Tumor cell invasion in blood vessels assessed by immunohistochemistry is related to decreased survival in patients with bladder cancer treated with radical cystectomy.

BACKGROUND: Lymphovascular invasion (VI) is an established prognostic marker for many cancers including bladder cancer. There is a paucity of data regarding whether the prognostic significance of lymphatic invasion (LVI) differs from blood vessel invasion (BVI). The aim was to examine LVI and BVI separately using immunohistochemistry (IHC), and investigate their associations with clinicopathological characteristics and prognosis. A secondary aim was to compare the use of IHC with assessing VI on standard HAS (hematoxylin-azophloxine-saffron) sections without IHC. METHODS: A retrospective, population -based series of 292 invasive bladder cancers treated with radical cystectomy (RC) with curative intent at Vestfold Hospital Trust, Norway were reviewed. Traditional histopathological markers and VI based on HAS sections were recorded. Dual staining using D2-40/CD31 antibodies was performed on one selected tumor block for each case. RESULTS: The frequency of LVI and BVI was 32 and 28%, respectively. BVI was associated with features such as higher pathological stages, positive regional lymph nodes, bladder neck involvement and metastatic disease whereas LVI showed weaker or no associations. Both BVI and LVI independently predicted regional lymph node metastases, LVI being the slightly stronger factor. BVI, not LVI predicted higher pathological stages. BVI showed reduced recurrence free (RFS) and disease specific (DSS) survival in uni-and multivariable analyses, whereas LVI did not. On HAS sections, VI was found in 31% of the cases. By IHC, 51% were positive, corresponding to a 64% increased sensitivity in detecting VI. VI assessed without IHC was significantly associated with RFS and DSS in univariable but not multivariable analysis. CONCLUSIONS: Our findings indicate that BVI is strongly associated with more aggressive tumor features. BVI was an independent prognostic factor in contrast to LVI. Furthermore, IHC increases VI sensitivity compared to HAS.

Vascular architectural patterns in clear cell renal cell carcinoma and clear cell papillary renal cell carcinoma.

Renal cell carcinomas (RCC) are well-vascularized tumors. Although clear cell RCC (CCRCC) show a characteristic vascular network, some cases show overlapping features with other RCC. We aimed to evaluate vascular architectural patterns, microvessel density (MVD), and endothelial cell density (ECD) in CCRCC compared to clear cell papillary RCC (ccpRCC). Thirty-four RCC (17 CCRCC and 17 ccpRCC) were included in the study. CD34 was used to evaluate vascular architectural patterns by microscopic estimation in all cases. CD34, ERG, and Bioquant Osteo 2019 Imaging Analysis Software were used to evaluate MVD and ECD in 17 CCRCC and 15 ccpRCC. Mean MVD was 526.63 in CCRCC vs. 426.18 in ccpRCC (p = 0.16); mean ECD was 937.50 in CCRCC vs. 1060.21 in ccpRCC (p = 0.25). CD34 highlighted four distinct vascular architectural patterns: pseudoacinar, Golgi-like, lacunae, and scattered. Lacunae and pseudoacinar was the most frequent combination in CCRCC; lacunae and Golgi-like was the predominant combination among ccpRCC. Pseudoacinar was most extensive in CCRCC and least in ccpRCC; Golgi-like was predominant in ccpRCC and uncommon in CCRCC. The extent of pseudoacinar and Golgi-like vascular architectural patterns was significantly different between CCRCC and ccpRCC (p < 0.05). Pathologists acquainted with these different vascular architectural patterns may utilize them as an additional tool in the distinction of CCRCC from ccpRCC.

Toward Raman Subcellular Imaging of Endothelial Dysfunction.

Multiple diseases are at some point associated with altered endothelial function, and endothelial dysfunction (ED) contributes to their pathophysiology. Biochemical changes of the dysfunctional endothelium are linked to various cellular organelles, including the mitochondria, endoplasmic reticulum, and nucleus, so organelle-specific insight is needed for better understanding of endothelial pathobiology. Raman imaging, which combines chemical specificity with microscopic resolution, has proved to be useful in detecting biochemical changes in ED at the cellular level. However, the detection of spectroscopic markers associated with specific cell organelles, while desirable, cannot easily be achieved by Raman imaging without labeling. This critical review summarizes the current advances in Raman-based analysis of ED, with a focus on a new approach involving molecular Raman reporters that could facilitate the study of biochemical changes in cellular organelles. Finally, imaging techniques based on both conventional spontaneous Raman scattering and the emerging technique of stimulated Raman scattering are discussed.

