TIMP3 is Regulated by Pericytes upon Shear Stress Detection Leading to a Modified Endothelial Cell Response.

OBJECTIVES: Atherosclerosis is a hallmark of cardiovascular disease. Shear stress on endothelial cells has been linked to atherogenesis and to fibrous cap thinning and rupture. Pericytes reside in the sub-endothelial space of vessels and have vasoprotective effects. They are subjected to shear stress when endothelial cell integrity is disrupted. The aim was to investigate the susceptibility and response of pericytes to shear stress. METHODS: Endothelial cells and pericytes were seeded in two dimensional monocultures and co-cultures, and in a novel three dimensional co-culture system and were subjected to no, low and high shear stress (0, 10, 30 dyne/cm2) for 48 h. The morphological response to flow was assessed by histology and the expression of extracellular matrix proteins was analysed using quantitative polymerase chain reaction, immunoblotting, and ELISA. RESULTS: While endothelial cells aligned into flow direction, pericytes aligned perpendicularly (p < .001), indicating that they must be capable of sensing flow. When pericytes were embedded into a 3D matrix they showed similar alignment and pericytes built long processes towards the lumen. Under shear stress endothelial cells upregulated "a disintegrin and metalloproteinase with thrombospondin motif 1" (ADAMTS-1) (p < .01) and pericytes upregulated "tissue inhibitor of matrix metalloproteinase" (TIMP) 3 (p < .05), an inhibitor of ADAMTS-1, meanwhile differential expression of extracellular matrix (ECM) proteins could be detected in co-cultures of both cells. For TIMP3 expression direct cell-cell contact between endothelial cells and pericytes was required. CONCLUSION: The experiments highlight that pericytes are able to sense direct flow thereby regulating ECM proteins known to be involved in vascular remodelling. Furthermore, pericytes counter-regulate endothelial ADAMTS-1 by protective TIMP3 expression to prevent matrix degradation and maintain vascular stability. For this protective effect direct cell contact was necessary. This observation might represent an adaptive, protective mechanism of pericytes to counteract endothelial damage in the onset of atherosclerosis.

Streptococcus mitis and Gemella haemolysans were simultaneously found in atherosclerotic and oral plaques of elderly without periodontitis-a pilot study.

OBJECTIVES: Local infections may contribute to the initiation and progression of several clinical diseases in humans. Atherosclerotic plaques of subjects suffering from periodontitis are colonized by periopathogens; however, the presence of bacteria in atherosclerotic plaques in patients without severe forms of periodontitis is of high relevance for the general population. MATERIALS AND METHODS: Patients who were electively treated for atherosclerotic lesions of the carotid artery and without clinical signs of periodontitis were eligible for the study. Oral and atherosclerotic plaques were sampled, processed, and analyzed for their microbial composition by 454-sequencing. RESULTS: Seventeen patients were included in the analyses, and 76 % of all atherosclerotic plaque specimens were positive for bacterial DNA. In the oral plaques, 76,532 sequences were identified representing 1 phylum, 17 classes, 112 families, and 263 genera. In atherosclerotic plaques, 6112 sequences representing 1 phylum, 4 classes, 8 families, and 36 genera were found. The bacterial DNAs of the species Gemella haemolysans and Streptococcus mitis were simultaneously found in atherosclerotic as well as oral plaque samples of 3 patients. CONCLUSIONS: These results indicated that in subjects without periodontitis, the transmission of oral bacteria to atherosclerotic plaques of the carotid artery is a feasible event. CLINICAL RELEVANCE: The prevention of transient bacteremia from the oral cavity requires high levels of oral health.

Development of implantable autologous small-calibre vascular grafts from peripheral blood samples.

