Home About us Contact
|
Home About us Contact | ||
|
|
||
Vascularization
Kinds of Vascularization Selected AbstractsSkin Repair Using a Porcine Collagen I/III Membrane,Vascularization and Epithelization PropertiesDERMATOLOGIC SURGERY, Issue 6 2010FALK WEHRHAN MD BACKGROUND Collagen membranes have been developed to overcome the problem of limited availability of skin grafts. Vascularization and restricted functional epithelization limit the success of bioartificial constructs. OBJECTIVE To compare the vascularization, epithelization, and integration of a porcine collagen I/III membrane with that of split-thickness skin grafts on skin wounds. MATERIALS AND METHODS In 21 adult pigs, full-thickness skin defects on the rear side of the ear healed by split-thickness skin grafting, by covering with the membrane, or by free granulation. Skin samples on postoperative days 1, 3, 7, 14, 21, and 28 were evaluated histologically (hematoxylin-eosin, Sirius Red) and using immunohistochemistry (cytokeratin 5/6, transforming growth factor beta receptor (TGF,R-III) and immunoblot (TGF,1,3, Smad2/3). Epithelial thickness and TGF,R-III-positive capillary area were quantitatively assessed. RESULTS Epithelization and vascularization in the membrane group were not significantly different from in the group treated with a split-thickness skin graft. Free granulation showed significantly slower epithelization and vascularization (p<.05). TGF,1 and Smad2/3 complex expression were high during free granulation. Matrix was distinguishable until day 7. CONCLUSIONS This membrane serves as a suitable full-thickness dermal substitute, because the membrane is vascularized faster than free granulation tissue and enables early epithelization. Geistlich Biomaterials (Wolhusen, Switzerland) provided the collagen membrane used in this study [source] Vascularization of the developing chick limb bud: role of the TGF, signalling pathwayJOURNAL OF ANATOMY, Issue 1 2003Neil Vargesson Abstract The developing vertebrate limb has fascinated developmental biologists and theoreticians for decades as a model system for investigating cell fate, cell signalling and tissue interactions. We are beginning to understand the mechanisms and signalling pathways that control and regulate the outgrowth and formation of the limb bud into a differentiated identifiable limb by late embryogenesis. However, the mechanisms underlying the development and maintenance of the vasculature of the developing limb are far from being completely understood. The vasculature supplies oxygen, nutrients and signals to developing tissues, allowing them to develop and grow. Moreover, a lot of evidence recently points to molecules involved in morphological development also controlling vascular development. Thus understanding how the vasculature forms and is patterned in the developing limb may further our understanding of limb development. In this review I outline how blood vessels are formed and maintained and how the developing chick limb is vascularized. I also review the role of the TGF, superfamily signalling pathway in the development of the chick limb vasculature: in particular, how antagonizing TGF, signalling in the developing chick limb has shed new light on the role vascular smooth muscle cells play in vessel calibre control and how this work has added to our understanding of TGF, superfamily signal transduction. [source] Vascularization of the Fleshy Comb in the Domestic ChickenANATOMIA, HISTOLOGIA, EMBRYOLOGIA, Issue 2005B. Vollmerhaus Up to now little is known about the vascularization of the chicken fleshy comb (crista carnosa). In order to evaluate the vascularization of the crista carnosa of the cook (breed White Leghorn), corrosion casts were created by injecting Plastoid into the internal carotid as described by Schummer (1951). Specimens were investigated by stereomicroscopy and scanning electron microscopy (SEM). Generally the dermis is highly vascularized by two capillary networks, which are localized beneath the epithelium and beneath the dermal papillas. The dense subepithelial network is characterized by the presence of sinusoid vessels. In the subcutaneous plexus numerous arteriovenous anastomoses of different types occur. Additionally there are arteriovenous anastomoses between the main vessels reaching the indentations of the comb. Our results show the presence of superficial and dense capillary networks and arteriovenous anastomoses are the anatomical basis for the functions of the chicken comb in mating behaviour and thermoregulation. Reference, Schummer, A. 1951: Simplified method for plastoid corrosion. Anat. Anaz. 98, 288,290. [source] Inhibition of angiogenesis by interleukin-4 gene therapy in rat adjuvant-induced arthritisARTHRITIS & RHEUMATISM, Issue 8 2006Christian S. Haas Objective Interleukin-4 (IL-4) can modulate neovascularization. In this study, we used a gene therapy approach to investigate the role of IL-4 in angiogenesis in rat adjuvant-induced arthritis (AIA), a model for rheumatoid arthritis. Methods Rats received an adenovirus producing IL-4 (AxCAIL-4), a control virus without insert, or control vehicle (phosphate buffered saline) intraarticularly before arthritis onset. At peak onset of arthritis, rats were killed. Vascularization was determined in the synovial tissue, and correlations with inflammation were assessed. Ankle homogenates were used in angiogenesis assays in vitro and in vivo, and protein levels of cytokines and growth factors were assessed by enzyme-linked immunosorbent assay. Synovial tissue expression of ,v integrins was determined by immunohistochemistry. Results IL-4 induced a reduction in synovial tissue vessel density, which was paralleled by a decrease in inflammation. AxCAIL-4 joint homogenates significantly (P < 0.05) inhibited