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.

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.

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.

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.

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.

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.

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.

Homograft patch repair in carotid artery rupture.

The case of a 24-year-old man with a rupture of the left common carotid artery and history of intravenous drug abuse is presented. Due to absence of a suitable autologous vein segment the carotid bulb was repaired with a human allograftpatch.

Severe obstructive calcifications affecting the descending and suprarenal abdominal aorta without coexisting peripheral atherosclerotic disease--coral reef aorta.

The case of a 58-year-old woman with leg claudication due to a very rare form of atherosclerosis affecting the descending thoracic and abdominal aorta--known as coral reef aorta--without involvement of the femoro-distal vessels is reported. The patient was treated with a polyester bifurcation graft from the proximal descending aorta to both common iliac arteries via a left dorsal mini-thoracotomy and a second left retroperitoneal approach. This unusual approach was chosen instead of direct aortic replacement in order to prevent paraplegia. In case of future visceral or left renal malperfusion the diseased artery can be connected to the prosthesis directly or by the use of an additional bypass graft. This would not be the case with a conventional axillo-bifemoral graft.

[Tissue engineering for heart valves and vascular grafts]. / Tissue Engineering für Herzklappen und Gefässe.

Current prosthetic substitutes for heart valves and blood vessels have numerous limitations such as limited durability (biological valves), susceptibility to infection, the necessity of lifelong anticoagulation therapy (prosthetic valves), and reduced patency in small-caliber grafts, for example. Tissue engineering using either polymers or decellularized native allogeneic or xenogenic heart valve/vascular matrices may provide the techniques to develop the ideal heart valve or vascular graft. The matrix scaffold serves as a basis on which seeded cells can organise and develop into the valve or vascular tissue prior to or following implantation. The scaffold is either degraded or metabolised during the formation and organisation of the newly generated matrix, leading to vital living tissue. This paper summarises current research and first clinical developments in the tissue engineering of heart valves and vascular grafts.

Differentiated corticosteroid production and regeneration after selective transplantation of cultured and noncultured adrenocortical cells in the adrenalectomized rat.

UNLABELLED: Syngeneic transplantation of adrenocytes was investigated in Lewis rats in regard to differentiated hormone secretion and cortex regeneration after bilateral adrenalectomy as an alternative to steroid substitution. METHODS: Purified cell suspensions of glomerulosa (density 1.061 +/- 0.001 g/ml) and fasciculata (density 1.034 +/- 0.003 g/ml) cells were obtained by density gradient separation and were transplanted under the kidney capsule either immediately or after a 29-day culture period. Animals were killed after transplantation of cultured glomerulosa (CG-Tx) or cultured fasciculata cells (CF-Tx), noncultured glomerulosa cells (G-Tx) or non-cultured fasciculata cells (F-Tx), or both cell types (GF-Tx) for morphological studies after 30, 120, and 360 days. Plasma samples were drawn for measurement of corticosterone and aldosterone as well as 24 hr-urine for sodium and potassium levels at day 3, 30, 120, and 360 after transplantation. RESULTS: In primary culture fasciculata cell number remained stationary although glomerulosa cell number increased to almost 10-fold. Vital cortex cells were demonstrated in each explanted graft by histochemistry but only group G-Tx, CG-Tx, and GF-Tx (purified cell suspensions of zona glomerulosa and fasciculata) showed neocortex-like structures. We found plasma (urine) corticosterone to decrease from preoperatively 256-304 ng/ml (226-239 ng/day) in untreated animals to levels about half as high 3 days after transplantation, increasing to normal values in all study groups 30 days after treatment (data given as range). Plasma aldosterone concentrations, 150-180 pg/ml in untreated rats, decreased to nondetectable levels for 1 week after bilateral adrenalectomy. At day 30 group GF-Tx, G-Tx, and CG-Tx showed comparable aldosterone plasma concentrations (104-122 pg/ml); however, levels in F-Tx and CF-Tx were 19-49 pg/ml, and did not increase significantly within the observation period. CONCLUSIONS: Cells derived from the zona glomerulosa maintain viability, produce both aldosterone and corticosterone, and regenerate a neocortex with cells that histologically resemble both zona glomerulosa and fasciculata cells. They are therefore suitable for adrenocortical transplantation. In contrast, cells derived from the zona fasciculata maintain viability, but do not regenerate zona glomerulosa and do not produce aldosterone. These results suggest that the cell migration model, in which zona glomerulosa cells can acquire the phenotype of zona fasciculata cells as they can migrate centripetally, is more likely the correct explanation of adrenocortical zonation.

