Higher Vessel Density Research Articles

Lymphatic Obstruction

Antrochoanal polyps (ACPs) originate from the inner wall of the maxillary sinus and either pass through the natural sinus ostia or cause pressure-induced destruction of the medial sinus wall. Eventually, they extend into the choanae and nasopharynx. Most authors who have studied the microstructure of ACPs, including the component stromal cells and surface epithelium, have not examined the transitional area between the sinus mucosa and the pedicle of the polyp. No explanation has been given for the absence of a cystic intrasinus portion of the polyp, in many cases refuting the therapy (most accepted) that polyps are caused by a mucous gland with a blocked acinus. We noted during endoscopic removal of the ACPs that the antral part of the polyp was cystic in only 5% of patients, and polypoid in 95%. The cystic intrasinus portion of the polyp is a cornerstone of the pathophysiology of ACPs, whether caused by inflammation, cicatrization, or allergy. This finding prompted us to examine the transitional area between the sinus mucosa and the pedicle of the polyp to verify the possibility that lymphatic obstruction--whether primary (areas of higher tissue pressure) or secondary (cicatrization or inflammation)-could be an etiologic factor in the formation of ACPs. The study material consisted of 25 ACPs and 25 chronic maxillary sinusitis mucosal biopsy specimens (control group). The detection of lymphatic vessels was based on the identification of lymph vessel endothelial hyaluronic acid receptor I (LYVE-I) in the endothelial cells of the lymphatic capillaries. This was the first lymph-specific hyaluronic acid receptor to be characterized, and is a uniquely powerful marker for lymph vessels, differentiating them from (blood) capillaries. The density of the lymphatic vessels was marked in 22 of the 25 ACP specimens, ie, 88% of the ACP cases, compared with 16% of the control group. This study resulted in two main findings. The first was the absence of intramaxillary cysts in the ACPs in 23 cases (92%). The second was the markedly high density of lymphatic vessels in the transitional area between the sinus mucosa and the pedicle of the ACPs, in comparison with the density in the control group. These two findings refute the "blocked acinus theory" and indicate that lymphatic obstruction, whether primary or secondary to chronic sinus infection, might play a leading role in the formation and further growth of ACPs.

Disentangling the effects of ontogeny and environmental factors on xylem anatomy in a semiarid chamaephyte

The hydraulic system of plants reflects a balance between efficiency and safety which is continuously adjusted along plant's life to guarantee survival under changing conditions. One of such adjustments is the formation of new conductive cells, so vessel traits along ring series reflect part of plant's hydraulic plasticity. In this work we disentangled the effects of ontogeny and environmental factors on wood anatomy of a semiarid chamaephyte, Linum suffruticosum. We sampled individuals on contrasting slope aspects in the Ebro valley (Spain), and measured ring width and vessel traits to obtain annually-resolved data series. General Additive Models (GAMs) were used to identify the variability associated to cambial age, slope aspect, and climatic factors: a shift towards a more efficient xylem configuration was observed at the eighth year of life; narrower rings characterized by higher vessel density and smaller vessel sizes were found at south-facing slopes; and low precipitation at the onset of the growing season resulted in safer vessel features. The combination of quantitative analysis of vessels along ring series and GAMs allowed a global understanding of xylem's functional plasticity; moreover, the need for adequately assessing ontogenetic and topographic effects when predicting plant distribution under global change scenarios was reinforced.

Mesenchymal stromal cells induce epithelial‐to‐mesenchymal transition in human colorectal cancer cells through the expression of surface‐bound TGF‐β