both endothelial cell (EC) migration and tube formation in vitro. Similarly, AxCAIL-4 inhibited capillary sprouting in the rat aortic ring assay, and vessel growth in the in vivo Matrigel plug assay. The angiostatic effect occurred despite high levels of vascular endothelial growth factor (VEGF), and was associated with down-regulation of the proangiogenic cytokines IL-18, CXCL16, and CXCL5 and up-regulation of the angiogenesis inhibitor endostatin. Of interest, AxCAIL-4 also resulted in decreased EC expression of the ,v and ,3 integrin chains. Conclusion In rat AIA, IL-4 reduces synovial tissue vascularization via angiostatic effects, mediates inhibition of angiogenesis via an association with altered pro- and antiangiogenic cytokines, and may inhibit VEGF-mediated angiogenesis and exert its angiostatic role in part via ,v,3 integrin. This knowledge of the specific angiostatic effects of IL-4 may help optimize target-oriented treatment of inflammatory arthritis. [source] Skin Repair Using a Porcine Collagen I/III Membrane,Vascularization and Epithelization PropertiesDERMATOLOGIC SURGERY, Issue 6 2010FALK WEHRHAN MD BACKGROUND Collagen membranes have been developed to overcome the problem of limited availability of skin grafts. Vascularization and restricted functional epithelization limit the success of bioartificial constructs. OBJECTIVE To compare the vascularization, epithelization, and integration of a porcine collagen I/III membrane with that of split-thickness skin grafts on skin wounds. MATERIALS AND METHODS In 21 adult pigs, full-thickness skin defects on the rear side of the ear healed by split-thickness skin grafting, by covering with the membrane, or by free granulation. Skin samples on postoperative days 1, 3, 7, 14, 21, and 28 were evaluated histologically (hematoxylin-eosin, Sirius Red) and using immunohistochemistry (cytokeratin 5/6, transforming growth factor beta receptor (TGF,R-III) and immunoblot (TGF,1,3, Smad2/3). Epithelial thickness and TGF,R-III-positive capillary area were quantitatively assessed. RESULTS Epithelization and vascularization in the membrane group were not significantly different from in the group treated with a split-thickness skin graft. Free granulation showed significantly slower epithelization and vascularization (p<.05). TGF,1 and Smad2/3 complex expression were high during free granulation. Matrix was distinguishable until day 7. CONCLUSIONS This membrane serves as a suitable full-thickness dermal substitute, because the membrane is vascularized faster than free granulation tissue and enables early epithelization. Geistlich Biomaterials (Wolhusen, Switzerland) provided the collagen membrane used in this study [source] Attenuation of retinal vascular development and neovascularization in transgenic mice over-expressing thrombospondin-1 in the lensDEVELOPMENTAL DYNAMICS, Issue 7 2006Zhifeng Wu Abstract Thrombospondin-1 (TSP1) is an endogenous inhibitor of angiogenesis and induces endothelial cell (EC) apoptosis. To study the role TSP1 plays during vascular development and neovascularization, we assessed the effects of ectopic TSP1 expression in the lens on retinal vascularization in transgenic mice. The TSP1 over-expressing mice showed abnormalities in the development of retinal vasculature. There was a dramatic decrease in the density of superficial and deep vascular plexuses of the retina in transgenic mice. The retinal vessels in TSP1 transgenic mice also appeared nonuniform and abnormal in maturation. We detected an increase in the number of EC undergoing apoptosis, which was compensated, in part, by an increase in cell proliferation in retinal vasculature of TSP1 transgenic mice. The TSP1 transgenic mice also exhibited increased levels of vessel obliteration and a limited preretinal neovascularization during oxygen-induced ischemic retinopathy (OIR). Our results indicate increased expression of TSP1 attenuates normal retinal vascularization and preretinal neovascularization during OIR. Therefore, modulation of TSP1 expression may provide an effective mechanism for regulation of ocular angiogenesis. Developmental Dynamics 235:1908,1920, 2006. © 2006 Wiley-Liss, Inc. [source] Vascular regression is required for mesenchymal condensation and chondrogenesis in the developing limbDEVELOPMENTAL DYNAMICS, Issue 3 2001Melinda Yin Abstract Vascular regression occurs during limb mesenchymal cell condensation and chondrogenesis, but it is unclear whether it is required for these processes or is a secondary phenomenon without major regulatory roles. To address this issue, beads presoaked with the potent angiogenic factor vascular endothelial growth factor (VEGF) were implanted in the vicinity of the prospective digit 2 in early chick embryo wing buds and the effects on angiogenesis and digit development were determined over time. We found that VEGF treatment caused a marked local increase in blood vessel number and density. Strikingly, this was accompanied by inhibition of digit 2 development as revealed by lack of expression of chondrogenic transcription factor Sox9 and absence of Alcian blue staining. Vascular distribution and skeletal development in adjacent areas remained largely unaffected. Inhibition of digit formation and excess vascularization were both reversible upon further embryonic growth and dissipation of VEGF activity. When supernumerary digits were induced at the anterior limb margin by retinoic acid treatment, their development was also preceded by vascular regression; interestingly, cotreatment with VEGF inhibited supernumerary digit development as well. Direct exposure of limb mesenchymal cells in micromass cultures to VEGF caused no obvious effects on condensation and chondrogenesis, indicating that VEGF effects are not due to direct action on skeletal cells. Our