Absence of lymphatic vessels in non-functioning bleb capsules of glaucoma drainage devices.

PURPOSE: To evaluate the presence and appearance of blood and lymphatic vessels in non-functioning bleb capsules of glaucoma drainage devices (GDD). MATERIALS AND METHODS: Non-functioning (n=14) GDD-bleb capsules of 12 patients were analyzed by immunohistochemistry for blood vessels (CD31, vascular endothelium), lymphatic vessels (lymphatic vessel endothelial hyaluronan receptor-1 [LYVE-1] and podoplanin) and macrophages (CD68). RESULTS: CD31+++ blood vessels and CD68+ macrophages were detected in the outer layer of all specimens. LYVE-1 immunoreactivity was registered in single non-endothelial cells in 8 out of 14 (57%) bleb capsule specimens. Podoplanin-immunoreactivity was detected in all cases, located in cells and profiles of the collagen tissue network of the outer and/or the inner capsule layer. However, a colocalization of LYVE-1 and podoplanin as evidence for lymphatic vessels was not detected. CONCLUSIONS: We demonstrate the presence of blood-vessels but absence of lymphatic vessels in non-functioning bleb capsules after GDD-implantation. While the absence of lymphatic vessels might indicate a possible reason for drainage device failure, this needs to be confirmed in upcoming studies, including animal experiments.

3D Bioprinting-Tunable Small-Diameter Blood Vessels with Biomimetic Biphasic Cell Layers.

Blood vessel damage resulting from trauma or diseases presents a serious risk of morbidity and mortality. Although synthetic vascular grafts have been successfully commercialized for clinical use, they are currently only readily available for large-diameter vessels (>6 mm). Small-diameter vessel (<6 mm) replacements, however, still present significant clinical challenges worldwide. The primary objective of this study is to create novel, tunable, small-diameter blood vessels with biomimetic two distinct cell layers [vascular endothelial cell (VEC) and vascular smooth muscle cell (VSMC)] using an advanced coaxial 3D-bioplotter platform. Specifically, the VSMCs were laden in the vessel wall and VECs grew in the lumen to mimic the natural composition of the blood vessel. First, a novel bioink consisting of VSMCs laden in gelatin methacryloyl (GelMA)/polyethylene(glycol)diacrylate/alginate and lyase was designed. This specific design is favorable for nutrient exchange in an ambient environment and simultaneously improves laden cell proliferation in the matrix pore without the space restriction inherent with substance encapsulation. In the vessel wall, the laden VSMCs steadily grew as the alginate was gradually degraded by lyase leaving more space for cell proliferation in matrices. Through computational fluid dynamics simulation, the vessel demonstrated significantly perfusable and mechanical properties under various flow velocities, flow viscosities, and temperature conditions. Moreover, both VSMCs in the scaffold matrix and VECs in the lumen steadily proliferated over time creating a significant two-cell-layered structure. Cell proliferation was confirmed visually through staining the markers of alpha-smooth muscle actin and cluster of differentiation 31, commonly tied to angiogenesis phenomena, in the vessel matrices and lumen, respectively. Furthermore, the results were confirmed quantitatively through gene analysis which suggested good angiogenesis expression in the blood vessels. This study demonstrated that the printed blood vessels with two distinct cell layers of VECs and VSMCs could be potential candidates for clinical small-diameter blood vessel replacement applications.

Enhanced Hemocompatibility and In Vivo Analytical Accuracy of Intravascular Potentiometric Carbon Dioxide Sensors via Nitric Oxide Release.

