In vitro recellularization of decellularized bovine carotid arteries using human endothelial colony forming cells

Background: Many patients suffering from peripheral arterial disease (PAD) are dependent on bypass surgery. However, in some patients no suitable replacements (i.e. autologous or prosthetic bypass grafts) are available. Advances have been made to develop autologous tissue engineered vascular grafts (TEVG) using endothelial colony forming cells (ECFC) obtained by peripheral blood draw in large animal trials. Clinical translation of this technique, however, still requires additional data for usability of isolated ECFC from high cardiovascular risk patients. Bovine carotid arteries (BCA) were decellularized using a combined SDS (sodium dodecyl sulfate) -free mechanical-osmotic-enzymatic-detergent approach to show the feasibility of xenogenous vessel decellularization. Decellularized BCA chips were seeded with human ECFC, isolated from a high cardiovascular risk patient group, suffering from diabetes, hypertension and/or chronic renal failure. ECFC were cultured alone or in coculture with rat or human mesenchymal stromal cells (rMSC/hMSC). Decellularized BCA chips were evaluated for biochemical, histological and mechanical properties. Successful isolation of ECFC and recellularization capabilities were analyzed by histology. Results: Decellularized BCA showed retained extracellular matrix (ECM) composition and mechanical properties upon cell removal. Isolation of ECFC from the intended target group was successfully performed (80% isolation efficiency). Isolated cells showed a typical ECFC-phenotype. Upon recellularization, co-seeding of patient-isolated ECFC with rMSC/hMSC and further incubation was successful for 14 (n = 9) and 23 (n = 5) days. Reendothelialization (rMSC) and partial reendothelialization (hMSC) was achieved. Seeded cells were CD31 and vWF positive, however, human cells were detectable for up to 14 days in xenogenic cell-culture only. Seeding of ECFC without rMSC was not successful. Conclusion: Using our refined decellularization process we generated easily obtainable TEVG with retained ECM- and mechanical quality, serving as a platform to develop small-diameter (< 6 mm) TEVG. ECFC isolation from the cardiovascular risk target group is possible and sufficient. Survival of diabetic ECFC appears to be highly dependent on perivascular support by rMSC/hMSC under static conditions. ECFC survival was limited to 14 days post seeding

Independent effects of sham laparotomy and anesthesia on hepatic microRNA expression in rats

Background: Studies on liver regeneration following partial hepatectomy (PH) have identified several microRNAs (miRNAs) that show a regulated expression pattern. These studies involve major surgery to access the liver, which is known to have intrinsic effects on hepatic gene expression and may also affect miRNA screening results. We performed two-third PH or sham laparotomy (SL) in Wistar rats to investigate the effect of both procedures on miRNA expression in liver tissue and corresponding plasma samples by microarray and qRT-PCR analyses. As control groups, non-treated rats and rats undergoing anesthesia only were used. Results: We found that 49 out of 323 miRNAs (15%) were significantly deregulated after PH in liver tissue 12 to 48 hours postoperatively (>20% change), while 45 miRNAs (14%) were deregulated following SL. Out of these miRNAs, 10 miRNAs were similarly deregulated after PH and SL, while one miRNA showed opposite regulation. In plasma, miRNA upregulation was observed for miR-133a and miR-133b following PH and SL, whereas miR-100 and miR-466c were similarly downregulated following anesthesia and surgery. Conclusions: We show that miRNAs are indeed regulated by sham laparotomy and anesthesia in rats. These findings illustrate the critical need for finding appropriate control groups in experimental surgery

Dual versus single vessel normothermic ex vivo perfusion of rat liver grafts using metamizole for vasodilatation