Mesenchymal stem/stromal cells (MSC) are multipotent precursors endowed with the ability to home to primary and metastatic tumor sites, where they can integrate into the tumor-associated stroma. However, molecular mechanisms and outcome of their interaction with cancer cells have not been fully clarified. In this study, we investigated the effects mediated by bone marrow-derived MSC on human colorectal cancer (CRC) cells in vitro and in vivo. We found that MSC triggered epithelial-to-mesenchymal transition (EMT) in tumor cells in vitro, as indicated by upregulation of EMT-related genes, downregulation of E-cadherin and acquisition of mesenchymal morphology. These effects required cell-to-cell contact and were mediated by surface-bound TGF-β newly expressed on MSC upon coculture with tumor cells. In vivo tumor masses formed by MSC-conditioned CRC cells were larger and characterized by higher vessel density, decreased E-cadherin expression and increased expression of mesenchymal markers. Furthermore, MSC-conditioned tumor cells displayed increased invasiveness in vitro and enhanced capacity to invade peripheral tissues in vivo. Thus, by promoting EMT-related phenomena, MSC appear to favor the acquisition of an aggressive phenotype by CRC cells.

Triple-negative, Basal Marker-expressing, and High-grade Breast Carcinomas are Characterized by High Lymphatic Vessel Density and the Expression of Podoplanin in Stromal Fibroblasts

Podoplanin, expressed in the lymphatic but not in the blood vessel endothelium, is widely used as a specific marker for lymphatic endothelial cells and lymphangiogenesis. The relation between lymphatic vessel density and breast cancer subtype or the expression of basal markers has not yet been investigated. We assessed lymphatic vessel density (LVD), blood vessel density, and the expression of podoplanin in stromal cancer-associated fibroblasts, in 156 invasive ductal breast cancers (T≥1, N≥1, M0). Afterwards, we assessed the relationship between the above-mentioned parameters and (i) breast cancer subtype (luminal vs. HER2 vs. triple negative), (ii) tumor grade (G1 vs. G2 vs. G3), (iii) the expression of cytokeratin (CK) 5/6, (iv) P-cadherin, (v) smooth muscle actin (SMA), and (vi) the pattern/intensity of stromal lymphocytic infiltration. We found a significantly higher LVD and podoplanin expression in stromal fibroblasts in (i) G3 tumors, (ii) triple-negative carcinomas, (iii) tumors expressing CK5/6, SMA, or P-cadherin, and (iv) neoplasms with stroma intensively infiltrated by lymphocytes. Moreover, we observed a significant inverse relationship between the expression of podoplanin in luminal A subtype, P-cadherin, CK5/6, and SMA-negative tumors and tumors without strong lymphocytic infiltration. A significantly higher percentage of tumors with strong lymphocytic infiltration was noted among G3 carcinomas. Breast carcinomas of different grades, subtypes, and basal marker expression are characterized by different composition of the stroma, that is different LVD, podoplanin expression in stromal fibroblasts, and the pattern/intensity of lymphocytic infiltration.

Atorvastatin Treatment of Rats with Ischemia-Reperfusion Injury Improves Adipose-Derived Mesenchymal Stem Cell Migration and Survival via the SDF-1α/CXCR-4 Axis

BackgroundAdipose-derived mesenchymal stem cells (ASCs) transplantation is a promising approach for myocardium repair. Promotion of ASCs migration and survival is the key for improving ASCs efficiency. SDF-1α is a critical factor responsible for ASCs migration and survival. Atorvastatin (Ator) is capable of up-regulating SDF-1α. Therefore, we're going to investigate whether ASCs migration and survival could be improved with atorvastatin.Methods In vitro study, cardiomyocytes were subjected to anoxia-reoxygenation injury and subsequently divided into different groups: group blank control, Ator, Ator plus L-NAME (A+L-NAME) and Ator plus AMD3100 (A+AMD3100).When migration analysis completed, cardiomyocytes were used for subsequent analyses. In vivo study, rats underwent ischemia-reperfusion injury were assigned into different groups corresponding to in vitro protocols. ASCs were transplanted on the seventh day of atorvastatin therapy. Seven days later, the rates of migration, differentiation and apoptosis were evaluated.ResultsCompared with other groups, ASCs migration in vitro was significantly improved in group Ator, which was dependent on SDF-1α/CXCR-4 coupling. Results of in vivo study were consistent with that of in vitro study, further supporting the notion that the efficacy of atorvastatin on ASCs migration improvement was related to SDF-1α/CXCR-4 axis. Higher vessel density in group Ator might be another mechanism responsible for migration improvement. Concomitantly, apoptosis was significantly reduced in group Ator, whereas no significant difference of differentiation was found.ConclusionMigration and survival of ASCs could be improved by atorvastatin under ischemia-reperfusion injury, which were ascribed to SDF-1α/CXCR-4 axis.

