[OCT, Triple H or anything else?] / OCT, Triple H oder doch etwas anderes?

Hyperammonemia is a life-threatening condition, the prognosis of which depends on a rapid reduction of ammonia. If a hepatic cause is excluded, the differential diagnosis is broad and even in adulthood includes hereditary metabolic diseases. Here, the case of a 25-year-old female patient with severe hyperammonemia refractory to standard therapy is described and the relevance of extracorporeal elimination of ammonia emphasized.

The Use of Intact Fish Skin Grafts in the Treatment of Necrotizing Fasciitis of the Leg: Early Clinical Experience and Literature Review on Indications for Intact Fish Skin Grafts.

BACKGROUND: Necrotizing fasciitis (NF) is a serious infectious disease that can initially place the patient's life in danger and, after successful surgical and antibiotic treatment, leaves extensive wounds with sometimes even exposed bones and tendons. Autologous skin grafts are not always possible or require adequate wound bed preparation. Novel intact fish skin grafts (iFSGs; Kerecis® Omega3 Wound, Kerecis hf, Isafjördur, Iceland) have already shown their potential to promote granulation in many other wound situations. Faster wound healing rates and better functional and cosmetic outcomes were observed due to their additionally postulated anti-inflammatory and analgesic properties. Therefore, iFSGs may also be essential in treating NF. We present our initial experience with iFSGs in treating leg wounds after NF and review the literature for the current spectrum of clinical use of iFSGs. CASE PRESENTATIONS: We present two male patients (aged 60 and 69 years) with chronic or acute postsurgical extensive leg ulcers six weeks and six days after necrotizing fasciitis, respectively. Both suffered from diabetes mellitus without vascular pathologies of the lower limbs. A single application of one pre-meshed (Kerecis® Graftguide) and one self-meshed 300 cm2 iFSG (Kerecis® Surgiclose) was performed in our operation room after extensive surgical debridement and single circles of negative wound pressure therapy. Application and handling were easy. An excellent wound granulation was observed, even in uncovered tibia bone and tendons, accompanied by pain relief in both patients. Neither complications nor allergic reactions occurred. The patients received autologous skin grafting with excellent functional and cosmetic outcomes. CONCLUSIONS: iFSGs have the potential to play a significant role in the future treatment of NF due to the fast promotion of wound granulation and pain relief. Our experience may encourage surgeons to use iFSGs in NF patients, although high-quality, large-sized studies are still required to confirm these results. The observed effects of iFSGs on wounds associated with NF may be transferred to other wound etiologies as well.

Development of a dual-component infection-resistant arterial replacement for small-caliber reconstructions: A proof-of-concept study.

Introduction: Synthetic vascular grafts perform poorly in small-caliber (<6mm) anastomoses, due to intimal hyperplasia and thrombosis, whereas homografts are associated with limited availability and immunogenicity, and bioprostheses are prone to aneurysmal degeneration and calcification. Infection is another important limitation with vascular grafting. This study developed a dual-component graft for small-caliber reconstructions, comprising a decellularized tibial artery scaffold and an antibiotic-releasing, electrospun polycaprolactone (PCL)/polyethylene glycol (PEG) blend sleeve. Methods: The study investigated the effect of nucleases, as part of the decellularization technique, and two sterilization methods (peracetic acid and γ-irradiation), on the scaffold's biological and biomechanical integrity. It also investigated the effect of different PCL/PEG ratios on the antimicrobial, biological and biomechanical properties of the sleeves. Tibial arteries were decellularized using Triton X-100 and sodium-dodecyl-sulfate. Results: The scaffolds retained the general native histoarchitecture and biomechanics but were depleted of glycosaminoglycans. Sterilization with peracetic acid depleted collagen IV and produced ultrastructural changes in the collagen and elastic fibers. The two PCL/PEG ratios used (150:50 and 100:50) demonstrated differences in the structural, biomechanical and antimicrobial properties of the sleeves. Differences in the antimicrobial activity were also found between sleeves fabricated with antibiotics supplemented in the electrospinning solution, and sleeves soaked in antibiotics. Discussion: The study demonstrated the feasibility of fabricating a dual-component small-caliber graft, comprising a scaffold with sufficient biological and biomechanical functionality, and an electrospun PCL/PEG sleeve with tailored biomechanics and antibiotic release.