In this letter, the innate ability of nitric oxide (NO) to inhibit platelet activation/adhesion/thrombus formation is employed to improve the hemocompatibility and in vivo accuracy of an intravascular (IV) potentiometric PCO2 (partial pressure of carbon dioxide) sensor. The catheter-type sensor is fabricated by impregnating a segment of dual lumen silicone tubing with a proton ionophore, plasticizer, and lipophilic cation-exchanger. Subsequent filling of bicarbonate and strong buffer solutions and placement of Ag/AgCl reference electrode wires within each lumen, respectively, enables measurement of the membrane potential difference across the inner wall of the tube, with this potential changing as a function of the logarithm of sample PCO2. The dual lumen device is further encapsulated within a S-nitroso-N-acetyl-DL-penicillamine (SNAP)-doped silicone tube that releases physiological levels of NO. The NO releasing sensor exhibits near-Nernstian sensitivity toward PCO2 (slope = 59.31 ± 0.78 mV/decade) and low drift rates (<2 mV/24 h after initial equilibration). In vivo evaluation of the NO releasing sensors, performed in the arteries and veins of anesthetized pigs for 20 h, shows enhanced accuracy (vs non-NO releasing sensors) when benchmarked to measurements of discrete blood samples made with a commercial blood gas analyzer. The accurate, continuous monitoring of blood PCO2 levels achieved with this new IV NO releasing PCO2 sensor configuration could help better manage hospitalized patients in critical care units.

Enhanced vascularization and biocompatibility of rat pancreatic decellularized scaffolds loaded with platelet-rich plasma.

The ultimate goal of pancreatic tissue engineering is to create a long-lived substitute organ to treat diabetes. However, the lack of neovascularization and the occurrence of immune response limit the efficacy of tissue-engineered pancreas after in vivo transplantation. Platelet-rich plasma (PRP) is an autologous platelet concentrate containing a large number of growth factors and immunoregulatory factors. The aim of this study was to evaluate rat pancreatic decellularized scaffold (PDS) loaded with PRP for vascularization, host inflammatory response and macrophage polarization in an animal model. The study results indicated that compared to PDS, PRP-loading PDS exhibited the enhanced mechanical properties and released growth factors in a slow and sustained manner to supplement the loss of growth factors during decellularization. In vitro, human umbilical vein endothelial cells (HUVECs) were seeded in PDS and PRP-loading PDS, and cultured in the circular perfusion system. When compared with PDS, PRP-loading PDS significantly promoted the colonization, proliferation and pro-angiogenic genes expression of cells on scaffolds. In vivo, PDS loaded with PRP then re-endothelialized with HUVECs were implanted subcutaneously in rats, which enhanced the angiogenesis of scaffolds, inhibited the host inflammatory response, and induced the polarization dominated by pro-regenerative M2 macrophages that also facilitated tissue vascular regeneration. Thus, the re-endothelialized PRP-loading PDS may represent a promising bioengineered pancreas with sustained vascularization and excellent biocompatibility.

Integrin ß4 Is an Effective and Efficient Marker in Synchronously Highlighting Lymphatic and Blood Vascular Invasion, and Perineural Aggression in Malignancy.

Lymphovascular invasion (LVI) and perineural invasion (PNI) are 2 important pathologic parameters and need to be accurately assessed in multiple malignancies. Integrin ß4, a member of the integrin family, has been reported to be positively expressed in vascular endothelia, peripheral nerves, and a collection of epithelia. However, little is known about the effectiveness of ß4 immunostaining on the recognition of LVI and PNI. Herein, we explored the applicability of ß4 immunostaining in stomach, thyroid, and breast cancers. Parallel immunostaining of D2-40, CD34, and S-100 was performed as controls for lymphatic endothelia, vascular endothelia, and neural fibers, respectively. The results demonstrated that ß4 concurrently stained the lymphatic and vascular endothelia, and the peripheral nerves. Both LVI and PNI were clearly and accurately outlined by ß4 immunostaining. ß4 was also expressed in the majority of tumor cells, enabling recognition of LVI and PNI encroached by small tumor clusters. In contrast to D2-40 and CD34, ß4 staining was not observed in stromal cells, and therefore it facilitated differentiation between the shrinkage cleft and LVI. According to our results, ß4 staining strikingly increased the diagnostic accuracy and interobserver concordance for LVI and PNI compared with hematoxylin and eosin staining alone. Finally, the applicability of ß4 was confirmed in 9 other types of malignancies, including cancers of the colon, prostate, esophagus, lung, kidney, uterus, tongue, bladder, and liver. Collectively, ß4 is a reliable marker for synchronous detection and diagnosis of LVI and PNI.

Artificial small-diameter blood vessels: materials, fabrication, surface modification, mechanical properties, and bioactive functionalities.