Metaphyseal bone formation induced by a new injectable β-TCP-based bone substitute: A controlled study in rabbits

Adequate filling of bone defects still poses a challenge in every day clinical work. As many bone defects are irregularly shaped the need for appropriate scaffolds reaching the complete defect surface are great. The purpose of this pre-clinical pilot study was to investigate the handling, biocompatibility, biodegradation and osteoconductivity of a new pasty bone substitute (pure phase β-TCP, hyaluronic acid, methylcellulose) in bone tissue. In an unilateral tibial defect model the peri-implant and bone tissue response to the new pasty bone substitute was tested in New Zealand white rabbits for up to 24 weeks compared to empty controls. Analysis included HR-pQCT scans, histomorphometric evaluation and quantification of vascularization of un-decalcified histological slices. After 1 week the experimental group presented significantly higher new bone volume fraction (p = 0.021) primarily consisting of immature bone matrix and higher vessel density compared to controls (p = 0.013). After 4 weeks bone formation was not significantly different to controls but was distributed more evenly throughout the defect. Bone matrix was now mineralized and trabeculae were thicker than in controls (p = 0.002) indicating faster intramedullary bone maturation. Controls presented extensive periosteal bone formation, major fibrous tissue influx and high vascularization. After 12 and 24 weeks there was no new bone detectable. There were no severe signs of inflammation at all time points. The substitute showed an early induction of bone formation. It promoted accelerated intramedullary bone repair and maturation and prevented periosteal bone formation indicating its potential use for reconstructive surgery of bone defects.

Abstract 1476: Mesenchymal stromal cells induce epithelial-to-mesenchymal transition in human colorectal cancer cells through the expression of surface-bound TGF-β.

Abstract Mesenchymal stem/stromal cells (MSC) are multipotent precursors endowed with the ability to home to primary and metastatic tumor site. Because of this feature, they are currently being used in preclinical models and in clinical trials as potential vehicles for delivery of targeted anti-cancer therapies. Clinical applications of MSC however, require a deeper understanding of their biology and, in particular, of their interaction with cancer cells. In this study we have investigated the effects mediated by human bone marrow-derived MSC on human colorectal cancer (CRC) cells in vitro and in vivo. We found that MSC induced upregulation of epithelial-to-mesenchymal (EMT)-related genes and downregulation of E-cadherin in tumor cells. These effects required cell-to-cell contact and were mediated by expression of surface-bound TGF-β, induced in MSC upon coculture with tumor cells. CRC cells exposed to MSC formed larger tumor masses in vivo, characterized by higher vessel density and increased mesenchymal marker expression, and displayed enhanced invasive capacity. Thus, by promoting cell expansion, EMT-related phenomena and tumor angiogenesis, MSC appear to favor the acquisition of an aggressive phenotype by CRC cells. These findings raise concerns regarding the safety of MSC administration for clinical purposes in patients with cancer. Citation Format: Valentina Mele, Manuele Giuseppe Muraro, Diego Calabrese, Nunzia Amatruda, Francesca Amicarella, Dennis Pfaff, Brynn Kvinlaug, Chiara Bocelli-Tyndall, Ivan Martin, Therese J. Resink, Luigi M. Terracciano, Giulio Cesare Spagnoli, Giandomenica Iezzi. Mesenchymal stromal cells induce epithelial-to-mesenchymal transition in human colorectal cancer cells through the expression of surface-bound TGF-β. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1476. doi:10.1158/1538-7445.AM2013-1476

Quantification of microvascular cerebral blood flux and late‐stage tumor compartmentalization in 9L gliosarcoma using flow enhanced MRI