[Age-related vascular changes exemplified by the carotid artery]. / Alterungsbedingte Gefäßveränderungen am Beispiel der Arteria carotis.

One of the main risk factors for the presence of carotid stenosis and carotid-related stroke is age. The aim of this review article is to present the current state of knowledge on age-related vascular changes using carotid stenosis as an example.Vascular aging (vascular senescence) is a decrease of structural and functional properties of the vessel wall that takes place on different levels. At the multicellular level an increase in vessel volume and diameter as well as intima media thickness occurs with age mainly due to atherosclerotic changes in the vessel wall. At the cellular and extracellular levels there is a decrease in elastin fibers, smooth muscle cells, and total cellularity, an increase in lipid, cholesterol, and calcium phosphate deposition as well as neovascularization. The causes of vascular aging at the molecular level include, in particular oxidative stress, chronic inflammatory response, mitochondrial dysfunction, epigenetic changes, dysregulation of the expression of non-coding RNAs (ncRNAs), and the increase in senescence. Age-related loss of tissue healing and repair capacity make plaques more vulnerable and, in the case of the carotid artery, more susceptible to ischemic stroke.Increasing knowledge of the influence of aging on the epigenetics and ncRNAs in atherosclerotic plaques can in the future more accurately quantify individual patient risk and contribute to the development of targeted therapeutic strategies; however, further studies are needed in this field to understand the full extent of vascular aging and its associated diseases so that these can then be specifically targeted.

Out of sight for the endoscopist? Gastrointestinal bleeding after aortic repair.

BACKGROUND AND STUDY AIM: Secondary Aortoenteric Fistulas (sAEF) are difficult to diagnose and usually result in fatal gastrointestinal (GI) bleeding following aortic repair. Outcomes are largely dependent on a timely diagnosis, but AEFs remain challenging to identify endoscopically and are usually diagnosed on computed tomography (CT) scans. The aim of our study was optimize diagnosis of AEF by identifying patients developing GI bleeding after aortic repair, investigate their clinical course and identify factors specific to different bleeding sources. METHODS: A retrospective, single-center study capturing all patients developing upper or lower GI bleeding after aortic surgery between January 2009 and March 2020 was performed. Electronic health records were screened for diagnostic codes of the relevant procedures. Bleeding was classified into three groups: AEF with demonstrable fistula, ischemic - macroscopic ulceration plus histological confirmation or imaging and "other" due to other recognized conventional cause, such as peptic ulcer disease. RESULTS: 47 GI bleeding episodes in 39 patients were identified. Of these, 10 episodes (21%) were caused by AEF, 16 (34%) by ischemic ulceration and 21 (45%) due to other causes. Patients with AEF exhibited more frequent hemodynamic instability requiring vasopressors and had higher mortality, while ischemic ulcerations were associated with more recent operation or hypotensive episode. CONCLUSIONS: GI bleeding complications are uncommon following aortic surgery. AEF and ischemic ulceration are however frequent bleeding causes in this cohort. In patients presenting with fulminant bleeding, primary CT-scanning should be considered.

Circulating microRNAs in Symptomatic and Asymptomatic Carotid Stenosis.

Background: Specific microRNAs (miRs) have been implicated in the pathophysiology of atherosclerosis and may represent interesting diagnostic and therapeutic targets in carotid stenosis. We hypothesized that the levels of specific circulating miRs are altered in patients with symptomatic carotid stenosis (sCS) in comparison to those in patients with asymptomatic carotid stenosis (aCS) planned to undergo carotid endarterectomy (CEA). We also studied whether miR levels are associated with plaque vulnerability and stability over time after CEA. Methods: Circulating levels of vascular-enriched miR-92a, miR-126, miR-143, miR-145, miR-155, miR-210, miR-221, miR-222, and miR-342-3p were determined in 21 patients with sCS and 23 patients with aCS before CEA and at a 90-day follow-up. Transcranial Doppler ultrasound for detection of microembolic signals (MES) in the ipsilateral middle cerebral artery was performed prior to CEA. Carotid plaques were histologically analyzed. Results: Mean levels of miRs were not considerably different between groups and were only marginally higher in sCS than aCS concerning miR-92a, miR-210, miR-145, and miR-143 with the best evidence concerning miR-92a. After adjustment for vascular risk factors and statin pre-treatment, the effect sizes remained essentially unchanged. At follow-up, however, these modest differences remained uncorroborated. There were no relevant associations between miR-levels and MES or histological plaque vulnerability features. Conclusions: This study does not provide evidence for strong associations between specific circulating miRs and symptomatic state in a collective of comprehensively characterized patients with carotid stenosis. Further work is needed to elucidate the role of circulating miRs as targets in advanced carotid atherosclerosis.