Cardiovascular diseases, especially ones involving narrowed or blocked blood vessels with diameters smaller than 6 millimeters, are the leading cause of death globally. Vascular grafts have been used in bypass surgery to replace damaged native blood vessels for treating severe cardio- and peripheral vascular diseases. However, autologous replacement grafts are not often available due to prior harvesting or the patient's health. Furthermore, autologous harvesting causes secondary injury to the patient at the harvest site. Therefore, artificial blood vessels have been widely investigated in the last several decades. In this review, the progress and potential outlook of small-diameter blood vessels (SDBVs) engineered in vitro are highlighted and summarized, including material selection and development, fabrication techniques, surface modification, mechanical properties, and bioactive functionalities. Several kinds of natural and synthetic polymers for artificial SDBVs are presented here. Commonly used fabrication techniques, such as extrusion and expansion, electrospinning, thermally induced phase separation (TIPS), braiding, 3D printing, hydrogel tubing, gas foaming, and a combination of these methods, are analyzed and compared. Different surface modification methods, such as physical immobilization, surface adsorption, plasma treatment, and chemical immobilization, are investigated and are compared here as well. Mechanical requirements of SDBVs are also reviewed for long-term service. In vitro biological functions of artificial blood vessels, including oxygen consumption, nitric oxide (NO) production, shear stress response, leukocyte adhesion, and anticoagulation, are also discussed. Finally, we draw conclusions regarding current challenges and attempts to identify future directions for the optimal combination of materials, fabrication methods, surface modifications, and biofunctionalities. We hope that this review can assist with the design, fabrication, and application of SDBVs engineered in vitro and promote future advancements in this emerging research field.

Unique molecular networks: Formation and role of elastin cross-links.

Elastic fibers are essential assemblies of vertebrates and confer elasticity and resilience to various organs including blood vessels, lungs, skin, and ligaments. Mature fibers, which comprise a dense and insoluble elastin core and a microfibrillar mantle, are extremely resistant toward intrinsic and extrinsic influences and maintain elastic function over the human lifespan in healthy conditions. The oxidative deamination of peptidyl lysine to peptidyl allysine in elastin's precursor tropoelastin is a crucial posttranslational step in their formation. The modification is catalyzed by members of the family of lysyl oxidases and the starting point for subsequent manifold condensation reactions that eventually lead to the highly cross-linked elastomer. This review summarizes the current understanding of the formation of cross-links within and between the monomer molecules, the molecular sites, and cross-link types involved and the pathological consequences of abnormalities in the cross-linking process.

Interobserver Agreement in Vascular Invasion Scoring and the Added Value of Immunohistochemistry for Vascular Markers to Predict Disease Relapse in Stage I Testicular Nonseminomas.

Vascular invasion has been identified as an informative risk factor for relapse in stage I testicular nonseminomas, used to tailor treatment. We investigated interobserver agreement in vascular invasion reporting and studied the potential additional value of immunohistochemistry for vascular markers for predicting relapse. Patients (n=52) with stage I testicular nonseminomas undergoing surveillance (1993-2006) were included (median follow-up of 66 mo). Two formalin-fixed paraffin-embedded blocks with >1 cm tissue and tumor/normal parenchyma interface were stained with hematoxylin and eosin and CD31, FVIII, and D2-40. Slides were assessed by 3 independent testicular germ cell tumor-dedicated pathologists, and agreement was assessed using Cohen κ statistic. Sensitivity, specificity, and accuracy of vascular invasion scoring in predicting relapse were calculated. Agreement among testicular germ cell tumor-dedicated pathologists was moderate (κ=0.49 to 0.54), as was performance in predicting disease relapse (particularly, specificity of 86%). Immunohistochemistry increased overall sensitivity (71%), but decreased specificity (71%) in predicting relapse. All patients (n=8) with both blood and lymphatic vascular invasion developed a relapse. In multivariable analysis (including age, tumor size, rete testis invasion, and serum tumor markers), only vascular invasion had an independent impact in predicting relapse. Assessment of vascular invasion by testicular germ cell tumor-dedicated pathologists is good and is clinically meaningful, predicting disease relapse. Immunohistochemistry for vascular markers improves sensitivity of detecting disease relapse and allows for the identification of high-risk patients with both blood and lymphatic vascular invasion simultaneously, potentially of interest for tailored chemotherapy.

