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.

Percutaneous dilatational tracheostomy (PDT) in trauma patients: a safe procedure.

PURPOSE: Percutaneous dilatational tracheostomy (PDT) is a standard procedure routinely performed on intensive care units. While complication rates and long-term outcomes have been studied in different patient populations, there are few studies known to these authors involving PDT in trauma patients and the complications which may result. METHODS: Between March 2007 and August 2013, all instances and peri-procedural complications during PDT occurring on the trauma intensive care unit, a unit specialized in the care of injured patients and especially polytrauma patients, were documented. PDTs were performed by a surgeon with the assistance and supervision of another, using bronchoscopic guidance performed by the respiratory medicine department. RESULTS: 289 patients were included in the study, 225 men and 64 women with a mean age of 49 ± 21 years. Complications occurred in 37.4 % of cases. The most common complication, bleeding, occurred in 26.3 % of patients ranging from little to severe bleeding. Fracture of tracheal cartilage occurred in 6 % of PDT cases. Additional complications such as dislocation of the guidewire, hypotension, and oxygen desaturation were observed. Most complications did not require treatment. The second tracheal intercartilaginous space was successfully intubated in 82 % of cases. CONCLUSIONS: PDT is a safe procedure in trauma patients. When considering the severity of complications such as major blood loss, pneumothorax, or death, this evidence suggests that PDT is safer in trauma patients compared to other patient cohorts.

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.

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.

Adipogenic differentiation of laser-printed 3D tissue grafts consisting of human adipose-derived stem cells.

Laser-assisted bioprinting (LaBP) allows the realization of computer-generated 3D tissue grafts consisting of cells embedded in a hydrogel environment. In this study, human adipose-derived stem cells (hASCs) were printed in a free-scalable 3D grid pattern by means of LaBP. We demonstrate that neither the proliferation ability nor the differentiation behaviour of the stem cells was affected by the LaBP procedure. Furthermore, the 3D grafts were differentiated down the adipogenic lineage pathway for 10 days. We verify by quantitative assessments of adipogenic markers that the 3D grafts resemble cell lineages present in natural adipose tissue. Additionally, we provide the proof that even pre-differentiated hASCs could be utilized for the generation of 3D tissue grafts. These results indicate that the biofabrication of living grafts resembling their complex native origin is within reach.

[Jehova's Witnesses and severe injury with impending hemorrhaging : how complex is the treatment?]. / Zeugen Jehovas und Schwerverletzung mit drohender Hämorrhagie : Wie komplex ist die Behandlung?

Hemorrhage is frequently seen during the early phases of polytrauma management and intensive care treatment of the severely injured. Traumatic coagulopathy as well as the sometimes overlooked hyperfibrinolysis may lead to further complications. Therefore, transfusion of blood products and coagulation factors is often crucial. Jehova's Witnesses reject transfusions of blood and blood products due to religious convictions. In this case report a therapeutic approach of a multiple trauma patient suffering from traumatic brain injury, blunt chest trauma and liver laceration is described, who has been treated without blood products. As one main focus, ethical as well as legal aspects are discussed. Beside therapeutic concepts, such as the administration of coagulation factors, recombinant erythropoietin and iron, ethical and legal aspects remain part of the controversial discussion.

[Viability of autologous fat grafts harvested with the Coleman technique and the tissu trans system (shippert method): a comparative study]. / Vitalitätsvergleich von Fetttransplantaten welche mit der Coleman Technik und dem Tissu Trans System (Shippert Methode) gewonnen wurden.

Various methods for harvesting and refining autologous fat grafts have been described. One of the standard procedures, the Coleman technique, is based on manual aspiration to reduce the negative presssure and the centrifugation of the grafts. The Shippert technique uses automatic liposuction with reduced negative pressure and abstains from centifugation in order not to reduce viability of the graft by exposing it to centrifugal forces. This study intends to compare the viability of fat grafts processed with the above-mentioned methods.Fat grafts were obtained in 9 patients by using both the Tissu Trans system (Shippert technique) and the Coleman technique. To evaluate the impact of centrifugation forces, the grafts harvested with the Coleman technique were treated with standard adjustment of the centrifuge and also with doubled g-force. Viability of fat grafts was analysed with the WST-8 test and with annexin V/PI assay FACS analysis.The viability of fat grafts processed by the Coleman technique was significantly higher compared to the Shippert technique on applying the WST-8 test. Applying the annexin V/PI analysis, the viability of fat grafts was almost equal with both techniques. Whereas the fat grafts processed with the Tissu Trans system are injected without condensation, the grafts refined with the Coleman technique were concentrated 3 times by centrifugation compared to the primary liposuctioned graft volumes.The Coleman technique allows the preparation of a fat graft containing more viable cells than the Shippert technique. This is in part due to the condensation of the graft by centrifugation using the Coleman technique. The factor of condensation of the grafts harvested and refined with the Coleman technique exceeds the factor of increased fat graft viability in comparison to the Shippert technique. The Tissu Trans system is more than twice as fast and easier to use with a preferential use for large volume grafts like in breast augmentation, whereas the Coleman technique produces a more condensed graft, favouring it for fat grafting to the face where less volume is needed.

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.

Involvement of lignification and membrane permeability in the tomato root response to boron toxicity.