INTRODUCTION: At present the generation of a small-calibre (≤5 mm) vascular replacement for artificial bypasses remains a challenge for tissue engineering. The biocompatibility of bioartificial vessel replacements is of decisive significance for function and depends on the materials used. A completely autologous vessel substitute must exhibit high biocompatibility and functionality. For this purpose we developed and optimised a technique for the engineering of an autologous bypass material from a fibrin scaffold and vascular cells isolated from the same sample of peripheral blood in a porcine model. MATERIALS AND METHODS: Fibrinogen, late outgrowth endothelial and smooth muscle cells were isolated from peripheral blood samples (n=14, 100 mL each). Fibroblasts were isolated from porcine aortic adventitial tissue (n=4). Tubular seeded fibrin segments were obtained using an injection moulding technique with the simultaneous incorporation of the in vitro expanded cells into the fibrin matrix. The segments were cultivated under dynamic conditions with pulsatile perfusion in a bioreactor. Morphological and functional characterization was done. RESULTS: Artificial vascular segments with a length of 150 mm were reproducibly obtained with a hierarchical arrangement of incorporated cells similar to the structure of the vascular wall. By additional seeding of fibroblasts, suturable segments with biomechanical properties suitable for implantation into the arterial system were obtained. CONCLUSIONS: Implantable bioartificial vascular grafts can be generated from blood. After cultivation under dynamic conditions the vascular segments possess a structure similar to that of the vascular wall and exhibit biomechanical properties sufficient for implantation as arterial substitutes.

Risk factors associated with surgical site infections following vascular surgery at a German university hospital.

Surgical site infection (SSI) after vascular surgery is a serious complication increasing morbidity, mortality, and costs for healthcare systems. A 4-year retrospective cohort study was performed in a university hospital with patients who had undergone arterial vascular surgery below the aortic arch. Investigated variables included demographics and clinical data. Forty-four of 756 patients experienced SSI, 29 of which were superficial, five were deep, and 10 had organ/space infections. Coagulase-negative staphylococci (22%), enterococci (20%), and Staphylococcus aureus (18%) were the most common pathogens. Independent risk factors for SSIs were femoral grafting [odds ratio (OR) 6·7], peripheral atherosclerotic disease, Fontaine stages III-IV (OR 4·1), postoperative drainage >5 days (OR 3·6), immunosuppression (OR 2·8), duration of operation >214 min (OR 2·8), and body mass index >29 (OR 2·6). The application of perioperative antibiotic prophylaxis was an independent protective factor (OR 0·2). Patients with certain risk factors for SSIs warrant special attention for infection prevention.

Prevention of vascular graft infections with antibiotic graft impregnation prior to implantation: in vitro comparison between daptomycin, rifampin and nebacetin.

OBJECTIVE: To compare the in vitro efficacy of graft impregnation with nebacetin versus rifampin versus daptomycin against vascular graft infections caused by Staphylococcus epidermidis and Staphylococcus aureus and nebacetin versus rifampin against Pseudomonas aeruginosa and Escherichia coli. MATERIALS: Twenty-three Dacron-grafts (1 cm2) for each micro-organism were microbiologically tested and eight grafts per antibiotic underwent viability tests against human umbilical vein endothelial cells (ECs). Fifteen grafts (5/antibiotic agent) underwent 15 min impregnation and contamination with 4 ml bacterial solution (optical density (OD (600 nm)): 0.20 ± 0.02). After 24-h-incubation, all grafts were washed with phosphate-buffered saline and underwent sonification to release viable adherent bacteria. OD (600 nm) of the solution was measured. Afterwards, six 1:10 dilution steps took place and colony-forming units (CFUs) were counted. RESULTS: Nebacetin showed comparable efficacy to daptomycin against Gram-positive bacteria. Both eradicated more efficiently S. epidermidis than rifampin (daptomycin:0, rifampin:5 ± 7.3, nebacetin:0 CFU ml(-1), P = 0.0003). All antibiotics showed comparable antibacterial activity against S. aureus. Nebacetin was more efficient than rifampin to eradicate Gram-negative organisms (P. aeruginosa: rifampin:1308 ± 252, nebacetin:8 ± 8 CFU ml(-1), P = 0.01, E. coli: rifampin:294 ± 159, nebacetin:0.2 ± 0.5 CFU ml(-1), P = 0.001), while only rifampin was toxic against ECs (daptomycin:30.88 ± 5.44, rifampin:5.13 ± 5.08, nebacetin:28.50 ± 3.82 ECs/field, P = 0.0003). CONCLUSIONS: Nebacetin showed excellent in vitro antibacterial activity against both Gram-positive and -negative pathogens representing an effective candidate for vascular graft impregnation.

[Traumatic dissection of carotid arteries caused by high energy motorcycle accident]. / Traumatische Karotisdissektion nach Motorradunfall.