Accumulation of Amyloid Beta (Aß) Peptide on Blood Vessel Walls in the Damaged Brain after Transient Middle Cerebral Artery Occlusion.

It is well known that amyloid beta (Aß) peptides are generated in blood vessels, released into the brain during thrombosis, and temporarily accumulate in this organ after injury. Here we demonstrate that 24 h after transient middle cerebral artery occlusion (tMCAO), one of the standard models of focal ischemic stroke, Aß peptide accumulates in the brain, concentrating on the blood vessel walls. Because Aß oligomers are known to induce significant damage to brain cells, they act as an additional damaging factor during ischemic stroke. Considering that they have been shown to form ion channels in cells, affecting osmotic balance, we used an Aß peptide channel blocker, tromethamine (2-amino-2-(hydroxymethyl) propane-1,3-diol), to prevent this additional injury. Tromethamine injected 0.1 g/100 g body weight intraperitoneally at 5 min before tMCAO decreased water content in the damaged hemisphere, as measured by dry brain weight. Congo red staining, which binds only to Aß oligomer plaques (amyloid), showed that there was no significant presence of plaques. Therefore, we suggest that Aß peptide oligomers are responsible for some of the brain damage during stroke and that blockage of the ion channels that they form could be beneficial in treating this complex neurological syndrome.

Microfluidic Fabrication of Biomimetic Helical Hydrogel Microfibers for Blood-Vessel-on-a-Chip Applications.

Nature has created many perfect helical microstructures, including DNA, collagen fibrils, and helical blood vessels, to achieve unique physiological functions. While previous studies have developed a number of microfabrication strategies, the preparation of complex helical structures and cell-laden helical structures for biomimetic applications remains challenging. In this study, a one-step microfluidics-based methodology is presented for preparing complex helical hydrogel microfibers and cell-laden helical hydrogel microfibers. Several types of complex helical structures, including multilayer helical microfibers and superhelical hollow microfibers with helical channels, are prepared by simply tuning the flow rates or modifying the geometry of microfluidic device. With the decent perfusability, the hollow microfibers may simulate the structural characteristics of helical blood vessels and create swirling blood flow in a blood-vessel-on-chip setup. Such hydrogel-based helical microstructures may potentially be used in areas such as blood vessel tissue engineering, organ-on-chips, drug screening, and biological actuators.

Engineering the vasculature of decellularized rat kidney scaffolds using human induced pluripotent stem cell-derived endothelial cells.

Generating new kidneys using tissue engineering technologies is an innovative strategy for overcoming the shortage of donor organs for transplantation. Here we report how to efficiently engineer the kidney vasculature of decellularized rat kidney scaffolds by using human induced pluripotent stem cell (hiPSCs)-derived endothelial cells (hiPSC-ECs). In vitro, hiPSC-ECs responded to flow stress by acquiring an alignment orientation, and attached to and proliferated on the acellular kidney sections, maintaining their phenotype. The hiPSC-ECs were able to self-organize into chimeric kidney organoids to form vessel-like structures. Ex vivo infusion of hiPSC-ECs through the renal artery and vein of acellular kidneys resulted in the uniform distribution of the cells in all the vasculature compartments, from glomerular capillaries to peritubular capillaries and small vessels. Ultrastructural analysis of repopulated scaffolds through transmission and scanning electron microscopy demonstrated the presence of continuously distributed cells along the vessel wall, which was also confirmed by 3D reconstruction of z-stack images showing the continuity of endothelial cell coverage inside the vessels. Notably, the detection of fenestrae in the endothelium of glomerular capillaries but not in the vascular capillaries was clear evidence of site-specific endothelial cell specialisation.

Assessment of the static upright balance index and brain blood oxygen levels as parameters to evaluate pilot workload.