Measurements of tumor microvasculature are important to obtain an understanding of tumor angiogenesis and for the evaluation of therapies. In this work, we characterize the evolution of the microvascular flux at different stages of tumor growth in the 9L rat brain tumor model. The absolute quantification of cerebral blood flux is achieved with MRI at 7 T using the flow enhanced signal intensity (FENSI) method. FENSI flux maps were obtained between 5 and 14 days after glioma cell inoculation. Based on cerebral blood flux maps, we highlighted two main stages of tumor growth, below and above 3 mm, presenting distinct flux patterns and vascular properties. No significant difference emerged from the group analysis performed on the data collected at an early developmental stage (tumor size < 3 mm) when compared with healthy tissue. At a late developmental stage (tumor size > 3 mm), we observed a significant decrease in the cerebral blood flux inside the gliosarcoma (-33%, p < 0.01) and compartmentalization of the tumor (p < 0.05). FENSI flux maps delineated a low-flux tumor core (58 ± 17 μL/min/cm(2) ) and higher vascularized regions around the tumor periphery (85 ± 21 μL/min/cm(2) ). Histology was performed on 11 animals to finely probe the intratumor heterogeneity and microvessel density, and the results were compared with the information derived from FENSI flux maps. The hyper- and hypoperfused tumor regions revealed with FENSI at the late tumor developmental stage correlated well with the ratios of high and low blood vessel density (R(2) = 0.41) and fractional vascular surface (R(2) = 0.67) observed with fluorescence microscopy [cluster of differentiation 31 (CD31) staining].

ROXAS – an efficient and accurate tool to detect vessels in diffuse-porous species

Wood-anatomical parameters form a valuable archive to study past limitations on tree growth and act as a link between dendrochronology and ecophysiology. Yet, analysing these parameters is a time-consuming procedure and only few long chronologies exist. To increase measurement efficiency of wood-anatomical parameters, novel tools like the automated image-analysis system ROXAS were developed. So far, ROXAS has only been applied to measure large earlywood vessels in ring-porous species.In this study, we evaluate if ROXAS is also suitable for efficient and accurate detection and measurement of vessels in diffuse-porous European beech. To do so, we compared the outcome of ROXAS with that of the established measurement programme Image-Pro Plus in terms of efficiency and accuracy.The two methods differed substantially in efficiency with automatic measurements using ROXAS being 19 times faster than with Image-Pro Plus. Although the procedures led to similar patterns in annual variation of mean vessel area and vessel density, the absolute values differed. Image-Pro Plus measured systematically lower mean vessel areas and higher vessel densities than ROXAS. This was attributed to the species-specific technical settings in ROXAS, leading to more realistic results than those obtained using the default settings in Image-Pro Plus. A shortcoming of ROXAS was, however, that small vessels (&lt;100 μm²) could not be detected with sufficient accuracy. Yet, based on thin sections of European beech, it is generally difficult to distinguish such small vessels from parenchyma cells. Moreover, these small vessels do not contribute substantially to conductive efficiency. Therefore, we do not foresee any problems for most studies if the lower vessel area threshold to be measured is set to 100 μm². Overall, ROXAS proved to be useful for measuring vessel parameters in diffuse-porous tree species, allowing accurate and efficient analyses of large numbers of samples.

Anti-Thymocyte Globulin Induces Neoangiogenesis and Preserves Cardiac Function after Experimental Myocardial Infarction