Injuries of internal carotid arteries caused by high energy trauma are rare but often combined with poor outcome. Blunt trauma to the head and neck as well as the use of newer motorcycle helmets together with crash circumstances should promptly lead to a differentiated polytrauma management with expansion of radiologic diagnostics. This could lead to a reduction of overlooked dissections and an increase in promptly and correctly treated injuries.

Recommendations for reporting treatment of aortic graft infections.

The purpose of these recommendations is to provide a standard format for reporting treatment results and standardised epidemiologic data after aortic vascular graft infection to improve the comparison of clinical outcomes between different therapeutic approaches and different study populations. Analytical reporting standards for patients' characteristics, type and extent of the disease, type of treatment and study design are described. Adherence to these recommendations will improve clinical relevance, quality and comparability of future studies dealing with aortic vascular graft infections.

Effects of the ABO-mismatch between donor and recipient of cryopreserved arterial homografts.

AIM: Cryopreserved arterial homograft (CAH) is a well-established substitute material for in situ reconstruction of vascular infections. However, their degeneration remains serious complication. Although several studies propose ABO-mismatching between CAH-donor and -recipient as the main reason, the results are controversial. We compared the outcome between ABO-compatible and ABO-incompatible CAH recipients to evaluate the contribution of ABO-mismatching. METHODS: Between January 2004 and December 2007, a retrospective review in 32 patients who underwent CAH-implantation was performed. The patients were divided in ABO-incompatible (group A: 17/32 patients; 53%) and ABO-compatible (group B: 15/32 patients; 47%) to CAH donor. Leucocytes, platelets and C-reactive protein (CRP) levels were recorded during the in-hospital stay. These were correlated with the surface of implanted homograft (SIH). Mid-term survival- and freedom-from-reoperation (FFR) rates were also calculated. RESULTS: In both groups, peak of leucocytes and CRP was recorded on third postoperative day (POD3) and regarding platelets lowest values on POD1. Interestingly, a second CRP-peak was reported on POD8 in group A (A: 172±104mg/L vs. B: 75±55mg/L, P=0.01). No relationship between second CRP-peak and SIH was found. After 27 months median follow-up (range, 5-49 months), survival- (65% vs. 84%, P=0.28) and FFR-rates (94% vs. 93%, P=0.98) remained comparable. CONCLUSION: We consider that the second CRP-peak expresses an early cytoimmunologic response of ABO-incompatible recipients against CAH. However, we did not find any relationship between ABO-incompatibility and poor mid-term outcome in terms of reoperation or mortality. Longer surveillance of our patients is mandatory.

A biological alternative to alloplastic grafts in dialysis therapy: evaluation of an autologised bioartificial haemodialysis shunt vessel in a sheep model.

OBJECTIVES: To evaluate bioartificial haemodialysis access grafts in a sheep model with respect to patency and morphology. MATERIAL AND METHODS: Bovine internal thoracic arteries (n=28) were decellularised. Fourteen grafts (DC grafts) were directly implanted as cervical AV shunts, the remaining were re-seeded with endothelial cells (ECs) derived from blood samples of the later ovine recipient (EC grafts) first. Following simulated punctures and duplex ultrasound scans to determine patency, grafts were explanted for immunohistochemical characterisation after 3 and 6 months, respectively. DC grafts underwent biomechanical testing for compliance (C), suture retention strength (SRT), and burst pressure (BP) before (n=6) and after (n=6) implantation. RESULTS: Following 3 and 6 months, the majority of EC (n=6/6; n=6/7) and DC grafts (n=5/6; n=5/7) were patent and not relevantly stenosed (peak systolic velocity: EC grafts=76 cm s(-1)±4; DC grafts=77 cm s(-1)±5). Simulated haemodialysis punctures revealed significantly shorter bleeding times in all bioartificial grafts than in native jugular veins (P>0.001). Comparing native carotid arteries with DC grafts prior to and post-implantation, the latter differed significantly with respect to C (P>0.001; P=0.005), whereas only pre-implant DC grafts differed regarding BP (P=0.002); no differences were observed for SRT. Histology revealed complete endothelial surface coverage of EC, but not DC grafts. Furthermore, DC grafts exhibited areas of pronounced tissue calcification. CONCLUSION: The preclinical development of a bioartificial haemodialysis access graft with promising mechanical and morphological properties in a sheep model is feasible.

Carotid endarterectomy with or without concomitant aortocoronary bypass in elderly patients: an analysis of 599 procedures.