Perfusion promotes endothelialized pore formation in high concentration fibrin gels otherwise unsuitable for tube development.

Vascularization of tissue engineered implants is crucial for their survival and integration in the recipient's body. Pre-vascularized, fibrin-based implants offer a solution since low concentration fibrin hydrogels (1 mg/mL) have been shown to promote tube formation of endothelial cells in co-culture with adipogenic stem cells. However, higher fibrinogen concentrations (> 20 mg/mL) enabling the fabrication of stable implants are necessary.We here characterized fibrin gels of 1-30 mg/mL for their rheological properties and whether they support tube formation of endothelial cell-adipogenic stem cell co-cultures for up to 7 days. Moreover, 20 mg/mL gels containing preformed channels and endothelial cell-adipogenic stem cell co-culture were perfused continuously in a customized flow chamber with 3.9 dyn/cm2 for 12 days and analyzed for capillary formation.Rheology of fibrin gels showed increasing stability proportional to fibrinogen concentration with 20 mg/mL gels having a storage module of 465 Pa. Complex tube networks stable for 7 days were observed at 1-5 mg/mL gels whereas higher concentrations showed initial sprouting only. However, perfusion of 20 mg/mL fibrin gels resulted in endothelialized pore formation in several layers of the gel with endothelial cell-adipogenic stem cell co-culture.Thus, perfusion supports the formation of capillary-like structures in fibrin gels that are too dense for spontaneous tube formation under static conditions. Future studies are necessary to further increase pore density and to investigate proper nutrition of tissue-specific target cells in the scaffold.

Distinct systemic cytokine networks in symptomatic and asymptomatic carotid stenosis.

Inflammatory processes are crucial in atherosclerosis and atherothrombosis. This study aimed to identify a cytokine-pattern that is associated with plaque-vulnerability or symptomatic state in comprehensively investigated patients with symptomatic (sCS) and asymptomatic carotid stenosis (aCS). Twenty-two patients with sCS and twenty-four patients with aCS undergoing carotid endarterectomy (CEA) were considered. A cytokine-panel was measured in plasma-specimens prior to surgery and at a 90 day follow-up. Doppler-ultrasound detecting microembolic signals (MES) in the ipsilateral middle cerebral artery was performed. Carotid plaques were analysed regarding histopathological criteria of plaque-vulnerability and presence of chemokine receptor CXCR4. Correction for multiple comparisons and logistic regression analysis adjusting for vascular risk factors, grade of stenosis, antithrombotic and statin pretreatment were applied. In sCS-patients higher plasma-levels of Fractalkine (CX3CL1), IFN-α2, IL-1ß, IL-2, IL-3, IL-7 were found compared to aCS-patients. CXCR4-expression on inflammatory cells was more evident in sCS- compared to aCS-plaques and was associated with vulnerability-criteria. In contrast, plasma-cytokine-levels were not related to CXCR4-expression or other vulnerability-criteria or MES. However, in both groups distinct inter-cytokine correlation patterns, which persisted at follow-up and were more pronounced in the sCS-group could be detected. In conclusion, we identified a distinct cytokine/chemokine-network in sCS-patients with elevated and closely correlated mediators of diverse functions.

Prospective evaluation of preoperative lung ultrasound for prediction of perioperative outcome and myocardial injury in adult patients undergoing vascular surgery (LUPPO study).