OBJECTIVE: To investigate the potential for static upright balance function and brain-blood oxygen parameters to evaluate pilot workload. METHODS: Phase 1: The NASA Task Load Index (NASA-TLX) was used to compare the workloads of real flights with flight simulator simulated flight tasks in 15 pilots (Cohort 1). Phase 2: To determine the effects of workload, 50 cadets were divided equally into simulated flight task load (experimental) and control groups (Cohort 2). The experimental group underwent 2 h of simulated flight tasks, while the control group rested for 2 h. Their static upright balance function was evaluated using balance index-1 (BI-1), before and after the tasks, with balance system posturography equipment and cerebral blood oxygen parameters monitored with near infrared spectroscopy (NIRS) in real time. Sternberg dual-task and reaction time tests were performed in the experimental and control groups before and after the simulated flight tasks. RESULTS: (Phase1) There was a significant correlation between the workload caused by real flight and simulated flight tasks (P<0.01), indicating that NASA-TLX scales were also a tool for measuring workloads of the stimulated flight tasks. (Phase 2) For the simulated flight task experiments, the NASA-TLX total scores were significantly different between the two groups (P<0.001) and (pre-to-post) changes of the BI-1 index were greater in the experimental group than in controls (P<0.001). The cerebral blood oxygen saturation levels (rsO2) (P<0.01) and ΔHb reductions (P<0.05) were significantly higher in the experimental, compared to the control group, during the simulated flight task. In contrast to the control group the error rates (P = 0.002) and accuracy (P<0.001) changed significantly in the experimental group after the simulated flight tasks. CONCLUSIONS: The simulated flight task model could simulate the real flight task load and static balance and NIRS were useful for evaluating pilots' workload/fatigue.

Carbon debris and fiber cleaving: Effects on potassium-titanyl-phosphate laser energy and chorioallantoic membrane model vessel coagulation.

OBJECTIVES/HYPOTHESIS: Photoangiolytic precision afforded by the 532-nm potassium-titanyl-phosphate (KTP) laser relies on predictable energy delivery. Inadequate energy output can cause vessel rupture, and excessive energy can cause thermal damage. The quality of the cleaved surface and carbon deposits from ablated tissue are two factors that could negatively impact fiber performance. The effects of these on energy output and blood vessel coagulation were assessed using a chorioallantoic membrane (CAM) model. STUDY DESIGN: Comparative analysis. METHODS: Laser fibers with carbon debris, optimal fiber cleaving, and suboptimal cleaving were inspected at three times magnification, and the light dispersion pattern of each fiber was rated. The average energy output from consecutive pulses through each fiber configuration was recorded. The effect of these fiber conditions on clinical efficacy was estimated by measuring vessel coagulation versus rupture in the CAM model. Repeated measures analysis of variance compared results. RESULTS: Carbon debris and suboptimal cleaving resulted in decreased energy output in comparison to optimal cleaving ([-Δ244 mJ, d = 4.31, P < .001] and [-Δ195 mJ, d = 6.04, P < .001]). Optimal cleaving resulted in immediate coagulation of vessels. Fibers with suboptimal cleaving and carbon debris had unpredictable outcomes, requiring multiple pulses for coagulation or causing vessel rupture. CONCLUSIONS: KTP laser fiber function is significantly affected by fiber tip condition. Carbon debris and suboptimal cleaving create significant attenuation of energy, which results in an unpredictable angiolytic effect, as demonstrated by increased vessel rupture in the CAM model. Optimal recleaving of KTP laser fibers restores prior energy output and predictable coagulation. Care should be taken to avoid carbon debris on laser-fiber tips and to cleave fibers properly. LEVEL OF EVIDENCE: NA Laryngoscope, 129:2244-2248, 2019.

A hydrogel derived from acellular blood vessel extracellular matrix to promote angiogenesis.

The biocompatibility and bioactivity of injectable acellular extracellular matrix nominates its use as an optimal candidate for cell delivery, serving as a reconstructive scaffold. In this study, we investigated the feasibility of preparing a blood vessel matrix (BVM) hydrogel, which revealed its pro-angiogenic effects in vitro and its therapeutic effects in an in vivo skin flap model. Aortic and abdominal aortic arteries from pigs were acellularized by Triton-X 100 and confirmed by hematoxylin and eosin and 4,6-diamidino-2-phenylindole staining. Different concentrations of blood vessel matrix hydrogel were generated successfully through enzymatic digestion, neutralization, and gelation. Hematoxylin and eosin staining, Masson's trichrome staining, collagen type I immunohistochemistry staining, and enzyme-linked immunosorbent assays showed that type I collagen and some growth factors were retained in the hydrogel. Scanning electron microscopy demonstrated the different diametric fibrils in blood vessel matrix hydrogels. A blood vessel matrix hydrogel-coated plate promoted the tube formation of human umbilical vein endothelial cells in vitro. After injection into skin flaps, the hydrogel improved the flap survival rate and increased blood perfusion and capillary density. These results indicated that we successfully prepared a blood vessel matrix hydrogel and demonstrated its general characteristics and angiogenic effects in vitro and in vivo.