RationaleAcute myocardial infarction (AMI) followed by ventricular remodeling is the major cause of congestive heart failure and death in western world countries.ObjectiveOf relevance are reports showing that infusion of apoptotic leucocytes or anti-lymphocyte serum after AMI reduces myocardial necrosis and preserves cardiac function. In order to corroborate this therapeutic mechanism, the utilization of an immunosuppressive agent with a comparable mechanism, such as anti-thymocyte globulin (ATG) was evaluated in this study.Methods and ResultsAMI was induced in rats by ligation of the left anterior descending artery. Initially after the onset of ischemia, rabbit ATG (10 mg/rat) was injected intravenously. In vitro and in vivo experiments showed that ATG induced a pronounced release of pro-angiogenic and chemotactic factors. Moreover, paracrine factors released from ATG co-incubated cell cultures conferred a down-regulation of p53 in cardiac myocytes. Rats that were injected with ATG evidenced higher numbers of CD68+ macrophages in the ischemic myocardium. Animals injected with ATG evidenced less myocardial necrosis, showed a significant reduction of infarct dimension and an improvement of post-AMI remodeling after six weeks (infarct dimension 24.9% vs. 11.4%, p<0.01). Moreover, a higher vessel density in the peri-infarct region indicated a better collateralization in rats that were injected with ATG.ConclusionsThese data indicate that ATG, a therapeutic agent successfully applied in clinical transplant immunology, triggered cardioprotective effects after AMI that salvaged ischemic myocardium by down-regulation of p53. This might have raised the resistance against apoptotic cell death during ischemia. The combination of these mechanisms seems to be causative for improved cardiac function and less ventricular remodeling after experimental AMI.

High density of peritumoral lymphatic vessels measured by D2-40/podoplanin and LYVE-1 expression in gastric cancer patients: an excellent prognostic indicator or a false friend?

BackgroundOne of the most important prognostic indicators in gastric cancer is the presence of metastases in lymph nodes. Even now, little is known about lymphangiogenesis in neoplastic tissue, and little is also known about the transmission of a neoplastic cell from the tumor mass into a lymphatic vessel.MethodsThis study examined the relationships between the density of lymphatic vessels (LVD) stained immunohistochemically with lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) and D2-40 (podoplanin) antibodies, the expression of vascular endothelial growth factor (VEGF)-C/D, selected clinical and pathomorphological factors, and the 5-year overall survival of gastric cancer patients.ResultsStatistical analysis showed no impact of increased intratumoral or peritumoral LVD on gastric cancer patient survival, irrespective of the protein used to stain lymphatic vessels. Analysis showed that the probability of overall survival was decreased in the cases with enhanced VEGF-D immunoreactivity (P = 0.0045).ConclusionThe study showed that the studied markers cannot be used to determine the required extent of the surgical procedure, as they have no statistically significant correlation with the degree of progression of the cancer, the stage of the disease assessed according to the TNM 5th classification of malignant tumors, clinicopathological features, and patient survival. VEGF-D is the only marker that can be regarded as an unfavorable prognostic indicator for patients with advanced gastric cancer.

Prognostic Significance of EpCAM-Positive Disseminated Tumor Cells in Rectal Cancer Patients With Stage I Disease

Here we evaluated the prevalence and prognostic impact of epithelial cell adhesion molecule (EpCAM)-positive disseminated tumor cells (DTCs) in stage I rectal cancer. Further we tested the association of these single tumor cells or small tumor cell groups with the extent of peritumoral lymphangiogenesis. A total of 845 regional lymph nodes (LN) of 44 patients classified as negative on conventional histopathology were retrospectively reanalyzed with immunohistochemistry (IHC) using the monoclonal antibody Ber-Ep4 directed against EpCAM for the detection of DTCs. The degree of lymphangiogenesis in the primary tumors was assessed by IHC of the primary tumor tissue using the monoclonal antibody D2-40, which reacts with the lymphatic endothelium. The IHC results were correlated with clinico-pathologic parameters and clinical follow-up data. EpCAM-positive DTCs in LNs were detected in 8 (18.2%) of the 44 patients. During a median follow-up of 59 months, 3 (37.5%) of the 8 patients with EpCAM-positive DTCs relapsed, whereas none of the DTC-negative patients developed tumor recurrence (P=0.004). Survival analysis revealed a significant effect of the prevalence of DTCs on overall survival (P=0.0009) and on recurrence-free survival (P=0.0001). Finally, the prevalence of EpCAM-positive DTCs in perirectal LNs was significantly correlated with a high density of peritumoral lymphatic vessels (P=0.015). Our results show that DTCs may occur in stage I of rectal cancer and are associated with poor prognosis. Their occurrence seems to be linked to a high density of newly formed lymphatic vessel at the primary tumor site. According to our data, patients with DTCs in their LN might benefit from adjuvant therapy.