BACKGROUND: Myocardial injury after non-cardiac surgery (MINS) is a frequent perioperative event in vascular surgery, associated both with worse outcome and subsequent cardiovascular events. Current guidelines advocate troponin (hs-cTnT) and NT-proBNP measurements in selected patients before surgery, but accurate preoperative identification of patients at risk for MINS is an unmet clinical need. Focused lung ultrasound (LUS) might help to select patients at increased risk for MINS, because it can visualize B-line artifacts correlating to cardiopulmonary disease. Therefore, we investigated whether quantification of B-line artifacts improves perioperative risk predictive accuracy for MINS. METHODS: In this prospective single-center observational study, 136 consecutive open vascular surgery patients underwent conventional preoperative assessment expanded by lung ultrasound. Lung ultrasound B-lines were counted in each of 28 bilateral scan fields of the anterior and lateral chest. Improvement of risk predictive accuracy was quantified with area under receiver operating characteristic (ROC) curve analysis and net reclassification improvement (NRI). RESULTS: We included 118 patients into the final analysis. Twenty-three (19%) patients fulfilled the criteria for the primary endpoint MINS. Three or more bilateral positive B-line fields were calculated as the best ROC-derived cutoff associated with an increased incidence of MINS (odds ratio: 4.4; 95% confidence interval [CI]: 1.5 to 12.7; P=0.007). Adding LUS to hs-cTnT measurements improved risk predictive accuracy for MINS (NRI: 0.36, P=0.043). CONCLUSIONS: Lung ultrasound in combination with hs-cTnT showed a better test accuracy than hs-cTnT alone and might guide clinicians to identify vascular patients at increased risk for MINS.

Methylation in pericytes after acute injury promotes chronic kidney disease.

The origin and fate of renal myofibroblasts is not clear after acute kidney injury (AKI). Here, we demonstrate that myofibroblasts were activated from quiescent pericytes (qPericytes) and the cell numbers increased after ischemia/reperfusion injury-induced AKI (IRI-AKI). Myofibroblasts underwent apoptosis during renal recovery but one-fifth of them survived in the recovered kidneys on day 28 after IRI-AKI and their cell numbers increased again after day 56. Microarray data showed the distinctive gene expression patterns of qPericytes, activated pericytes (aPericytes, myofibroblasts), and inactivated pericytes (iPericytes) isolated from kidneys before, on day 7, and on day 28 after IRI-AKI. Hypermethylation of the Acta2 repressor Ybx2 during IRI-AKI resulted in epigenetic modification of iPericytes to promote the transition to chronic kidney disease (CKD) and aggravated fibrogenesis induced by a second AKI induced by adenine. Mechanistically, transforming growth factor-ß1 decreased the binding of YBX2 to the promoter of Acta2 and induced Ybx2 hypermethylation, thereby increasing α-smooth muscle actin expression in aPericytes. Demethylation by 5-azacytidine recovered the microvascular stabilizing function of aPericytes, reversed the profibrotic property of iPericytes, prevented AKI-CKD transition, and attenuated fibrogenesis induced by a second adenine-AKI. In conclusion, intervention to erase hypermethylation of pericytes after AKI provides a strategy to stop the transition to CKD.

Sequential Surgical Procedures in Vascular Surgery Patients Are Associated With Perioperative Adverse Cardiac Events.

Patients at elevated cardiovascular risk are prone to perioperative cardiovascular complications, like myocardial injury after non-cardiac surgery (MINS). We have demonstrated in a mouse model of atherosclerosis that perioperative stress leads to an increase in plaque volume and higher plaque vulnerability. Regulatory T cells (Tregs) play a pivotal role in development and destabilization of atherosclerotic plaques. For this exploratory post-hoc analysis we identified 40 patients recruited into a prospective perioperative biomarker study, who within the inclusion period underwent sequential open vascular surgery. On the basis of protein markers measured in the biomarker study, we evaluated the perioperative inflammatory response in patients' plasma before and after index surgery as well as before and after a second surgical procedure. We also analyzed available immunohistochemistry samples to describe plaque vulnerability in patients who underwent bilateral carotid endarterectomy (CEA) in two subsequent surgical procedures. Finally, we assessed if MINS was associated with sequential surgery. The inflammatory response of both surgeries was characterized by postoperative increases of interleukin-6,-10, Pentraxin 3 and C-reactive protein with no clear-cut difference between the two time points of surgery. Plaques from CEA extracted during the second surgery contained less Tregs, as measured by Foxp3 staining, than plaques from the first intervention. The 2nd surgical procedure was associated with MINS. In conclusion, we provide descriptive evidence that sequential surgical procedures involve repeat inflammation, and we hypothesize that elevated rates of cardiovascular complications after the second procedure could be related to reduced levels of intraplaque Tregs, a finding that deserves confirmatory testing and mechanistic exploration in future populations.