results are the first to provide evidence that vascular regression is required for mesenchymal condensation and chondrogenesis. A model of how patterning mechanisms and vascular regression may intersect and orchestrate limb skeletogenesis is proposed. © 2001 Wiley-Liss, Inc. [source] Adaptations of amphibious fish for surviving life out of waterFISH AND FISHERIES, Issue 3 2005Martin D J Sayer Abstract There are a small number of fish species, both marine and freshwater, that exhibit a truly amphibious habit that includes periods of aerial exposure. The duration of emersion is reflected in the level of physical and physiological adaptation to an amphibious lifestyle. Fish that are only briefly out of water retain predominantly aquatic attributes whereas there are semi-terrestrial species that are highly adapted to prolonged periods in the aerial habitat. Desiccation is the main stressor for amphibious fish and it cannot be prevented by physiological means. Instead, amphibious fish resist excessive water loss by means of cutaneous modification and behavioural response. The more terrestrially adapted fish species can tolerate considerable water loss and may employ evaporation to aid thermoregulation. The amphibious habit is limited to fish species that can respire aerially. Aerial respiration is usually achieved through modification to existing aquatic pathways. Freshwater air-breathers may respire via the skin or gills but some also have specialized branchial diverticula. Marine species utilize a range of adaptations that may include modified gills, specialized buccopharyngeal epithelia, the intestine and the skin. Areas of enhanced respiratory activity are typified by increased vascularization that permits enhanced perfusion during aerial exposure. As with other adaptations the mode of nitrogenous elimination is related to the typical durations of emersion experienced by the fish. Intertidal species exposed on a regular cycle, and which may retain some contact with water, tend to remain ammoniotelic while reducing excretion rates in order to prevent excessive water loss. Amphibious fish that inhabit environments where emersion is less predictable than the intertidal, can store nitrogen during the state of emersion with some conversion to ureotelism or have been shown to tolerate high ammonia levels in the blood. Finally, the more amphibious fish are more adapted to moving on land and seeing in air. Structural modifications to the pectoral, pelvic, dorsal and anal fins, combined with a well-developed musculature permit effective support and movement on land. For vision in air, there is a general trend for fish to possess close-set, moveable, protruberant eyes set high on the head with various physical adaptations to the structure of the eye to allow for accurate vision in both air and water. [source] Essential role for ERK2 mitogen-activated protein kinase in placental developmentGENES TO CELLS, Issue 11 2003Naoya Hatano Background:, Extracellular signal-regulated kinase 2 (ERK2) has been implicated in cell proliferation, differentiation, and survival. However, its role in vivo remains to be determined. Results:, Here we show that the targeted disruption of the mouse ERK2 gene results in embryonic lethality by E11.5 and severe abnormality of the placenta. In these animals, the labyrinthine layer of the placenta is very thin and few foetal blood vessels are observed. ERK2 mutants can be rescued by the transgenic expression of ERK2, demonstrating that these abnormalities are caused by ERK2-deficiency. Although ERK2-deficient fetuses are much smaller than wild-type littermates, this seems to be secondary to malfunction of the placenta. When the placental defect is rescued by tetraploid-aggregation, ERK2-deficient foetuses grow as well as littermate controls. Conclusion:, These observations indicate that ERK2 is essential for placental development and suggest that ERK2 in the trophoblast compartment may be indispensable for the vascularization of the labyrinth. [source] The role of selective angiographic embolization of the musculo-skeletal system in haemophiliaHAEMOPHILIA, Issue 4 2009E. C. RODRIGUEZ-MERCHAN Summary., The incidence of haemarthrosis as a result of a spontaneous periarticular aneurysm in haemophilia is very low. In these circumstances, angiographic embolization might be considered as a promising therapeutic and coagulation factor saving option in joint bleeds not responding to replacement of coagulation factor to normal levels. Moreover, embolization should be considered as a possible treatment for postoperative pseudoaneurysms complicating total knee arthroplasty in haemophilia. However, the pathological process of aneurysmal bleeding and clotting factor replacement is entirely different. While embolization is the treatment of choice for some periarticular complications that may occur, it is by no means a panacea for all resistant periarticular bleeds in haemophilia or for postoperative bleeding which usually settles with clotting factor replacement. Another use of arterial embolization is for the treatment of haemophilic tumours of the pelvis, because they can act as a focus for infection and cause cutaneous fistulas. When they present perforations and infections of endogenous origin, their course is usually fatal. Suitable treatment has been investigated on numerous occasions, most of the literature agreeing that the only curative treatment is surgical resection. However, surgical resection after performing arterial embolization to reduce the vascularization of the pseudotumour is a good alternative, thereby reducing the size of the pseudotumour and the risk of bleeding complications during surgery. It is important to bear in mind that despite its efficacy, arterial embolization is an invasive procedure