (embryonic dedifferentiation) by conventional and double fluorescence immunohistochemistry and reverse transcription polymerase chain reaction. Looking at these markers, additional cell culture experiments with human aortic (HA)-VSMC were done under stimulation with IL-1beta, IL-6, and TNF-alpha. Cytokeratins and beta-catenin were expressed significantly higher in atherosclerotic than in normal carotids primarily localized in VSMC of the shoulder/cap region of atherosclerotic lesions. Additionally, heterogeneous cellular coexpression of CD31 and/or CD34 was observed in subregions of progressive atherosclerotic lesions by VSMC. The expression of those differentiation markers by stimulated HA-VSMC showed a time and cytokine dependency in vitro. Our findings show that (1) VSMC of progressive atheromas have the ability of differentiation, (2) that transdifferentiation and dedifferentiation phenomena are topographically diverse localized in the subregions of advanced atherosclerotic lesions, and (3) are influenced by inflammatory cytokines like IL-1beta, IL-6, and TNF-alpha.

Reflux patterns in the ovarian and hypogastric veins in patients with varicose veins and signs of pelvic venous incompetence.

Patients with varicose veins who also had clinical and/or duplex ultrasound findings suspicious of pelvic venous incompetence (PVI) underwent selective retrograde catheter phlebography of the pelvic veins. One hundred and one patients (all female, mean age 49.3 years) underwent selective phlebography of the pelvic veins. In 68 cases (67.3%) a varicose vein recurrence after previous stripping of the greater saphenous vein was present, and about half the patients (n=45, 44.6%) were multipara ( > or =2 episodes of childbirth). The presence and extent of any reflux was documented and the ovarian and pelvic veins affected by the reflux were recorded. Retrograde selective phlebography demonstrated a PVI in 75 patients (74.2%). The left ovarian vein and the right hypogastric vein were most frequently affected by reflux (n = 41, 54.6% each). The left hypogastric vein was incompetent in 35 patients (46.6%) and the right ovarian vein in 3 cases (4%). In about half the patients with pelvic venous incompetence, reflux was demonstrated in more than one of the main pelvic veins (n=38, 50.6%). Fifty-one (68%) of the 75 patients with pelvic venous incompetence had varicose vein recurrence after previous stripping of the greater saphenous vein. Extension of the reflux into varicose veins of the groin or lower leg was demonstrated in 44 patients (58.6%). Thirty-nine patients (52%) received treatment for their pelvic venous incompetence (coil embolisation, sclerotherapy or videoscopic ovarian vein ligation). Pelvic venous reflux was present in 75% of our study population. Combined reflux in more than one pelvic vein was common and in about 60% of cases the pelvic reflux was shown to feed varicose veins of the legs. Therefore, typical clinical and/or duplex findings should lead to a strong suspicion of pelvic venous incompetence and reduce the need for selective retrograde catheter phlebography in this selected group of patients.

Overexpression of MMP9 and tissue factor in unstable carotid plaques associated with Chlamydia pneumoniae, inflammation, and apoptosis.

Tissue remodeling by matrix metalloproteinases (MMPs) and plasminogen activators such as tissue factor (TF) is postulated to be involved in the pathogenesis of atherosclerosis. The in situ expression of MMP9 and TF in unstable atherosclerotic plaques has not been examined in detail. Moreover, interference of tissue remodeling by vascular inflammation, apoptosis, and Chlamydia pneumoniae inside plaque subregions is unclear. A total of 40 autopsy carotid arteries (controls) and 20 atherosclerotic carotid endarterectomy specimens (with type VI lesions, according to the American Heart Association classification) from stroke patients were analyzed for expression of MMP9 and TF using in situ techniques. The data on tissue remodeling were correlated with the presence of inflammatory cells (T cells, B-cells, macrophages), apoptosis, and the presence of C. pneumoniae using immunohistochemistry and Western blot analyses. We found a significant overexpression of MMP9 and TF in progressive atherosclerotic carotid arteries, especially in the shoulder and cap subregions (both p < 0.05). Expression of MMP9 and TF correlated significantly with T-cell and macrophage infiltrates as well as with apoptosis (p < 0.05). C. pneumoniae infection was significantly associated with elevated TF expression (p < 0.01) but not with MMP9. MMP9 and TF are thus significantly overexpressed in progressive atherosclerotic plaques, and their relevant subregions (shoulder and cap) are involved in plaque instability. This process is associated with local inflammatory cell infiltrates and apoptosis, which might be influenced by infectious agents such as C. pneumoniae.