Ineffectiveness of a marine sanctuary zone to protect burrunan dolphins (Tursiops australis sp.nov.) from commercial tourism in Port Phillip Bay, Australia

Over the past two decades, considerable growth in commercial cetacean-based tourism has exposed coastal cetacean populations to high vessel density. Since 1989, Port Phillip Bay, Victoria, Australia, has hosted a thriving dolphin-swim tourism industry comprised of three licensed vessels. This study assessed the effectiveness of the Ticonderoga Bay Sanctuary Zone, a protected area enacted under the Wildlife (Whales) Regulations 1998, to serve the resident dolphins as an area of ‘respite’ and ‘refuge’ from anthropogenic stress, including commercial tourism. Research was conducted onboard 104 dolphin-swim tours, with both tour operation and dolphin school behaviour recorded using a combination of continuous observations and 1-min scan samples. During all observed encounters within the sanctuary zone, tour operations contravened the site-specific minimal approach distance regulation. By also contravening generic permit conditions (compliance range 0–70%) to the level of unsatisfactory compliance consistent with that documented outside the sanctuary zone, tour operations did not exercise any additional caution during a dolphin encounter within the sanctuary zone. Recommendations for this industry include a shift from sole reliance on passive management strategies to a judicious management plan that includes enforcement to support the governing regulations.

Relationship Between High Density of Peritumoral Lymphatic Vessels and Biological Behavior of Cervical Cancer

To investigate the relationship between lymphangiogenesis and lymphatic metastasis in cervical squamous carcinoma. Eighty cases of invasive cervical squamous cancer were selected as objects of our study. Double immunohistochemical staining with antibodies against lymphatic vessel endothelial hyaluronan receptor 1 and Ki-67 was used to label the lymphatic vessels and mark the proliferative lymphatic vessels in cervical squamous cancer. The peritumoral lymphatic vessel density and intratumoral lymphatic vessel density was assessed. The lymphatic vessels proliferation index was evaluated by calculating Ki-67 proliferation index (PI) to reflect the lymphangiogenesis of cervical squamous cancer. Then the correlation between lymphangiogenesis and clinicopathologic features of cervical squamous cancer was analyzed. The LVD of cervical cancer (15.23 ± 3.6) was clearly higher than that of the adjacent normal cervical subepithelial tissues (9.9 ± 2.5, P < 0.001). The peritumoral lymphatic vessel density of cervical cancer (18.75 ± 4.3) was significantly higher than the intratumoral lymphatic vessel density of cervical cancer (11.71 ± 4.9, P < 0.001). Lymphatic PI (LPI) of cervical cancer (0.258 ± 0.07) was higher than that of the adjacent normal cervical subepithelial tissues (0.068 ± 0.08, P < 0.001). The peritumoral lymphatic vessel PI of cervical cancer (0.324 ± 0.06) was notably higher than the intratumoral lymphatic vessel PI of cervical cancer (0.232 ± 0.06, P < 0.001). Peritumoral lymphatic vessel density and peritumoral lymphatic vessel were clearly associated with the lymph node metastasis (P = 0.001 and P = 0.002, respectively) and lymphovascular space invasion (P = 0.024 and P = 0.01, respectively). The high density of peritumoral lymphatic vessels is a potential predictor of more aggressive phenotype of cervical squamous cancer.

MENSTRUAL BLOOD STEM CELLS IMPROVED MYOCARDIAL SURVIVAL AFTER RAT MYOCARDIAL INFARCTION BY PARACRINE

ObjectivesIn the study, we hypothesised menstrual blood stem cells (MBSCs) protect ischaemic myocardium through paracrine effect.MethodsMBSCs were isolated and expanded from human female menstrual blood. Then we infused the MBSCs...