Dual Pharmacological Inhibition of Angiopoietin-2 and VEGF-A in Murine Experimental Sepsis.

BACKGROUND: Sepsis is a pathological host response to infection leading to vascular barrier breakdown due to elevated levels of angiopoietin-2 (Angpt-2) and vascular endothelial growth factor-A (VEGF-A). Here, we tested a novel heterodimeric bispecific monoclonal IgG1-cross antibody of Angpt-2 and VEGF - termed "A2V." METHODS: Cecal ligation and puncture was used to induce murine polymicrobial sepsis. Organs and blood were harvested for fluorescence immunohistochemistry and RT-PCR, and survival was recorded. In vitro endothelial cells were stimulated with plasma from septic shock patients costimulated with A2V or IgG antibody followed by immunocytochemistry and real-time transendothelial electrical resistance. RESULTS: Septic mice treated with A2V had a reduced induction of the endothelial adhesion molecule ICAM-1, leading to a trend towards less transmigration of inflammatory cells (A2V: 42.2 ± 1.0 vs. IgG 48.5 ± 1.7 Gr-1+ cells/HPF, p = 0.08) and reduced tissue levels of inflammatory cytokines (e.g., IL-6 mRNA: A2V 9.4 ± 3.2 vs. IgG 83.9 ± 36.7-fold over control, p = 0.03). Endothelial permeability was improved in vivo and in vitro in stimulated endothelial cells with septic plasma. Survival was improved by 38% (p = 0.02). CONCLUSION: Dual inhibition of Angpt-2 and VEGF-A improves murine sepsis morbidity and mortality, making it a potential therapeutic against vascular barrier breakdown.

Conventional culture diagnostics vs. multiplex PCR for the detection of causative agents of vascular graft infections - results of a single centre observational pilot study.

Background: Timely diagnosis of vascular graft infections is of major importance in vascular surgery. The detection of causative microorganisms is needed for specific medical treatment, but conventional culture is often slow, insensitive and inconclusive due to antibiotic pre-treatment. Detection of bacterial DNA by polymerase chain reaction (PCR) might bypass these problems. We hypothesised that multiplex PCR (mPCR) is feasible, fast and sensitive to detect causative microorganisms in vascular graft infections. Patients and methods: We performed a pilot observational prospective study comparing conventional culture and a commercial mPCR. Inclusion criteria were: confirmed graft infection, suspicious imaging, clinical suspicion, anastomotic aneurysm and repeated graft occlusion. Diagnostic methods were performed using identical samples. Time to result, microorganisms and antibiotic resistance in both groups were compared using Student's t-test or nonparametric tests. Results: 22 samples from 13 patients were assessed and 11 samples were negative for bacteria. Some showed multiple germs. In total, we found 15 different organisms. 13 samples matched, 9 had non-concordant results. Out of the mismatches 3 microorganisms identified in PCR were not detected by culture. Time to result with PCR was shorter (median 5 h vs. 72 h, p < 0.001) than with culture. No resistance genes were detected by mPCR, but conventional culture allowed susceptibility testing and revealed resistance in 5 samples. Conclusions: mPCR seems to be a feasible and quick tool to detect causes of vascular graft infections within 24 h and might be helpful in antibiotic pre-treated patients. The detection of antibiotic resistance with mPCR needs improvement for clinical practice.

Adrenomedullin Is Associated With Surgical Trauma and Impaired Renal Function in Vascular Surgery Patients.