To gain an insight into the role of lignification and membrane permeability in the root response to boron (B) toxicity, lignification-related enzymes and a number of physiological and oxidative stress parameters were analyzed in two tomato (Solanum lycopersicum L.) cultivars (Kosaco and Josefina) subjected to 0.05 (control), 0.5 and 2mM B during 16 days. 2mM B supply inhibited root growth and increased the root B concentration in both tomato cultivars. Although excess B increased the hydrogen peroxide (H2O2) concentration in Kosaco, no major changes were observed in other oxidative-stress-related parameters. High levels of B supply also induced higher lignin deposition in Kosaco roots but did not in Josefina ones. The latter result was associated with an increase of the polyphenol oxidase (PPO), guaiacol peroxidase (GPOX) and soluble syringaldazine peroxidase (SPOX) activity in Kosaco roots. Boron toxicity did not induce lipid peroxidation but increased the leakage of K(+) and the passive efflux of B in tomato roots. We conclude that high concentrations of B do not cause major oxidative or membrane damage in tomato roots. The data also indicate that high levels of B supply induce a higher lignin deposition in Kosaco roots but not in Josefina ones. This phenomenon suggests that lignification is not an essential factor reducing root growth in tomato plants, however, it proves that exist a high genotypic variation in response to excess B at root level.

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.

[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.

Microtubule-dependent matrix metalloproteinase-2/matrix metalloproteinase-9 exocytosis: prerequisite in human melanoma cell invasion.

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that cleave and degrade a wide spectrum of extracellular matrix components. By enhancing turnover of extracellular matrix, MMP activity is also known to play a key role in tumor cell invasion. Because extracellular protease activity requires efficient release of these proteases to the cellular surface, we investigated storage, transport, and exocytosis of MMP-2 and MMP-9 in human melanoma cells using immunofluorescence, electrical, and biochemical techniques. Immunolabeling of melanoma cells with antibodies specific for MMP-2 and MMP-9 led to the identification of two distinct populations of small cytoplasmatic vesicles containing MMP-2 or MMP-9, respectively. In combination with alpha-tubulin-specific antibodies, both vesicle populations were found to be aligned along the microtubular network. Moreover, the molecular motor protein kinesin is shown to be localized on most of these vesicles, providing evidence that the identified vesicles are actively propelled along microtubules toward the plasma membrane. The functional relevance of these findings is demonstrated using low dosage (5.9 nmol/L) of paclitaxel to affect the microtubular function of melanoma cells. Although cell proliferation is not altered, paclitaxel treatment impairs secretion of MMP-2/MMP-9 and significantly reduces invasive activity in our new cell invasion assay. In conclusion, we demonstrate in melanoma cells that microtubule-dependent traffic of MMP-containing vesicles and exocytosis are critical steps for invasive behavior and therefore are potential targets for specific antitumor drugs.

Acellularized porcine heart valve scaffolds for heart valve tissue engineering and the risk of cross-species transmission of porcine endogenous retrovirus.

OBJECTIVE: Acellularized porcine heart valve scaffolds have been successfully used for heart valve tissue engineering, creating living functioning heart valve tissue. However, there is concern about the possibility of porcine endogenous retrovirus transmission. In this study we investigated whether acellularized porcine heart valve scaffold causes cross-species transmission of porcine endogenous retrovirus in a sheep model. METHODS: Acellularized porcine pulmonary valve conduits (n = 3) and in vitro autologous repopulated porcine pulmonary valve conduits (n = 5) were implanted into sheep in the pulmonary valve position. Surgery was carried out with cardiopulmonary bypass support. The animals were killed 6 months after the operation. Blood samples were collected regularly up to 6 months after the operation and tested for porcine endogenous retrovirus by means of polymerase chain reaction and reverse transcriptase-polymerase chain reaction. In addition, explanted tissue-engineered heart valves were tested for porcine endogenous retrovirus after 6 month in vivo. RESULTS: Porcine endogenous retrovirus DNA was detectable in acellularized porcine heart valve tissue. However, 6 months after implantation of in vitro and in vivo repopulated acellularized porcine heart valve scaffolds, no porcine endogenous retrovirus sequences were detectable in heart valve tissue and peripheral blood. CONCLUSION: Acellularized porcine matrix scaffolds used for creation of tissue-engineered heart valves do not transmit porcine endogenous retrovirus.

Triple valve replacement in a patient with severe carcinoid heart disease.

We report on the case of a 34-year-old male patient suffering from end-stage carcinoid heart disease with severe tricuspid, pulmonary and mitral valve regurgitation. In addition, a persisting foramen ovale was present. The primary carcinoid tumor was never discovered. However, urine 5-hydroxy-indole-acetic-acid (5-HIAA) were consistently elevated after the first diagnosis of carcinoid disease and after eight years of medication with Octreotide and Interferon alpha-2b our patient developed significant cardiac insufficiency mainly due to severe valvular dysfunction. Ultimately, mechanical tricuspid, mitral and pulmonary valve replacement was performed. Twelve hours following the operation the patient had to be returned to the operating room for persisting intrathoracic hemorrhage. He recovered uneventfully and was discharged from hospital on day 37. Twelve months following triple valve replacement the cardiac status recovered from preoperative NYHA-IV to NYHA-I.