Ultrasound elastography for the age determination of venous thrombi. Evaluation in an animal model of venous thrombosis.

The exact age determination of venous thrombi is important if thrombolytic therapy or surgical thrombectomy is considered. Clinical symptoms as well as duplex-ultrasound and phlebography are unreliable in this respect and do not allow an exact age estimation. Ultrasound elastography can provide information about the elastic properties of thrombi. Since thrombus elasticity decreases with age due to the organisation process, it should be possible to use elastography to stage the degree of organisation and thereby determine the age of venous thrombi. Experimental venous thrombi aging 1, 3, 6, 9, 12 and 15 days were created in a porcine model by laparoscopic ligation of the infrarenal Vena cava in combination with transfemoral infusion of thrombin. The thrombosed iliac veins were explanted and embedded in gelatine, after that they underwent examination by ultrasound elastography. In addition, histological evaluation of the thrombi was performed. Elastography demonstrated a decline in thrombus elasticity between days 6 and 12 with the 12-day-old thrombi being about 3 times harder then the 6-day-old thrombi. This correlated with the histological findings, which demonstrated a marked increase in fibroblast and collagen production in the clots during this time, with the 12- and 15-day thrombi showing signs of advanced organisation. In conclusion, in an experimental setting, ultrasound elastography was helpful in determining the exact age of venous thrombi. The differences in elasticity were most pronounced between days 6 and 12, which is also the most relevant time frame when considering invasive therapies in human venous thrombosis.

Validity of the macroscopic identification of neovascularization at the saphenofemoral junction by the operating surgeon.

PURPOSE: The intraoperative classification of recurrent veins at the saphenofemoral junction as neovascularization is based on certain macroscopic criteria. The validity of this neovascularization identification by the surgeon was evaluated by histologic and immunohistochemical examinations of the resected veins as the gold standard. METHODS: One hundred-four patients (mean age 57 +/- 10.9 years) with 130 affected extremities underwent redo surgery of the saphenofemoral junction for recurrent varicose veins at a mean of 12.0 +/- 7.9 years after the initial stripping of the greater saphenous vein. The surgeon used intraoperative findings to classify the recurrent veins as neovascularization (group A), no neovascularization (group B), or unclear findings (group C). The resected veins then underwent histologic and immunohistochemical examinations and were again classified into the same three groups (a, neovascularization; b, no neovascularization; c, unclear) according to the histologic results. RESULTS: The operating surgeon correctly recognized 16 (41%) of 39 histologically proven neovascularizations, whereas 22 (56%) were not identified. In contrast, 66 (84%) of 79 cases with no neovascularization were correctly identified during the operation. A definite classification by macroscopic criteria was not possible in 4 cases (3%) and the histologic examinations revealed unclear findings in 12 cases (9%). The macroscopic neovascularization identification had a sensitivity of 42.1% and a specificity of 85.7%. The positive predictive value reached 59.3% and the negative predictive value, 75%. CONCLUSION: The validity of intraoperative neovascularization identification is poor, but the surgeon is able to recognize non-neovascularization recurrences with relatively high accuracy. The etiologic classification of recurrent veins at the saphenofemoral junction should not be solely based on macroscopic criteria.

Detection of Chlamydia pneumoniae but not of Helicobacter pylori in symptomatic atherosclerotic carotids associated with enhanced serum antibodies, inflammation and apoptosis rate.

BACKGROUND AND PURPOSE: Numerous seroepidemiological and pathological studies linked Chlamydia pneumoniae and Helicobacter pylori with atherosclerosis. However, analyses of these infectious agents in the pathogenesis of stroke are either lacking or contradictory. Therefore, we evaluated the detection rate of C. pneumoniae and H. pylori in normal carotids vs. atherosclerotic carotids and compared these findings with serology, plaque morphology, inflammatory cell infiltrates and apoptosis rate. METHODS: The study was performed on 40 morphological normal carotids from autopsy and 20 advanced atherosclerotic carotids from endarterectomy after stroke. Serum IgG antibody titre was measured by enzyme immunoassay (H. pylori) and microimmunofluorescence (MIF) technique (C. pneumoniae). Immunohistochemistry (IHC) and Western blotting were performed to identify C. pneumoniae, H. pylori, to characterize plaque morphology (macrophages and smooth muscle cells) and the inflammatory infiltrate (T- and B cells) and to detect apoptosis (TUNEL staining). RESULTS: C. pneumoniae was found significantly more frequently in atherosclerotic than in normal carotids (P=0.001), which correlated with elevated C. pneumoniae IgG-antibody titres (P=0.048). Although H. pylori was not detected in carotids, elevated H. pylori antibody titres were significantly associated with the degree of atherosclerosis (P=0.001). The C. pneumoniae infected carotids displayed a slightly enhanced infiltrate of T cells and apoptosis rate, but no morphological changes. CONCLUSION: C. pneumoniae but not H. pylori, was detected by IHC primarily in symptomatic carotids, without specific morphological differences. Correlation of C. pneumoniae in-situ-detection and IgG antibodies suggested a possible connection between respiratory-tract and endovascular infection. The C. pneumoniae associated T-lymphocytes and apoptosis rate indicate an immune-mediated inflammatory process, involving vascular walls.