BACKGROUND:: Patients undergoing vascular surgery are prone to perioperative organ injury because of both higher prevalence of cardiovascular risk factors and the extent of surgery. Early detection of organ failure is essential to facilitate appropriate medical care. Midregional pro-adrenomedullin (MR-proADM) has been investigated in acute medical care settings to guide clinical decision-making regarding patient pathways and to identify patients prone to imminent cardiovascular or inflammatory complications. In this study, we evaluated the impact of perioperative MR-proADM levels as an early marker of perioperative cardiovascular and inflammatory stress reactions and kidney injury. METHODS:: The study was conducted as a monocentric, prospective, noninterventional trial at Hannover Medical School, Germany. A total of 454 consecutive patients who underwent open vascular surgery were followed from the day prior to until 30 days after surgery. The composite primary end point was defined as the occurrence of major adverse cardiac events (MACEs), acute kidney injury (AKI), or systemic inflammatory response syndrome (SIRS). Measurements were correlated with both medical history and postoperative MACE, AKI, or SIRS using univariate and multivariate regression analysis. RESULTS:: One hundred thirty-nine (31%) of the patients reached the primary end point within the study interval. Midregional pro-adrenomedullin change was associated with the combined primary end point and with the intensity of surgical trauma. Midregional pro-adrenomedullin change was increased in patients reaching the secondary end points, SIRS (optimal cutoff: 0.2 nmol/L) and AKI (optimal cutoff: 0.7 nmol/L), but not in patients with MACEs. CONCLUSION:: Increased levels of MR-proADM within the perioperative setting (1) were linked to the invasiveness of surgery and (2) identified patients with ongoing loss of renal function. Increased MR-proADM levels may therefore identify a subgroup of patients prone to excessive cardiovascular stress but did not directly correlate with adverse cardiac events. Consistently low levels of MR-proADM may identify a subgroup of patients with acceptable low risk to guide discharge from high-density care units.

Molecular Regulation of Acute Tie2 Suppression in Sepsis.

OBJECTIVES: Tie2 is a tyrosine kinase receptor expressed by endothelial cells that maintains vascular barrier function. We recently reported that diverse critical illnesses acutely decrease Tie2 expression and that experimental Tie2 reduction suffices to recapitulate cardinal features of the septic vasculature. Here we investigated molecular mechanisms driving Tie2 suppression in settings of critical illness. DESIGN: Laboratory and animal research, postmortem kidney biopsies from acute kidney injury patients and serum from septic shock patients. SETTING: Research laboratories and ICU of Hannover Medical School, Harvard Medical School, and University of Groningen. PATIENTS: Deceased septic acute kidney injury patients (n = 16) and controls (n = 12) and septic shock patients (n = 57) and controls (n = 22). INTERVENTIONS: Molecular biology assays (Western blot, quantitative polymerase chain reaction) + in vitro models of flow and transendothelial electrical resistance experiments in human umbilical vein endothelial cells; murine cecal ligation and puncture and lipopolysaccharide administration. MEASUREMENTS AND MAIN RESULTS: We observed rapid reduction of both Tie2 messenger RNA and protein in mice following cecal ligation and puncture. In cultured endothelial cells exposed to tumor necrosis factor-α, suppression of Tie2 protein was more severe than Tie2 messenger RNA, suggesting distinct regulatory mechanisms. Evidence of protein-level regulation was found in tumor necrosis factor-α-treated endothelial cells, septic mice, and septic humans, all three of which displayed elevation of the soluble N-terminal fragment of Tie2. The matrix metalloprotease 14 was both necessary and sufficient for N-terminal Tie2 shedding. Since clinical settings of Tie2 suppression are often characterized by shock, we next investigated the effects of laminar flow on Tie2 expression. Compared with absence of flow, laminar flow induced both Tie2 messenger RNA and the expression of GATA binding protein 3. Conversely, septic lungs exhibited reduced GATA binding protein 3, and knockdown of GATA binding protein 3 in flow-exposed endothelial cells reduced Tie2 messenger RNA. Postmortem tissue from septic patients showed a trend toward reduced GATA binding protein 3 expression that was associated with Tie2 messenger RNA levels (p < 0.005). CONCLUSIONS: Tie2 suppression is a pivotal event in sepsis that may be regulated both by matrix metalloprotease 14-driven Tie2 protein cleavage and GATA binding protein 3-driven flow regulation of Tie2 transcript.

Low Immunogenic Endothelial Cells Maintain Morphological and Functional Properties Required for Vascular Tissue Engineering.