AIM: Carotid endarterectomy (CEA) has been established as an effective treatment of carotid artery disease. Controversial remains the performance of CEA in elderly patients. Aim of this study is to report the mid-term (30 days) neurological outcome in patients older than 75 years after CEA with or without simultaneous aortocoronary bypass (CABG). METHODS: 599 patients undergoing CEA from January 2000 to December 2007 were enrolled. Isolated CEA was performed in 398/599 (66%) patients (group A). In 201/599(34%) patients (group B) was performed a combined procedure (CEA/CABG). 90/398(23%) patients of group A (group A1) and 49/201(24%) patients of group B (group B1) were >75 years old. 308/398 (77%) patients of group A (group A2) and 152/201 (76%) patients of group B (group B2) were <75 years old. Mortality, TIA and stroke rates as well as pre- and postoperative Rankin scale (RS) were reported. RESULTS: In isolated CEAs, mortality was higher in group A1 (A1:1.1% vs A2:0%, P=0.51). We found no significant differences in rates of TIA (A1:4.4% versus A2:3.2%, P=0.79) or stroke (A1:2.2% versus A2:1.9%, P=0.98). In CEA/CABG, mortality was 0% in group B1 and 5.9% in group B2 (P=0.17). No significant differences in rates of TIA (B1:2% versus B2:3%, P=0.76) or stroke (B1:2% versus B2:5%, P=0.70) were reported. Preoperative RS was the only positive predictor for postoperative stroke in groups A1 (P=0.02) and B1 (P=0.001). CONCLUSION: CEA is an appropriate and safe procedure in elderly patients. Under consideration should be the performance of CEA in elderly patients with high preoperative RS.

Significance of infection markers and microbiological findings during tissue processing of cryopreserved arterial homografts for the early postoperative course.

BACKGROUND: To evaluate homograft implantation for the urgent treatment of vascular infections on the basis of the course of infection using microbiological findings in perioperatively obtained specimens and during homograft processing. PATIENTS AND METHODS: 85 patients were treated with cryopreserved homografts from 2004-2007. The microbiological findings of the decontamination process of homografts in the tissue bank were evaluated. The perioperative infection profile (microorganisms, CRP, leukocytes, body temperature) of the patients was analysed. RESULTS: Complete microbiological and clinical follow-up for the postoperative course was available for 35 patients, who were treated with homografts from the same tissue bank and finally included into this study. 55 cryopreserved homografts were implanted. 35/55 (64%) homografts were positive for microorganisms before decontamination. 3/35 (9%) homografts remained positive after the decontamination. 33 patients were operated for prosthetic graft infection and 2 for an infiltration of a large vessel from neighbouring malignant disease. The most common infection agent was Staphylococcus aureus. Thirty-day mortality was 20% (7/35). Only in 4/35 (11%) patients were the microorganisms of the intraoperative swabs also detected during the postoperative course. The microorganisms were ORSA, Enterococcus faecium, Enterobacter aerogenes and Burkholderia cepacia. The patient with ORSA infection died on POD 11 from multiple organ failure and all other patients recovered. None of the postoperative swabs showed the homograft predecontamination microorganisms. Interestingly, a significant association (P = 0.003) between C-reactive protein increase two weeks after surgery and donor-recipient ABO mismatch was found. CONCLUSIONS: The implantation of homografts following the established decontamination is an alternative urgent therapeutic option in vascular infections with encouraging outcomes. The absence of the predecontamination focus in the postoperative specimens of patients, suggests that the postoperative course and outcomes show no strong relation to potential homograft contamination prior to the decontamination process.

New developments in tissue engineering of vascular prosthetic grafts.

Despite remarkable developments in vascular medicine in the last decades and intensive research on the improvement of bypass materials, an ideal bypass graft comparable to autologous veins or arteries is still not available for peripheral vascular and coronary artery bypass grafting. This article reviews established bypass materials and provides an overview over interesting new technologies particularly those associated with tissue-engineering and those already adopted clinically.

Human iliac vein replacement with a tissue-engineered graft.