Background: Normothermic ex vivo liver perfusion (NEVLP) is a promising strategy to increase the donor pool in liver transplantation. Small animal models are essential to further investigate questions regarding organ preservation and reconditioning by NEVLP. A dual vessel small animal NEVLP (dNEVLP) model was developed using metamizole as a vasodilator and compared to conventional portovenous single vessel NEVLP (sNEVLP). Methods: Livers of male Wistar rats were perfused with erythrocyte-supplemented culture medium for six hours by either dNEVLP via hepatic artery and portal vein or portovenous sNEVLP. dNEVLP was performed either with or without metamizole treatment. Perfusion pressure and flow rates were constantly monitored. Transaminase levels were determined in the perfusate at the start and after three and six hours of perfusion. Bile secretion was monitored and bile LDH and GGT levels were measured hourly. Histopathological analysis was performed using liver and bile duct tissue samples after perfusion. Results: Hepatic artery pressure was significantly lower in dNEVLP with metamizole administration. Compared to sNEVLP, dNEVLP with metamizole treatment showed higher bile production, lower levels of transaminases during and after perfusion as well as significantly lower necrosis in liver and bile duct tissue. Biochemical markers of bile duct injury showed the same trend. Conclusion: Our miniaturized dNEVLP system enables normothermic dual vessel rat liver perfusion. The administration of metamizole effectively ameliorates arterial vasospasm allowing for six hours of dNEVLP, with superior outcome compared to sNEVLP

Surface modification of decellularized bovine carotid arteries with human vascular cells significantly reduces their thrombogenicity

Background: Since autologous veins are unavailable when needed in more than 20% of cases in vascular surgery, the production of personalized biological vascular grafts for implantation has become crucial. Surface modification of decellularized xenogeneic grafts with vascular cells to achieve physiological luminal coverage and eventually thromboresistance is an important prerequisite for implantation. However, ex vivo thrombogenicity testing remains a neglected area in the field of tissue engineering of vascular grafts due to a multifold of reasons. Methods: After seeding decellularized bovine carotid arteries with human endothelial progenitor cells and umbilical cord-derived mesenchymal stem cells, luminal endothelial cell coverage (LECC) was correlated with glucose and lactate levels on the cell supernatant. Then a closed loop whole blood perfusion system was designed. Recellularized grafts with a LECC > 50% and decellularized vascular grafts were perfused with human whole blood for 2 h. Hemolysis and complete blood count evaluation was performed on an hourly basis, followed by histological and immunohistochemical analysis. Results: While whole blood perfusion of decellularized grafts significantly reduced platelet counts, platelet depletion from blood resulting from binding to re-endothelialized grafts was insignificant (p = 0.7284). Moreover, macroscopic evaluation revealed thrombus formation only in the lumen of unseeded grafts and histological characterization revealed lack of CD41 positive platelets in recellularized grafts, thus confirming their thromboresistance. Conclusion: In the present study we were able to demonstrate the effect of surface modification of vascular grafts in their thromboresistance in an ex vivo whole blood perfusion system. To our knowledge, this is the first study to expose engineered vascular grafts to human whole blood, recirculating at high flow rates, immediately after seeding

Angiogenic miRNAs, the angiopoietin axis and related TIE2-expressing monocytes affect outcomes in cholangiocarcinoma

Background: Tumour angiogenesis is modulated on both an epigenetic and protein level and has potential implications for immune cell responses. However, the importance of related angiogenic biomarkers in cholangiocarcinoma (CCA) is unknown. This study assessed human CCA samples for the expression of angiogenesisassociated microRNAs, angiopoietins (Angs) and monocytes expressing the Angreceptor, TIE2, with regards to prognostic significance after liver resection. Methods: Angiogenic miRNAs were analysed in frozen samples of intrahepatic CCA (iCC; n = 43) and hilar CCA (HC; n = 45). Ang-1 and Ang-2, as well as TIE2- expressing monocytes (TEMs), were detected in paraffin-embedded iCC sections (n = 88). MiRNA expression and the abundance of TEMs and Angs were correlated with clinicopathological characteristics and survival. Results: MiR-126 was downregulated in 76.7% of all CCA samples, with high relative expression associated with smaller tumours and reduced lymph node metastasis. High Ang-1 expression was associated with less lymphangiosis carcinomatosa and better histological grading (all p < 0.05). The absence of TEMs in iCC correlated with elevated CA19-9 levels. High relative miR-126 and low miR-128 levels were associated with improved survival in iCC and HC, respectively (all p < 0.05). High miR-126, low miR-128 and TEMs were independent prognostic factors for recurrence-free and overall survival (all p < 0.05). Conclusions: These results suggest that angiogenic miRNAs, Angs and TEMs are of prognostic value in CCA. In addition to the possible functional links between angiogenic miRNA expression profiles, Angs and immune-cell responses by TEMs, these data have clinical implications as novel diagnostic tools