with a reported rate of complications up to 25% (16% minor, 7% serious, 2% death). [source] Inducible nitric oxide synthase expression in laryngeal neoplasia: Correlation with angiogenesisHEAD & NECK: JOURNAL FOR THE SCIENCES & SPECIALTIES OF THE HEAD AND NECK, Issue 1 2002Alessandro Franchi MD Abstract Background The nitric oxide (NO) pathway plays a relevant role in angiogenesis and tumor progression in squamous cell carcinoma (SCC) of the head and neck. The aim of this study was to assess whether the NO pathway may be correlated with angiogenesis in the transition from laryngeal dysplasia to invasive carcinoma. Methods We investigated the expression of the inducible NO synthase (iNOS) in 26 laryngeal precancerous lesions and 35 squamous cell carcinomas with respect to microvessel density. In addition, we determined iNOS activity and cGMP levels in specimens from SCCs. Results There was a significant increase of iNOS levels detected immunohistochemically passing from hyperplastic/mild dysplastic to moderate/severe dysplastic lesions to SCC (p = .04). Accordingly, Northern and Western analyses demonstrated higher iNOS mRNA and protein levels in SCCs than dysplastic mucosa. iNOS expression was significantly correlated with microvessel counts both in the group of preneoplastic lesions (p = .02) and in the group of SCCs (p = .01). In addition, iNOS activity was correlated with iNOS immunohistochemical expression (p = .1) and was significantly associated with increased vascularization (p = .03) in SCCs. Similarly, iNOS expression was significantly correlated with cGMP levels in SCC (p = .02) and increased tumor vascularization correlated with higher cGMP levels (rs = .4; p = .01). Conclusions Our data indicate that the NO pathway may play a relevant role in the angiogenesis associated with the progression from laryngeal dysplasia to laryngeal SCC. © 2002 John Wiley & Sons, Inc. Head Neck 24: 16,23, 2002. [source] The angiogenic makeup of human hepatocellular carcinoma does not favor vascular endothelial growth factor/angiopoietin-driven sprouting neovascularization,,HEPATOLOGY, Issue 5 2008Wenjiao Zeng Quantitative data on the expression of multiple factors that control angiogenesis in hepatocellular carcinoma (HCC) are limited. A better understanding of the mechanisms underlying angiogenesis in HCC will improve the rational choice of anti-angiogenic treatment. We quantified gene and protein expression of members of the vascular endothelial growth factor (VEGF) and angiopoietin systems and studied localization of VEGF, its receptors VEGFR-1 and VEGFR-2, Angiopoietin (Ang)-1 and Ang-2, and their receptor, in HCC in noncirrhotic and cirrhotic livers. We employed real-time reverse transcription polymerase chain reaction (RT-PCR), western blot, and immunohistology, and compared the outcome with highly angiogenic human renal cell carcinoma (RCC). HCC in noncirrhotic and cirrhotic livers expressed VEGF and its receptors to a similar extent as normal liver, although in cirrhotic background, VEGFR-2 levels in both tumor and adjacent tissue were decreased. Ang-1 expression was slightly increased compared with normal liver, whereas Tie-2 was strongly down-regulated in the tumor vasculature. Ang-2 messenger RNA (mRNA) levels were also low in HCCs of both noncirrhotic and cirrhotic livers, implying that VEGF-driven angiogenic sprouting accompanied by angiopoietin-driven vascular destabilization is not pronounced. In RCC, VEGF-A levels were one order of magnitude higher. At the same time, endothelially expressed Ang-2 was over 30-fold increased compared with expression in normal kidney, whereas Ang-1 expression was decreased. Conclusion: In hepatocellular carcinoma, tumor vascularization is not per se VEGF/angiopoietin driven. However, increased CD31 expression and morphological changes representative of sinusoidal capillarization in tumor vasculature indicate that vascular remodeling is taking place. This portends that therapeutic intervention of HCC at the level of the vasculature is optional, and that further studies into the molecular control thereof are warranted. (HEPATOLOGY 2008.) [source] Correlation of hypoxic signalling to histological grade and outcome in cartilage tumoursHISTOPATHOLOGY, Issue 5 2010Stephane Boeuf Boeuf S, Bovée J V M G, Lehner B, Hogendoorn P C W & Richter W (2010) Histopathology56, 641,651 Correlation of hypoxic signalling to histological grade and outcome in cartilage tumours Aims:, The molecular mechanisms underlying the progression of central chondrosarcoma are so far poorly understood. The aim of this study was to identify genes involved in the progression of these tumours by comparison of gene expression and correlation of expression profiles to histological grade and clinical outcome. Methods and results:, Array-based gene expression profiling of 19 chondrosarcoma samples was performed. Beside differences in the expression of cartilage matrix molecules, high-grade chondrosarcoma showed enhanced expression of the matrix metalloproteinase MMP-2 and of the hypoxia-inducible molecule galectin 1. Immunohistochemical analysis of galectin 1 and of further hypoxia-associated proteins was performed on 68 central and peripheral tumour samples. Hypoxia-inducible factor 1, (HIF-1,) activation was significantly elevated in high-grade central chondrosarcoma. A negative correlation of carbonic anhydrase IX expression to metastasis-free survival was independent of histological grade. Conclusions:, The expression patterns identified in this study point towards a substantial role for angiogenic and hypoxic signalling in chondrosarcoma progression. The constitutive activation of the transcription factor HIF-1, in high-grade chondrosarcoma could play a central role in the regulation of cell metabolism and vascularization