The limited availability of native vessels suitable for the application as hemodialysis shunts or bypass material demands new strategies in cardiovascular surgery. Tissue-engineered vascular grafts containing autologous cells are considered ideal vessel replacements due to the low risk of rejection. However, endothelial cells (EC), which are central components of natural blood vessels, are difficult to obtain from elderly patients of poor health. Umbilical cord blood represents a promising alternative source for EC, but their allogeneic origin corresponds with the risk of rejection after allotransplantation. To reduce this risk, the human leukocyte antigen class I (HLA I) complex was stably silenced by lentiviral vector-mediated RNA interference (RNAi) in EC from peripheral blood and umbilical cord blood and vein. EC from all three sources were transduced by 93.1% ± 4.8% and effectively, HLA I-silenced by up to 67% compared to nontransduced (NT) cells or transduced with a nonspecific short hairpin RNA, respectively. Silenced EC remained capable to express characteristic endothelial surface markers such as CD31 and vascular endothelial cadherin important for constructing a tight barrier, as well as von Willebrand factor and endothelial nitric oxide synthase important for blood coagulation and vessel tone regulation. Moreover, HLA I-silenced EC were still able to align under unidirectional flow, to take up acetylated low-density lipoprotein, and to form capillary-like tube structures in three-dimensional fibrin gels similar to NT cells. In particular, addition of adipose tissue-derived mesenchymal stem cells significantly improved tube formation capability of HLA I-silenced EC toward long and widely branched vascular networks necessary for prevascularizing vascular grafts. Thus, silencing HLA I by RNAi represents a promising technique to reduce the immunogenic potential of EC from three different sources without interfering with EC-specific morphological and functional properties required for vascular tissue engineering. This extends the spectrum of available cell sources from autologous to allogeneic sources, thereby accelerating the generation of tissue-engineered vascular grafts in acute clinical cases.

Outgrowing endothelial and smooth muscle cells for tissue engineering approaches.

In recent years, circulating progenitors of endothelial cells and smooth muscle cells were identified in the peripheral blood. In our study, we evaluated the utilization of both cell types isolated and differentiated from peripheral porcine blood in terms for their use for tissue engineering purposes. By means of density gradient centrifugation, the monocyte fraction from porcine blood was separated, split, and cultivated with specific culture media with either endothelial cell growth medium-2 or smooth muscle cell growth medium-2 for the differentiation of endothelial cells or smooth muscle cells. Obtained cells were characterized at an early stage of cultivation before the first passage and a late stage (fourth passage) on the basis of the expression of the antigens CD31, CD34, CD45, nitric oxide synthase, and the contractile filaments smooth-muscle alpha-actin (sm-alpha-actin) and smoothelin. Functional characterization was done based on the secretion of nitric oxide, the formation of a coherent monolayer on polytetrafluoroethylene, and capillary sprouting. During cultivation in both endothelial cell growth medium-2 and smooth muscle cell growth medium-2, substantially two types of cells grew out: early outgrown CD45-positive cells, which disappeared during further cultivation, and in 85% (n = 17/20) of cultures cultivated with endothelial cell growth medium-2 colony-forming late outgrowth endothelial cells. During cultivation with smooth muscle cell growth medium-2 in 80% (n = 16/20) of isolations colony-forming late outgrowth smooth muscle cells entered the stage. Cultivation with either endothelial cell growth medium-2 or smooth muscle cell growth medium-2 had selective effect on the late outgrown cells to that effect that the number of CD31-positive cells increased from 34.8% ± 13% to 83.9% ± 8% in cultures cultivated with endothelial cell growth medium-2 and the number of sm-α-actin+ cells increased from 52.6% ± 18% to 88% ± 5% in cultures cultivated with smooth muscle cell growth medium-2, respectively. Functional analyses revealed significantly higher levels of nitric oxide secretion, endothelialization capacity, and capillary formation in not expanded cultures cultivated with endothelial cell growth medium-2 in comparison to later stages of cultivation and mature aortic cells. Blood seems to be a reliable and feasible source for the isolation of both endothelial and smooth muscle cells for application in tissue engineering approaches. Whereas, early co-cultures of early and late outgrowth cells provide functional advantages, the differentiation of cells can be directed selectively by the used culture medium for the expansion of highly proliferative late outgrowth endothelial cells and late outgrowth smooth muscle cells, respectively.

