Distinct microhemodynamic efficacy of arteriogenesis and angiogenesis in critically ischemic skin flaps.

Angiogenesis and arteriogenesis are regenerative vascular mechanisms dedicated to cope with critical ischemia after the interruption of the anatomical axial blood supply. The aim of the present study was to visualize, quantify and monitor the orchestration of these mechanisms and their microhemodynamic efficacy. A murine skin flap model was used that allowed for repetitive investigation of identical vascular structures by intravital microscopy. In the conduit arterioles, diameter and relative length increased to 133 ± 20% and 260 ± 80% over 7 days, respectively (both P<0.01), which reduced vascular resistance in this segment to 82 ± 35%. After 1 week, a peak in accumulation of activated leukocytes could be observed in the postcapillary venules (P<0.01) without relevant hemodynamic changes. Thereafter, the arteriolar remodeling was replaced by angiogenesis. Functional capillary density was increased to 141 ± 10% (P<0.01) and capillary diameter to 123 ± 6% (P<0.01) after 14 days. Both mechanisms of vascular regeneration were associated with increases in the capillary perfusion index, to 194 ± 42% (P<0.05) after 7 days and 366 ± 21% after 14 days (P>0.01). Immunohistochemical analysis revealed a correlation of arteriogenesis with eNOS upregulation and of angiogenesis with VEGF upregulation in the corresponding vessels. In conclusion, arteriogenesis was the initial regenerative mechanism leading to arteriolar remodeling, reduction in vascular resistance, and increase in capillary perfusion over the first 7 days. Thereafter, capillary perfusion was improved by angiogenesis in terms of an increase in functional capillary density.

Paracrine effects of mesenchymal stem cells enhance vascular regeneration in ischemic murine skin.

New theories on the regeneration of ischemic vasculature have emerged indicating a pivotal role of adult stem cells. The aim of this study was to investigate homing and hemodynamic effects of circulating bone marrow-derived mesenchymal stem cells (MSCs) in a critically ischemic murine skin flap model. Bone marrow-derived mesenchymal stem cells (Lin(-)CD105(+)) were harvested from GFP(+)-donor mice and transferred to wildtype C57BL/6 mice. Animals receiving GFP(+)-fibroblasts served as a control group. Laser scanning confocal microscopy and intravital fluorescence microscopy were used for morphological analysis, monitoring and quantitative assessment of the stem cell homing and microhemodynamics over two weeks. Immunohistochemical staining was performed for GFP, eNOS, iNOS, VEGF. Tissue viability was analyzed by TUNEL-assay. We were able to visualize perivascular homing of MSCs in vivo. After 4 days, MSCs aligned along the vascular wall without undergoing endothelial or smooth muscle cell differentiation during the observation period. The gradual increase in arterial vascular resistance observed in the control group was abolished after MSC administration (P<0.01). At capillary level, a strong angiogenic response was found from day 7 onwards. Functional capillary density was raised in the MSC group to 197% compared to 132% in the control group (P<0.01). Paracrine expression of VEGF and iNOS, but not eNOS could be shown in the MSC group but not in the controls. In conclusion, we demonstrated that circulating bone marrow-derived MSCs home to perivascular sites in critically ischemic tissue, exhibits paracrine function and augment microhemodynamics. These effects were mediated through arteriogenesis and angiogenesis, which contributed to vascular regeneration.

Endobiliary Radiofrequency Ablation for Malignant Biliary Obstruction over 32-Month Follow-Up.

Hilar cholangiocellular carcinoma (CCC) is a malignant neoplasm of epithelial origin occurring at the confluence of the right and left hepatic bile ducts. Typically, these tumors are small, poorly differentiated, exhibit aggressive biologic behavior with non-specific symptoms and tend to obstruct the intrahepatic bile ducts. Surgery is the only available curative option. Unfortunately, in less than half of the patients a complete resection is possible with poor survival rate in unresectable cases. In this report, we present the case of a 58-year-old woman with a history of unresectable hilar cholangiocarcinoma. Initially she was treated with intraductal dilatation of malignancy and placement of a plastic stent and chemotherapy (Gemcitabin® and Platinol®). Two years later she underwent a second-line chemotherapy with Gemcitabin® and Oxyplatin® because of tumor progression. Despite a second line chemotherapy and placement of an uncovered self-expandible metal stent (ucSEMS) that was extended later on by stent-in stent technique, there was tumor progression which led to a complex course with relapsing obstructive cholangiosepsis and cholestasis. Because of tumor ingrowth, endobiliary radiofrequency ablation of the malignant stenosis was performed in repeated sessions. This case illustrates that radiofrequency ablation of solitary malignant biliary obstruction is feasible, safe and allows an improvement of quality of life in non-operable patients.

The choice of anesthesia influences oxidative energy metabolism and tissue survival in critically ischemic murine skin.