Prognostic significance of macrophage invasion in hilar cholangiocarcinoma

Background: Tumor-associated macrophages (TAMs) promote tumor progression and have an effect on survival in human cancer. However, little is known regarding their influence on tumor progression and prognosis in human hilar cholangiocarcinoma. Methods: We analyzed surgically resected tumor specimens of hilar cholangiocarcinoma (n = 47) for distribution and localization of TAMs, as defined by expression of CD68. Abundance of TAMs was correlated with clinicopathologic characteristics, tumor recurrence and patients’ survival. Statistical analysis was performed using SPSS software. Results: Patients with high density of TAMs in tumor invasive front (TIF) showed significantly higher local and overall tumor recurrence (both ρ < 0.05). Furthermore, high density of TAMs was associated with decreased overall (one-year 83.6 % vs. 75.1 %; three-year 61.3 % vs. 42.4 %; both ρ < 0.05) and recurrence-free survival (one-year 93.9 % vs. 57.4 %; three-year 59.8 % vs. 26.2 %; both ρ < 0.05). TAMs in TIF and tumor recurrence, were confirmed as the only independent prognostic variables in the multivariate survival analysis (all ρ < 0.05). Conclusions: Overall survival and recurrence free survival of patients with hilar cholangiocarcinoma significantly improved in patients with low levels of TAMs in the area of TIF, when compared to those with a high density of TAMs. These observations suggest their utilization as valuable prognostic markers in routine histopathologic evaluation, and might indicate future therapeutic approaches by targeting TAMs

Notch Signaling Pathway in Pancreatobiliary Tumors

Background and Objectives: The Notch signaling pathway plays an important role both in the development of the ductal systems of the pancreas and the bile ducts as well as in cancer development and progression. The aim of this study was to examine the expression of central proteins of the Notch signaling pathway in pancreatobiliary tumors and its influence on patient survival. Materials and Methods: We compared the receptors (Notch1, Notch4), activating splicing factors (ADAM17), and target genes (HES1) of the Notch pathway and progenitor cell markers with relevance for the Notch signaling pathway (CD44, MSI1) between pancreatic adenocarcinomas (PDAC, n = 14), intrahepatic cholangiocarcinoma (iCC, n = 24), and extrahepatic cholangiocarcinoma (eCC, n = 22) cholangiocarcinomas via immunohistochemistry and ImageJ software-assisted analysis. An Immunohistochemistry (IHC)-score was determined by the percentage and intensity of stained (positive) cells (scale 0-7) and normal and malignant tissue was compared. In the IHC results, patients' (gender, age) and tumor (TNM Classification of Malignant Tumors, Union Internationale contre le Cancer (UICC) stages, grading, and lymphangitic carcinomatosa) characteristics were correlated to patient survival. Results: For eCC, the expression of CD44 (p = 0.043, IHC-score 3.94 vs. 3.54) and for iCC, the expression of CD44 (p = 0.026, IHC-score 4.04 vs. 3.48) and Notch1 (p < 0.001, IHC-score 2.87 vs. 1.78) was significantly higher in the tumor compared to non-malignant tissue. For PDAC, the expression of ADAM17 (p = 0.008, IHC-score 3.43 vs. 1.73), CD44 (p = 0.012, IHC-score 3.64 vs. 2.27), Notch1 (p = 0.012, IHC-score 2.21 vs. 0.64), and Notch4 (p = 0.008, IHC-score 2.86 vs. 0.91) was significantly higher in the tumor tissue. However, none of the analyzed Notch-signaling related components showed an association to patient survival. Conclusion: A significant overexpression of almost all studied components of the Notch signaling pathway can be found in the tumor tissue, however, without a significant influence on patient survival. Therefore, further studies are warranted to draw conclusions on Notch pathway's relevance for patient survival