Mast cell chymase degrades fibrinogen and fibrin.

BACKGROUND: The accumulation of immunoreactants and fibrinoid necrosis of postcapillary vessel walls are common pathological features of cutaneous immune complex vasculitis. In more advanced lesions, these immunoreactants are subject to proteolysis. Mast cell chymase is a powerful enzyme that can degrade several substrates including the extracellular matrix. Heparin can influence the catalytic properties of chymase. OBJECTIVES: To study the effects of recombinant human (rh) chymase on fibrinogen, coagulation and fibrinolysis, and to relate these effects to the pathogenesis of vasculitis. METHODS: The colocalization of chymase and fibrin in vasculitis specimens was analysed by immunohistochemical double staining. Fibrinogen and fibrin were treated with rh-chymase and the effects were studied in vitro by sodium dodecylsulfate polyacrylamide gel electrophoresis and a variety of clotting and fibrin gel experiments. The effects of rh-chymase on vasculitis cryosections were analysed by direct immunofluorescence. RESULTS: Chymase-positive mast cells were associated with fibrin-positive vessels in vasculitis cryosections. Rh-chymase degraded the alpha-, beta- and gamma-chains of fibrinogen, while heparin enhanced the degradation of the beta-chain. Rh-chymase pretreatment of fibrinogen prolonged thrombin-induced clotting time. Fibrinogen degradation products induced by rh-chymase increased the clotting time of human plasma. Rh-chymase degraded fibrin gel prepared from fibrinogen or human plasma. Immunofluorescence staining positivity of fibrin in vasculitis cryosections decreased after pretreatment with rh-chymase for 24 h, and heparin enhanced this effect. CONCLUSIONS: Mast cell chymase may constitute a previously unrecognized endogenous anticoagulant and fibrinolytic enzyme, and may be involved in the clearance of fibrin from vessel walls in aged vasculitis lesions.

Electrochemical fabrication of a biomimetic elastin-containing bi-layered scaffold for vascular tissue engineering.

Biomimetic tissue-engineered vascular grafts (TEVGs) have immense potential to replace diseased small-diameter arteries (<4 mm) for the treatment of cardiovascular diseases. However, biomimetic approaches developed thus far only partially recapitulate the physicochemical properties of the native vessel. While it is feasible to fabricate scaffolds that are compositionally similar to native vessels (collagen and insoluble elastic matrix) using freeze-drying, these scaffolds do not mimic the aligned topography of collagen and elastic fibers found in native vessels. Extrusion-based scaffolds exhibit anisotropic collagen orientation but these scaffolds are compositionally dissimilar (cannot incorporate insoluble elastic matrix). In this study, an electrochemical fabrication technique was employed to develop a biomimetic elastin-containing bi-layered collagen scaffold which is compositionally and structurally similar to native vessels and the effect of insoluble elastin incorporation on scaffold mechanics and smooth muscle cell (SMC) response was investigated. Further, the functionality of human umbilical vein endothelial cells (HUVECs) on the scaffold lumen surface was assessed via immunofluorescence. Results showed that incorporation of insoluble elastin maintained the overall collagen alignment within electrochemically aligned collagen (ELAC) fibers and this underlying aligned topography can direct cellular orientation. Ring test results showed that circumferential orientation of ELAC fibers significantly improved scaffold mechanics. Real-time PCR revealed that the expression of α-smooth muscle actin (Acta2) and myosin heavy chain (MyhII) was significantly higher on elastin containing scaffolds suggesting that the presence of insoluble elastin can promote contractility in SMCs. Further, mechanical properties of the scaffolds significantly improved post-culture indicating the presence of a mature cell-synthesized and remodeled matrix. Finally, HUVECs expressed functional markers on collagen lumen scaffolds. In conclusion, electrochemical fabrication is a viable method for the generation of a functional biomimetic TEVG with the potential to be used in bypass surgery.