in these tumours and may, for this reason, represent a potential target for chondrosarcoma therapy. [source] Structure, lymphatic vascularization and lymphocyte migration in mucosa-associated lymphoid tissueIMMUNOLOGICAL REVIEWS, Issue 1 2003Giacomo Azzali Summary:, In this review, we consider the morphological aspects and topographical arrangement of gut-associated lymphoid tissue (GALT) (solitary and aggregate lymph nodules or Peyer's patches) and of vermiform appendix in the human child and in some mammals. The spatial arrangement of the vessels belonging to apparatus lymphaticus periphericus absorbens (ALPA) and of blood vessels within each lymphoid follicle as well as the ultrastructural characteristics of the lymphatic endothelium with high absorption capacity are considered. Particular attention is also paid to the morphological and biomolecular mechanisms inducing lymphocyte transendothelial migration to the bloodstream by means of lymphatic vessels as well as their passage from blood into lymphoid tissue through the high endothelial venules (HEVs). The preferential transendothelial passage of lymphocytes and polymorphonuclear neutrophils within ALPA vessels of the interfollicular area does not occur following the opening of intercellular contacts, but rather it occurs by means of ,intraendothelial channels'. In HEVs, on the contrary, the hypothesis is plausible that lymphocyte transendothelial migration into lymphoid tissue occurs through a channel-shaped endothelial invagination entirely independent of interendothelial contacts. The lymph of ALPA vessels of the single Peyer's patch is conveyed into precollector lymphatic vessels and into prelymph nodal collectors, totally independent of the ALPA vessels of the gut segments devoid of lymphoid tissue. The quantitative distribution of T lymphocytes in the lymph of mucosal ALPA vessels suggests a prevalent function of fluid uptake, whereas a reservoir and supply function is implicated for the vessels of interfollicular area. The precollector lymphatic vessels and prelymph nodal collectors are considered to be vessels with low absorption capacity, whose main function is lymph conduction and flow. [source] A new technique for the study of periapical bone lesions: ultrasound real time imagingINTERNATIONAL ENDODONTIC JOURNAL, Issue 2 2002E. Cotti Abstract Aim This study describes the use of a real time-ultrasound imaging technique (echography) for the study of periradicular lesions. Methodology Twelve patients with periapical lesions of endodontic origin, diagnosed with conventional clinical and radiographic examination, were examined further using echography at the site of the diagnosed lesions. Each lesion was echographically characterized and described by an expert echographist together with an endodontist. Once the major echographic features were identified, information on the size of the lesion, its content, and its vascular supply was obtained and recorded. A tentative differential diagnosis between a cyst and a granuloma was made based on the data. Results In all cases it was possible to obtain an echographic image. It was also possible to measure the lesions, to evaluate their content and to view their vascularization in different regions of the mouth. Conclusions Ultrasound real time imaging is a promising diagnostic technique in endodontology, but further work is required to refine the process. [source] Glioblastoma cells incorporate into tumor vasculature and contribute to vascular radioresistanceINTERNATIONAL JOURNAL OF CANCER, Issue 9 2010Candice A. Shaifer Abstract Glioblastoma multiforme (GBM) remains the most devastating neoplasm of the central nervous system and has a dismal prognosis. Ionizing radiation represents an effective therapy for GBM, but radiotherapy remains only palliative because of radioresistance. In this study, we demonstrate that glioma cells participate in tumor vascularization and contribute to vascular radioresistance. Using a 3-dimensional coculture system, we observed an intimate interaction of glioma cells with endothelial cells whereby endothelial cells form vascular structures, followed by the recruitment and vascular patterning of glioma cells. In addition, tumor cells stabilize the vascular structures and render them radioresistant. Blocking initial endothelial vascular formation with endothelial-specific inhibitors prevented tumor cells from forming any structures. However, these inhibitors exhibited minimum effects on vascular structures formed by tumor cells, due to the absence of the targeted receptors on tumor cells. Consistent with the in vitro findings, we show that glioma cells form perfused blood vessels in xenograft tumor models. Together, these data suggest that glioma cells mimic endothelial cells and incorporate into tumor vasculature, which may contribute to radioresistance observed in GBM. Therefore, interventions aimed at the glioma vasculature should take into consideration the chimeric nature of the tumor vasculature. [source] Gene silencing of transcription factor Gli2 inhibits basal cell carcinomalike tumor growth in vivoINTERNATIONAL JOURNAL OF CANCER, Issue 1 2008Jingmin Ji Abstract Basal cell carcinoma (BCC) belongs worldwide to the most frequent malignancy among Caucasians. The understanding of the molecular mechanisms of BCC formation, which is a prerequisite for the development of efficient new therapies, is still incomplete. The formation of sporadic BCCs in the skin is associated with uncontrolled hedgehog signaling, and the transcription factor Gli2 has been identified as a key mediator or effector of this signaling. There is indication in the literature that preventing Gli2 function may inhibit BCC formation and growth in vivo; however, the mechanism is unclear and difficult to study