The beneficial effects of Momordica charantia (bitter gourd) on wound healing of rabbit skin

Momordica charantia (MC; bitter gourd) is a traditional herbal commonly used for its antidiabetic, antioxidant, contraceptive and antibacterial properties. In the current study, the authors aim to observe the topical effect of MC cream on the wound-healing process in rabbits. Moreover, they compare the healing potential with conventional creams used therapeutically. Towards this aim, 28 New Zealand rabbits were divided into four groups and excision wounds (7 cm²) were made on their backs. Open wound dressing was carried out daily for 28 days among the experimental groups with the application of dekspanthenol (Bepanthen®; BP group, n = 7), nitrofurazon (Furacin®; FR group, n = 7) and olive oil extract of MC (MC group, n = 7). No application was made to the control group. At the end of day 28, areas of the skin with initial wound area were en bloc dissected and prepared for histopathological and stereological analysis. Inflammatory cells were abundant in the control group and cream application led to a decrease in the number of these cells, especially in the MC group. The highest number of fibroblasts was detected in the MC group. Furthermore, the MC group displayed the highest fractions of epidermis to papillary dermis, fibroblasts to reticular dermis and collagen fibres to reticular dermis. The MC group also presented a high density of blood vessels, moderate density of collagen fibres and mature fibroblasts. The BP group showed better epithelialisation compared with the FR group, but the latter provided more effective reorganisation of the dermis. Different cream supplements caused healthy and fast wound healing according to untreated controls and the results show that administration of the MC extract improves and accelerates the process of wound healing in rabbits in comparison with the BP and FR extracts.

Creating 3D Angiogenic Growth Factor Gradients in Fibrous Constructs to Guide Fast Angiogenesis

Fast angiogenesis in 3D fibrous constructs that mimic the morphology of the extracellular matrix remains challenging due to limited porosity in the densely packed constructs. We investigated whether mimicking the in vivo chemotaxis microenvironment for native blood vessel formation would stimulate angiogenesis in the fibrous constructs. The chemotaxis microenvironment was created by introducing 3D angiogenic growth factor gradients into the constructs. We have developed a technique that can quickly fabricate (∼40 min) such 3D gradients by simultaneously electrospinning polycaprolactone (PCL) fibers, encapsulating gradient amount of bFGF (stabilized by heparin) into poly(lactide-co-glycolide) (PLGA) microspheres, and electrospraying the microspheres into PCL fibers. Gradient formation was confirmed by fluorescence microscopy. Gradients with different steepnesses were obtained by modulating the initial concentration of the bFGF solution. All of the constructs were able to sustainedly release bioactive bFGF over a 28 day period. The release kinetics was dependent on the bFGF loading and steepness of the gradient. In vitro cell migration study demonstrated that bFGF gradients significantly increased the depth of cell migration. To assess the efficacy of bFGF gradients in inducing angiogenesis, we implanted constructs subcutaneously using mouse model. bFGF gradients significantly promoted cell penetration into the constructs. After 10 days of implantation, a high density of mature blood vessels (positive to both CD31 and α-SMA) were formed in the constructs. Vessel density was increased with the increase in steepness of the bFGF gradient. These gradient constructs may have potential to engineer vascularized tissues for various applications.

High Lymph Vessel Density and Expression of Lymphatic Growth Factors in Peritoneal Endometriosis

To investigate the occurrence of lymph vessels and lymphangiogenic growth factors in peritoneal lesions, we performed immunohistochemical staining of peritoneal lesions of 37 patients with antibodies against podoplanin (D2-40), lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), prospero homeobox protein 1 (Prox-1), vascular epithelial growth factor (VEGF)-C/VEGF-D. Overall, 10 lesions were double stained against D2-40 and von Willebrand factor. The lymph vessel density in peritoneal lesion was significantly higher in comparison with healthy peritoneum. All lymph vessel makers could be detected, whereby the lymph vessel density of LYVE-1- and Prox-1-positive lymph vessels was significantly higher than the lymph vessel density of D2-40-positive lymph vessels. Endometriotic epithelial cells and stromal cells (SCs) showed a moderate-to-strong VEGF-C/VEGF-D expression. The VEGF-C-/VEGF-D-positive macrophages in endometriotic SCs could be observed. The lymphatic vasculature seems to form a further component of peritoneal lesions and could be involved in the inflammatory process. These data demonstrated a further step in the clarification of the pathogenesis of endometriosis.