Regional hyperthermic fibrinolytic perfusion after unsuccessful venous thrombectomy of extensive deep venous thrombosis.

OBJECTIVE: Because of the dose-dependent increase in bleeding complications, the intraoperative administration of fibrinolytic agents is limited. This limitation impairs the efficacy of fibrinolytic therapy because low-dose fibrinolysis often fails in the treatment of complex deep venous thrombosis (DVT). The aim of this study was to investigate the efficacy and safety of intraoperative high-dose fibrinolytic therapy for extended DVT, which was performed with the regional hyperthermic fibrinolytic perfusion (RHFP) technique. METHODS: From January 1993 to June 2001, in 53 patients with extended DVT, unsuccessful venous thrombectomy (recanalization, <50%) was followed by RHFP with 0.5 mg/kg of body weight of recombinant tissue plasminogen activator. The extent of thrombosis was documented before, during (after the surgical thrombectomy), and after (between postoperative days 2 and 5) surgery with phlebography and was quantified with the Marder score. Intraoperative and postoperative complications were recorded prospectively. RESULTS: After RHFP, a recanalization was achieved in 64 of 146 venous segments (43.8%) that were still occluded despite thrombectomy. Eighty-two segments (56.2%) remained occluded. Compared with the preoperative phlebography, 32 patients (60.3%) had a successful recanalization (>50%). Eleven patients (20.8%) showed minimal and 10 patients (18.9%) no recanalization. No lethal complications occurred. One patient (1.9%) had pulmonary embolism develop, and two patients (3.8%) had bleeding complications develop. CONCLUSION: With the intraoperative use of hyperthermia-assisted high-dose fibrinolysis, improvement of the results of mechanical thrombectomy of extended DVT was possible. The RHFP protected against systemic side effects of the fibrinolysis and show a high safety of application.

Comparison of laparoscopic aortic clamps in a pulsatile circulation model.

PURPOSE: This study was designed to evaluate the fatigue characteristics and the safety and effectiveness of laparoscopic aortic clamps in a pulsatile circulation model. METHODS: A heart-lung machine was used to create a pulsatile circulation model with bovine aortas resembling the vessels being cross-clamped. Four different models (A-D) of laparoscopic aortic clamps were investigated, and three identical probes of each model underwent testing. Preliminary examinations were conducted to define the size and thickness of the bovine aortas that would allow effective cross-clamping and to detect gross material or functional deficits of the clamps. Then, the instruments were placed in the circulation model, which was set at a frequency of 82/min and a pressure of 200/120 mm Hg. Each clamp was subjected to these conditions for 120 hours and was opened and closed 40 times to stimulate real-life conditions. Clamping failures and mechanical defects were recorded, and the clamp parts were afterward examined with an electron microscope. RESULTS: Two clamp models had to be eliminated from the study after the preliminary examinations. All three probes of model B displayed mechanical defects after a few applications. All probes of model D were excluded because none effectively occluded the aortas. All probes of model A and one probe of model C provided effective cross-clamping during the 120-hour test phase and showed no signs of mechanical failure. Two probes of model C broke after 51 and 57 hours of testing, respectively. Both times, the defect occurred during application of the clamps. The detailed analysis of all instruments after the testing, including electron microscope imaging, revealed that several construction deficits and weak points were responsible for the mechanical failures. CONCLUSION: A surprisingly high incidence of clamping failures and mechanical deficits were encountered during the testing. Of the four clamps tested, only one (model A) seemed to be safe and effective enough for routine clinical use. These disappointing results demonstrate the need for further cooperation between vascular surgeons and instrument manufacturers to develop safe and effective laparoscopic vascular clamps.