in humans. Therefore, we used a mouse tumor allograft model to investigate the role of Gli2 in tumor formation. A constitutively Gli2 expressing mouse tumor cell line was stably transfected with Gli2-specific shRNA to induce Gli2 gene silencing or with control shRNA. Injecting the Gli2 gene silenced cells into nude mice for tumor formation we detected a strongly retarded tumor growth compared with control tumor cells. Investigating the mechanisms, we found that Gli2 gene silencing has led to the disruption of the tumor structure as demonstrated by staining tumor sections with hematoxylin. Two main reasons for the tumor destruction were identified. We found that apoptosis was markedly increased while vascularization was strongly decreased in these tumors. Thus, important functions of the transcription factor Gli2 in this tumor model are the prevention of apoptosis and the promotion of microvascularization. © 2007 Wiley-Liss, Inc. [source] Mammalian target of rapamycin is activated in human gastric cancer and serves as a target for therapy in an experimental modelINTERNATIONAL JOURNAL OF CANCER, Issue 8 2007Sven A. Lang Abstract The mammalian target of rapamycin (mTOR) has become an interesting target for cancer therapy through its influence on oncogenic signals, which involve phosphatidylinositol-3-kinase and hypoxia-inducible factor-1, (HIF-1,). Since mTOR is an upstream regulator of HIF-1,, a key mediator of gastric cancer growth and angiogenesis, we investigated mTOR activation in human gastric adenocarcinoma specimens and determined whether rapamycin could inhibit gastric cancer growth in mice. Expression of phospho-mTOR was assessed by immunohistochemical analyses of human tissues. For in vitro studies, human gastric cancer cell lines were used to determine S6K1, 4E-BP-1 and HIF-1, activation and cancer cell motility upon rapamycin treatment. Effects of rapamycin on tumor growth and angiogenesis in vivo were assessed in both a subcutaneous tumor model and in an experimental model with orthotopically grown tumors. Mice received either rapamycin (0.5 mg/kg/day or 1.5 mg/kg/day) or diluent per intra-peritoneal injections. In addition, antiangiogenic effects were monitored in vivo using a dorsal-skin-fold chamber model. Immunohistochemical analyses showed strong expression of phospho-mTOR in 60% of intestinal- and 64% of diffuse-type human gastric adenocarcinomas. In vitro, rapamycin-treatment effectively blocked S6K1, 4E-BP-1 and HIF-1, activation, and significantly impaired tumor cell migration. In vivo, rapamycin-treatment led to significant inhibition of subcutaneous tumor growth, decreased CD31-positive vessel area and reduced tumor cell proliferation. Similar significant results were obtained in an orthotopic model of gastric cancer. In the dorsal-skin-fold chamber model, rapamycin-treatment significantly inhibited tumor vascularization in vivo. In conclusion, mTOR is frequently activated in human gastric cancer and represents a promising new molecular target for therapy. © 2007 Wiley-Liss, Inc. [source] Increased plasma MMP9 in integrin ,1-null mice enhances lung metastasis of colon carcinoma cellsINTERNATIONAL JOURNAL OF CANCER, Issue 1 2005Xiwu Chen Abstract Inhibitors of matrix metalloproteinases (MMPs) were developed as anticancer agents based on the observation that MMPs facilitate local tumor spread and metastasis by promoting matrix degradation and cell migration. Unfortunately, these inhibitors were unsuccessful in the clinical treatment of several cancers, including lung cancer. A possible reason contributing to their failure is that MMP activity is critical for the generation of inhibitors of tumor angiogenesis, including angiostatin. Thus, MMPs might play opposing roles in tumor vascularization and invasion. To determine which effect of elevated MMP levels dominates in the progression of metastatic cancer, experimental lung metastasis assays were performed in integrin ,1-null mice, a genetic model for increased plasma levels of MMP9 and MMP9-generated angiostatin (Pozzi et al., Proc. Natl. Acad. Sci. USA 2000;97:2202,7). We show that while the number of lung colonies in integrin ,1-null mice was significantly increased compared to their wild-type counterparts, tumor volume was markedly reduced. In vivo treatment with the MMP inhibitor doxycycline resulted in a significant decrease in the number of lung colonies in both genotypes, but the tumors that formed were bigger and more vascularized. Increased tumor vascularization paralleled decreased plasma levels of MMP9 and consequent decreased angiostatin synthesis. These results demonstrate that while inhibition of MMPs prevents and/or reduces tumor invasion and lung metastasis, it has the paradoxical effect of increasing the size and vascularization of metastatic tumors due to decreased generation of inhibitors of endothelial cell proliferation. The continued growth of these large well-vascularized tumors may explain the poor efficacy of MMP inhibitors in lung cancer clinical trials. © 2005 Wiley-Liss, Inc. [source] New method of dynamic color doppler signal quantification in metastatic lymph nodes compared to direct polarographic measurements of tissue oxygenationINTERNATIONAL JOURNAL OF CANCER, Issue 6 2005Thomas Scholbach Abstract Tumor growth depends on sufficient blood and oxygen supply. Hypoxia stimulates neovascularization and is a known cause for radio- and chemoresistance. The objective of this study was to investigate the use of a novel ultrasound technique for the dynamic assessment of vascularization and oxygenation in metastatic lymph nodes. Twenty-four patients (age 44,78 years) with cervical lymph node metastases of squamous cell head and neck cancer were investigated by color duplex sonography and 17 (age 46,78 years) were investigated