Experimental study on the best concentration of SVFs for promoting survival rate of fat graft

To investigate the effect of different concentrations of human adipose stromal vascular fraction cells (SVFs) on the survival rate of fat transplantation. 0.3 ml fat tissue, derived and refined from clinical liposuction patients, was mixed with different concentrations of SVFs as 5 x 10(5)/ml in Group A, or 1 x 10(6)/ml in Group B, or 2 x 10(6)/ml in Group C, or completely medium in control group D. Then the mixture was injected randomly under the back skin of 6 nude mice. The transplanted fat tissue in four groups was harvested at 3 months after implantation. Wet weight of fat grafts was measured for macroscopic aspects. After HE staining, blood vessel density, viable adipocytes and fibrous proliferation were counted respectively for histological evaluation. The wet weight of fat grafts in group B (81.670 +/- 7.528) mg was significantly higher than that in group A, C, D [(60.000 +/- 6.325) mg, (68.330 +/- 7.528) mg, (48.330 +/- 7.528) mg, respectively, P < 0.05)], but the difference between group A and group C was not statistically significant (P > 0.05). The grafts in group A, B and C had significantly higher blood vessel density than those in the control group D, whereas blood vessel density was the highest in group B (P < 0.05) and there was no significant difference between group A and C (P > 0.05). Compared with group A, C and D, histological analysis revealed that the fat grafts in group B was consisted predominantly of adipose tissue with less fat necrosis and fibrosis (P < 0.05). However, fibrosis counts were significant lower in group A, B and C than those in group D (P < 0.05), and there was no significant difference between group A and C (P > 0.05). The human isolated SVFs has the advantages to improve the survival rate of fat transplantation, and the magnitude of 1 x 10(6)/ml is more practical and safe, indicating a wide clinical application in the future.

TGF-beta/Smad pathway and BRAF mutation play different roles in circumscribed and infiltrative papillary thyroid carcinoma

Poorly circumscribed growth pattern, extra-thyroid extension and high intratumoural lymph vessel density are significantly associated to nodal metastatization in papillary thyroid carcinoma (PTC). It was also shown that transforming growth factor beta (TGF-beta)/Smad-dependent pathway activity is associated with local invasion, nodal metastatization and BRAF-mutated PTCs. We analysed the immunoexpression of TGF-beta, Smad2/Smad3, Smad4 and Smad7 in a series of 42 cases of classic PTC and 33 cases of follicular variant of PTC with known clinico-pathological and follow-up data, as well as BRAF and RAS status. The 75 PTCs were divided into poorly circumscribed (PCPTC) (n = 53) and well circumscribed (WCPTC) (n = 22) according to their borders. Nodal metastases were not detected in any WCPTC regardless of the presence of immunoexpression for TGF-beta, Smad2/Smad3, Smad4 and Smad7 and occurrence of BRAF mutation (in 20% of WCPTCs). Increased cytoplasmatic expression of TGF-beta at the periphery of PCPTC was associated to morphological features of invasiveness, featuring the so-called epithelial-to-mesenchymal transition (EMT), and presence of nodal metastases, as well as to the occurrence of BRAF mutation which did not significantly alter, per se, the frequency of nodal metastases. The nuclear expression of Smad7 was more frequent in WCPTCs than in PCPTCs and was associated with unicentricity and absence of extra-thyroid extension, vascular invasion and nodal metastases. We conclude that nodal metastases are associated to poorly circumscribed, locally invasive PTCs that exhibit low levels of nuclear Smad7 and a peripheral EMT phenotype displaying TGF-beta overexpression, regardless of the occurrence of BRAF mutation.