The modification of a previously described technique to generate venous conduits in a lamb model from a decellularised matrix and autologous cells and its application to human tissue is described. A 49-year-old woman underwent surgery for a large malignant pelvic tumour (carcinoma of unknown primary) involving the right iliac artery and vein. The right iliac artery was reconstructed with a cryopreserved human arterial allograft. For iliac vein reconstruction a tissue-engineered neo-vein was developed utilising a decellularised cryopreserved vein allograft that was reseeded in a bioreactor with autologous endothelial cells derived from the recipient's great saphenous vein. Both interposition grafts were patent initially, after 3, 6, 12, and 24 months, but the tissue-engineered neo-vein had become obstructed due to evolving disease four month postoperatively. Tissue engineered neo-veins may be a therapeutic option in selected cases with symptomatic vein stenosis or obstruction not curable with interventional methods or standard prosthetic replacement.

Preclinical development of tissue-engineered vein valves and venous substitutes using re-endothelialised human vein matrix.

OBJECTIVE: The aim of the study was to evaluate the use of a decellularised scaffold and its re-endothelialisation in vitro in order to create human vascular substitutes containing venous valves. This research is clinically relevant particularly with regard to the development of venous (valve containing) transplants to replace a diseased femoral vein valve and/or obstructed veins. This technique may enable causal treatment of venous reflux and obstructions. MATERIALS AND METHODS: Valve-bearing segments of human allogeneic great saphenous veins (GSVs) were decellularised using sodium deoxycholic acid (SD) and treated with DNase I. Human venous endothelial cells (ECs) were enzymatically harvested from the GSV, expanded up to the 3rd passage using FCS (n=20) or human AB serum (hABS; n=8) supplemented media before used for re-seeding. In special bioreactors, 3D re-seeding of 28 decellularised GSV was performed with constant perfusion (A; n=8), bidirectional perfusion (B; n=8), bidirectional perfusion/reduced flow (C; n=2), static conditions (D; n=2), and bidirectional perfusion/reduced flow using hABS (E; n=8) instead of FCS. Decellularised GSV, scaled-up EC and 3D-seeded tissue-engineered valve containing neo-veins underwent immunohistochemical and PCR characterisation. RESULTS: Intact collagen and elastin networks as well as complete acellularity were shown after GSV decellularisation. In EC culture, supplementation with hABS led to a significantly higher expression of vWF compared to FCS (p=0.025). Additional EC markers such as CD 31, FLK-1 and VE-Cadherin were not altered. EC re-seeding using hABS supplemented medium (E) led to a confluent monolayer of cells that were immunohistochemically positive for FLK-1, CD 31, vWF and VE-Cadherin and by means of PCR after RNA preparation in 7 of 8 cases but was unsuccessful if FCS was used (A-D). In A-D cells presented as conglomerates positive for CD 31 and VE-Cadherin, suggesting sufficient intercellular contact but not cell-matrix contact. CONCLUSIONS: Treatment with SD and DNase enables complete decellularisation of human valve containing veins whereas 3D matrix components such as collagen and elastin remain preserved. The lumen of the scaffold including the valves can be successfully re-seeded with a human EC monolayer in a 3D bioreactor. There is substantial evidence that hABS and not FCS is essential for the completion of cell-matrix contacts in human veins.

The role of cerebrospinal fluid S100 and lactate to predict clinically evident spinal cord ischaemia in thoraco-abdominal aortic surgery.

OBJECTIVE: Detecting spinal cord ischemia early during replacement of the thoracoabdominal aorta remains a challenge. In a high risk population, we have re-evaluated the potential impact of ischaemia/damage markers (S100, lactate) in the peripheral blood and CSF for perioperative patient management. PATIENTS AND METHODS: Thirteen patients undergoing replacement of the thoracoabdominal aorta (6 female, age 63 (27-71)) with continuous CSF pressure monitoring and drainage were entered into the study. A total of 485 CSF (C) and serum (S) samples were collected and analysed for S100, lactate and glucose. RESULTS: Two patients suffered from spinal cord injury (SCI) (15%). During and early after surgery, there was a strong correlation between C-S100 levels (r=0.79) and C-lactate levels (r=0.77) with time in patients with SCI. In patients with SCI C-lactate levels increased soon after aortic cross-clamping, whereas C-S100 levels did not become significantly elevated until 6 hours after cross-clamping. CONCLUSION: An increase of C-lactate occurs much earlier than the increase in C-S100 in patients with SCI. Both parameters may be used to adjust protective and therapeutic measures intra- and postoperatively.