additionally with polarography. Sonography was performed after contrast enhancer infusion under defined conditions. Intranodal perfusion data (color hue, colored area) were measured automatically by a novel software technique. This allows an evaluation of blood flow dynamics by calculating perfusion intensity,velocity, perfused area, as well as the novel parameters tissue resistance index (TRI) and tissue pulsatility index (TPI),for each point of a complete heart cycle. Tumor tissue pO2 was measured by means of polarographic needle electrodes placed intranodally. The sonographic and polarographic data were correlated using Pearson's test. Sonography demonstrated a statistically significant inverse correlation between hypoxia and perfusion and significant TPI and TRI changes with different N-stages. The percentage of nodal fraction with less than 10 mmHg oxygen saturation was significantly inversely correlated with lymph node perfusion (r = ,0.551; p = 0.021). Nodes with a perfusion of less than 0.05 cm/sec flow velocity showed significantly larger hypoxic areas (p = 0.006). Significant differences of TPI and TRI existed between nodes in stage N1 and N2/N3 (p = 0.028 and 0.048, respectively). This new method of dynamic signal quantification allows a noninvasive and quantitative assessment of tumor and metastatic lymph node perfusion by means of commonly available ultrasound equipment. © 2004 Wiley-Liss, Inc. [source] Hyaluronan-binding peptide can inhibit tumor growth by interacting with Bcl-2INTERNATIONAL JOURNAL OF CANCER, Issue 1 2004Ninfei Liu Abstract Previous studies have indicated that proteins that bind hyaluronan can also inhibit the growth of tumor cells. To determine if synthetic peptides also possessed these properties, we tested a series of polypeptides containing structural motifs from different proteins for their ability to bind [3H]hyaluronan, and identified one compound termed P4 that had a particularly strong interaction. Further studies revealed that P4 also inhibited the growth of tumor cells in tissue culture as well as on the chorioallantoic membranes of chicken embryos. In addition, expression vectors for P4 caused tumor cells to grow slower in nude mice and reduced their vascularization. The P4 peptide also inhibited VEGF-induced angiogenesis in the chorioallantoic membranes of chicken embryos. Studies on cultured cells indicated that P4 induced apoptosis, which was blocked by a pan-caspase inhibitor. Confocal microscopy revealed that shortly after its uptake, P4 became associated with mitochondria. Immunoprecipitation indicated that P4 could bind to Bcl-2 and Bcl-xL, which are associated with mitochondria and regulate apoptosis. This was also supported by the fact that P4 induced the release of cytochrome c from preparations of mitochondria. Taken together, these results suggest that P4 binds to Bcl-2 and related proteins and this activates the apoptotic cascade. © 2003 Wiley-Liss, Inc. [source] The role of angiogenesis, vascular maturation, regression and stroma infiltration in dormancy and growth of implanted MLS ovarian carcinoma spheroidsINTERNATIONAL JOURNAL OF CANCER, Issue 4 2004Assaf Gilead Abstract MLS ovarian epithelial carcinoma multicellular spheroids xenografted subcutaneously in CD-1 nude mice displayed growth delay, or dormancy, of up to 52 days. In the study reported here, implanted MLS spheroids were used for testing the role of angiogenesis and vascular maturation in triggering the initiation of tumor progression. The kinetics and impact of neovascular maturation and functionality, in dormancy, and growth of MLS spheroid xenografts were studied noninvasively by BOLD contrast MRI. MR data were supported by histologic staining for biotinylated albumin as a blood pool marker and alpha-smooth muscle actin (alpha-SMA) as marker for perivascular mural cells. Although the tumor periphery showed higher levels of total and mature vasculature than normal skin, the fraction of mature out of the total vessels as detected by MRI vascular maturation index (VMIMRI) was significantly lower in the tumor both before and after tumor exit from dormancy. The neovasculature induced by the implanted spheroid was unstable and showed cycles of vessel growth and regression. Surprisingly, this instability was not restricted to the immature vessels, but rather included also regression of mature vessels. During dormancy, neovasculature was predominantly peripheral with no infiltration into the implanted spheroid. Infiltration of alpha-SMA positive stroma cells into the spheroid was associated with functional vascularization and tumor growth. Thus, stroma infiltration and vascular maturation are an important checkpoint linking the angiogenic switch with initiation of tumor progression. © 2003 Wiley-Liss, Inc. [source] Dynamic T1-weighted monitoring of vascularization in human carcinoma heterotransplants by magnetic resonance imaging,INTERNATIONAL JOURNAL OF CANCER, Issue 1 2003Fabian Kiessling Abstract Studies on tumor angiogenesis and antiangiogenic therapies are commonly performed with tumor heterotransplants in nude mice. To monitor therapeutic effects, improved noninvasive analyses of functional data are required, in addition to the assessment of tumor volume and histology. Here, we report on sequential monitoring of vascularization of human squamous cell carcinomas growing as heterotransplants in nude mice using MRI. Using a custom-developed animal coil in a conventional whole-body 1.5 T MRI scanner, dynamic T1w sequences were recorded after i.v. injection of Gd-DTPA in tumors grown for 17, 21, 25, 29 and 33 days. Amplitude and the exchange rate constant (kep) were calculated according to a 2-compartment model, discriminating intravascular and interstitial spaces, and correlated with tumor size and