Impact of chronic infection with chlamydia pneumoniae on incidence of cardiac allograft vasculopathy.

Chronic infection with Chlamydia pneumoniae (CP) is associated with development of coronary disease. However, little information exists concerning CP infection and impact on posttransplant cardiac allograft vasculopathy (CAV). A total of 202 patients were investigated 5.5+/-3.1 years after cardiac transplantation (46.5+/-11.0 years; 169 male, 33 female). Assessment of CAV was performed by annual coronary angiograms. Chlamydia serology (IgG/IgA) was performed using micro-immunofluorescence. Statistics comprised analysis of variance and Kaplan-Meier analysis. A total of 152 patients were CAV positive. Elevated titers were present in 45% (IgG) and 72.8% (IgA) of patients. Generally, serostatus was not associated with development of CAV when evaluated over the total postoperative interval. However, after month 14 there was a significant trend toward lower actuarial freedom from CAV in patients with elevated IgA titers. CP seems not to play a significant role in the development of CAV early after heart transplantation but might be a predicting risk factor after the first postoperative year.

Transplantation of H-2Kb-transgenic adrenocortical cells in the mouse having undergone an adrenalectomy: functional and morphological aspects.

BACKGROUND: A new model of cellular adrenocortical transplantation after bilateral adrenalectomy in the mouse was established. This model was used to study the effects of the expression of the transgenic MHC class I molecule H-2K(b) (Kb) on graft survival and morphologic features, corticosterone secretion, and the possibility of tolerance induction in the recipient. METHOD: A single cell suspension of purified adrenocortical cells was grafted under the kidney capsule of B10.Br (H-2k) mice having adrenalectomies. Syngeneic, fully MHC-mismatched, and MHC class I-incompatible Kb-transgenic mice served as donor strains. To analyze graft function, urinary excretion and serum levels of corticosterone were monitored over 100 days. Tolerance induction in the graft recipients of Kb-transgenic and third party skin grafts was tested on day 50 after adrenocortical transplantation. Histological sections of the adrenocortical grafts were obtained on day 100. RESULTS: Recipients of syngeneic and Kb-transgenic grafts displayed pretransplant corticosterone levels on days 20, 50, and 100 and ACTH-stimulated serum corticosterone levels similar to those of controls on day 100 after adrenocortical transplantation. In contrast, in recipients of fully MHC-mismatched grafts, corticosterone excretion was significantly reduced. In this group, 4 of 7 mice did not survive. Syngeneic skin grafts survived indefinitely in recipients of syngeneic and Kb-transgenic adrenocortical grafts, whereas Kb-transgenic and fully MHC-mismatched skin grafts were acutely rejected. Tissue sections of the adrenocortical grafts revealed vascularized cell conglomerates in syngeneic and Kb-transgenic grafts without infiltrations of mononuclear cells. Furthermore, a differentiation similar to adrenocortical organization was partly found. CONCLUSION: In conclusion, a model of cellular adrenocortical transplantation was established. The results show that syngeneic transplantation resulted in physiological corticosterone levels early after transplantation, whereas fully MHC-incompatible grafts were rejected. Recipients of Kb-transgenic grafts showed unimpaired adrenocortical function, but did not tolerize toward Kb-transgenic skin grafts. Possible mechanisms include a local immunomodulatory effect of glucocorticoids secreted by the graft and a low immunogenicity of the relatively small numbers of transplanted cells.