Renal function interferes with copeptin in prediction of major adverse cardiac events in patients undergoing vascular surgery.

OBJECTIVE: Precise perioperative risk stratification is important in vascular surgery patients who are at high risk for major adverse cardiovascular events (MACE) peri- and postoperatively. In clinical practice, the patient's perioperative risk is predicted by various indicators, e.g. revised cardiac index (RCRI) or modifications thereof. Patients suffering from chronic kidney disease (CKD) are stratified into a higher risk category. We hypothesized that Copeptin as a novel biomarker for hemodynamic stress could help to improve the prediction of perioperative cardiovascular events in patients undergoing vascular surgery including patients with chronic kidney disease. METHODS: 477 consecutive patients undergoing abdominal aortic, peripheral arterial or carotid surgery from June 2007 to October 2012 were prospectively enrolled. Primary endpoint was 30-day postoperative major adverse cardiovascular events (MACE). RESULTS: 41 patients reached the primary endpoint, including 63.4% aortic, 26.8% carotid, and 9.8% peripheral surgeries. Linear regression analysis showed that RCRI (P< .001), pre- (P< .001), postoperative Copeptin (P< .001) and Copeptin level change (P= .001) were associated with perioperative MACE, but CKD remained independently associated with MACE and Copeptin levels. Multivariate regression showed that increased Copeptin levels added risk predictive information to the RCRI (P= .003). Especially in the intermediate RCRI categories was Copeptin significantly associated with the occurrence of MACE. (P< .05 Kruskal Wallis test). Subdivision of the study cohort into CKD stages revealed that preoperative Copeptin was significantly associated with CKD stages (P< .0001) and preoperative Copeptin measurements could not predict MACE in patients with more severe CKD stages. CONCLUSION: Preoperative Copeptin loses its risk predictive potential for perioperative MACE in patients with chronic kidney disease undergoing vascular surgery.

Drug Repurposing Screen Identifies Foxo1-Dependent Angiopoietin-2 Regulation in Sepsis.

OBJECTIVE: The recent withdrawal of a targeted sepsis therapy has diminished pharmaceutical enthusiasm for developing novel drugs for the treatment of sepsis. Angiopoietin-2 is an endothelial-derived protein that potentiates vascular inflammation and leakage and may be involved in sepsis pathogenesis. We screened approved compounds for putative inhibitors of angiopoietin-2 production and investigated underlying molecular mechanisms. DESIGN: Laboratory and animal research plus prospective placebo-controlled randomized controlled trial (NCT00529139) and retrospective analysis (NCT00676897). SETTING: Research laboratories of Hannover Medical School and Harvard Medical School. PATIENTS: Septic patients/C57Bl/6 mice and human endothelial cells. INTERVENTIONS: Food and Drug Administration-approved library screening. MEASUREMENTS AND MAIN RESULTS: In a cell-based screen of more than 650 Food and Drug Administration-approved compounds, we identified multiple members of the 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitor drug class (referred to as statins) that suppressed angiopoietin-2. Simvastatin inhibited 3-hydroxy-3-methyl-glutaryl-CoA reductase, which in turn activated PI3K-kinase. Downstream of this signaling, PI3K-dependent phosphorylation of the transcription factor Foxo1 at key amino acids inhibited its ability to shuttle to the nucleus and bind cis-elements in the angiopoietin-2 promoter. In septic mice, transient inhibition of angiopoietin-2 expression by liposomal siRNA in vivo improved absolute survival by 50%. Simvastatin had a similar effect, but the combination of angiopoietin-2 siRNA and simvastatin showed no additive benefit. To verify the link between statins and angiopoietin-2 in humans, we performed a pilot matched case-control study and a small randomized placebo-controlled trial demonstrating beneficial effects on angiopoietin-2. CONCLUSIONS: 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors may operate through a novel Foxo1-angiopoietin-2 mechanism to suppress de novo production of angiopoietin-2 and thereby ameliorate manifestations of sepsis. Given angiopoietin-2's dual role as a biomarker and candidate disease mediator, early serum angiopoietin-2 measurement may serve as a stratification tool for future trials of drugs targeting vascular leakage.