In surgical animal studies anesthesia is used regularly. Several reports in the literature demonstrate respiratory and cardiovascular side effects of anesthesiologic agents. The aim of this study was to compare two frequently used anesthesia cocktails (ketamine/xylazine [KX] versus medetomidine/climazolam/fentanyl [MCF]) in skin flap mouse models. Systemic blood values, local metabolic parameters, and surgical outcome should be analyzed in critical ischemic skin flap models. Systemic hypoxia was found in the animals undergoing KX anesthesia compared with normoxia in the MCF group (sO(2): 89.2% +/- 2.4% versus 98.5% +/- 1.2%, P < 0.01). Analysis of tissue metabolism revealed impaired anaerobic oxygen metabolism and increased cellular damage in critical ischemic flap tissue under KX anesthesia (lactate/pyruvate ratio: KX 349.86 +/- 282.38 versus MCF 64.53 +/- 18.63; P < 0.01 and glycerol: KX 333.50 +/- 83.91 micromol/L versus MCF 195.83 +/- 29.49 micromol/L; P < 0.01). After 6 d, different rates of flap tissue necrosis could be detected (MCF 57% +/- 6% versus KX 68% +/- 6%, P < 0.01). In summary we want to point out that the type of anesthesia, the animal model and the goal of the study have to be well correlated. Comparing the effects of KX and MCF anesthesia in mice on surgical outcome was a novel aspect of our study.

Influence of Anion and Cation Structure of Ionic Liquids on Carboxylic Acids Extraction.

A recently proposed new mechanism and a model of reactive extraction of carboxylic acids by hydrophobic ionic liquids (ILs) was tested on five systems from published as well as from new equilibrium data on liquid-liquid extraction of butyric and lactic acids (BA and LA) from aqueous solutions. Two phosphonium and one ammonium ILs were used. The model describes experimental data for all systems with a good fit. The mechanism of acid extraction by ILs is very similar for all tested systems. This indicates a more general validity of the developed model. The model allows deeper understanding of regularities in carboxylic acid extraction by hydrophobic ILs. Stability constants of the first acid-IL bonds are by one to three orders of magnitude higher compared to that of acid-acid bonds. Values of stability constants related to two acid-IL bonds are sensitive to a cation and anion structure while stability constants for acid-acid bonds more distant from polar head of IL are not sensitive to IL structure. The stability constants of acid-IL bonds for LA and phosphonium ILs are by more than one order of magnitude lower compared to those for BA and are not influenced with an anion structure. The value of stability constant for the first BA-IL bond is for phosphonium IL with a decanoate anion only one third of those for IL with a phosphinate anion. Differences in the stability of acid-IL bonds for BA and LA can be attributed to hydrophobic interactions which almost do not occur in LA extraction. Ammonium IL also forms a less stable BA-IL bond than the phosphonium IL with the same phosphinate anion. A less stable BA-IL bond can favor the higher recovery of volatile acid from the solvent by vacuum evaporation where free acid is separated instead of acid salts as in classical processes what is a great advantage.

Proteolysis of mature HIV-1 p6 Gag protein by the insulin-degrading enzyme (IDE) regulates virus replication in an Env-dependent manner.

There is a significantly higher risk for type II diabetes in HIV-1 carriers, albeit the molecular mechanism for this HIV-related pathology remains enigmatic. The 52 amino acid HIV-1 p6 Gag protein is synthesized as the C-terminal part of the Gag polyprotein Pr55. In this context, p6 promotes virus release by its two late (L-) domains, and facilitates the incorporation of the viral accessory protein Vpr. However, the function of p6 in its mature form, after proteolytic release from Gag, has not been investigated yet. We found that the mature p6 represents the first known viral substrate of the ubiquitously expressed cytosolic metalloendopeptidase insulin-degrading enzyme (IDE). IDE is sufficient and required for degradation of p6, and p6 is approximately 100-fold more efficiently degraded by IDE than its eponymous substrate insulin. This observation appears to be specific for HIV-1, as p6 proteins from HIV-2 and simian immunodeficiency virus, as well as the 51 amino acid p9 from equine infectious anaemia virus were insensitive to IDE degradation. The amount of virus-associated p6, as well as the efficiency of release and maturation of progeny viruses does not depend on the presence of IDE in the host cells, as it was shown by CRISPR/Cas9 edited IDE KO cells. However, HIV-1 mutants harboring IDE-insensitive p6 variants exhibit reduced virus replication capacity, a phenomenon that seems to depend on the presence of an X4-tropic Env. Furthermore, competing for IDE by exogenous insulin or inhibiting IDE by the highly specific inhibitor 6bK, also reduced virus replication. This effect could be specifically attributed to IDE since replication of HIV-1 variants coding for an IDE-insensitive p6 were inert towards IDE-inhibition. Our cumulative data support a model in which removal of p6 during viral entry is important for virus replication, at least in the case of X4 tropic HIV-1.

Application of a new laser Doppler imaging system in planning and monitoring of surgical flaps.

There is a demand for technologies able to assess the perfusion of surgical flaps quantitatively and reliably to avoid ischemic complications. The aim of this study is to test a new high-speed high-definition laser Doppler imaging (LDI) system (FluxEXPLORER, Microvascular Imaging, Lausanne, Switzerland) in terms of preoperative mapping of the vascular supply (perforator vessels) and postoperative flow monitoring. The FluxEXPLORER performs perfusion mapping of an area 9 x 9 cm with a resolution of 256 x 256 pixels within 6 s in high-definition imaging mode. The sensitivity and predictability to localize perforators is expressed by the coincidence of preoperatively assessed LDI high flow spots with intraoperatively verified perforators in nine patients. 18 free flaps are monitored before, during, and after total ischemia. 63% of all verified perforators correspond to a high flow spot, and 38% of all high flow spots correspond to a verified perforator (positive predictive value). All perfused flaps reveal a value of above 221 perfusion units (PUs), and all values obtained in the ischemic flaps are beneath 187 PU. In summary, we conclude that the present LDI system can serve as a reliable, fast, and easy-to-handle tool to detect ischemia in free flaps, whereas perforator vessels cannot be detected appropriately.