histology. High-resolution imaging of small heterotransplants from 100 to 1,000 mm3 was achieved, clearly discriminating vital and necrotic areas. Preceding the development of necroses, which were hyperintense in T2w images and confirmed with histology, a local decrease of amplitude and kep values was observed. Significantly higher amplitudes were found in tumor periphery than in central parts, correlating well with the vascular pattern obtained by immunocytochemistry. Tumor size correlated negatively with amplitude, probably as a result of increasing necrotic areas, whereas the reason for the observed increase of kep value with tumor size remains unclear. These data demonstrate that dynamic MRI is an excellent method for noninvasive assessment of tumor vascularization in small animals using a clinical whole-body scanner with little technical modifications. This technique provides functional data characterizing essential features of tumor biology and is thus appropriate for monitoring antiangiogenic therapies. © 2002 Wiley-Liss, Inc. [source] Influence of environmental stress on skin tone, color and melanogenesis in Japanese skinINTERNATIONAL JOURNAL OF COSMETIC SCIENCE, Issue 1 2005K. Kikuchi Introduction It is needless to say that one of the most potent environmental stress for melanogenesis of the human skin is the effect of ultraviolet (UV) light from the sunlight. Characteristic skin aging as a result of this UV light is recognized as photoaging. Clinical features in photoaging are wrinkles, skin laxity, coarseness, leathery, yellowing, lentigenes, mottled pigmentation, telangiectasia, sebaceous hypertrophy and purpura. There is an apparent difference in clinical features in photoaging among different races, i.e. between Caucasians, African American and Asians that include Japanese. Not only photo skin type but also environmental factors, such as climate, latitude, altitude and their habit of sunbathing, smoking and skin care influence the characteristic development of their photoaging. Racial difference in photoaging Caucasians tend to develop skin laxity and fine wrinkles more than Asians [1]. Asians tend to produce coarser wrinkles than the Caucasians although their development is rather late in life. There is also a difference in the skin color. Pigmentation is an earliest and prominent skin changes in Asians [1] and it increases with age [2]. In contrast, pigmentation is not apparent in the Caucasians although redness probably because of an increase in cutaneous vascularization becomes prominent in middle aged Caucasians [2]. Chung reported that seborrheic keratosis is a major pigmentary lesion in men, whereas hyperpigmented macules are prominent features in women in Koreans [3]. Melanogenesis pigmentation disorders in Japanese Ephelides (freckles) are commonly found in those with photo skin type I who have fair skin and red eyes and blond hair. They are also found in the Japanese. Clinical feature reveals that multiple small pigmentary macules on sun-exposed areas mainly on the mid-portion of the face. These lesions seem to be familial, becoming apparent even in early childhood after sun exposure. Melasma is an acquired pigmentary disorder commonly found in middle aged Japanese women characterized by irregular brown macules and patches on the sun-exposed areas on the face typically as bilaterally present macules on the cheeks. An increase in sex hormones as a result of pregnancy and intake of contraceptive pills is one of the etiological factors to develop melasma. Sun exposure also worsens it. Nevus of Ota is also a common pigmentary disorder found in the Japanese. It is usually unilateral, blue-brown to slate-gray pigmentary macules on the eyelid and cheek that appear in early childhood or in puberty. Acquired dermal melanocytosis is also a pigmentary disorder, in which dermal melanocytes are found as shown in nevus of Ota, characterized by bilateral brown to blue-gray macules on the forehead, temple, eyelid and malar areas in middle aged Japanese women. This tends to be misdiagnosed as melasma. Solar lentigo is an acquired pigmented macule induced by sun exposure. Solar lentigines are usually multiple, circumscribed brown macules. There are two types of solar lentigo. One is a small macular type, characterized by multiple, small brown macules whose diameter is less than 5 mm, being similar to ephelides (freckles). The other type is a large macular type, characterized by a few round to oval, brown macules whose diameter is beyond 1 cm. Some of their surface are hyperkeratotic and become elevated to produce seborrheic keratosis. Again, the early sign of photoaging in Japanese is pigmentated spots and these pigmentation disorders increase with age. Among the pigmentary changes, nevus of Ota, acquired dermal melanocytosis, melasma and large macular type of solar lentigo are characteristic skin changes found in the Japanese in addition to ephelides and small macular type of solar lentigo. Seasonal changes of the various functional properties of the skin including skin color assessed by non-invasive bioengineering techniques [4]. When we consider skin tone, color and melanogenesis, UV light from the sunlight is the most potent environmental stress, although we cannot forget also the important influence of environmental relative humidity affects our skin functions as well as its appearance. We investigated seasonal influences on the various properties of the skin in 39 healthy Japanese females consisting of different age groups. Their skin is thought to be affected by the UV light in summer, and by the exposure to the dry and cold air in winter. Materials and methods Biophysical, non-invasive measurements, including transepidermal water loss (TEWL) as a parameter for the barrier function of the stratum corneum (SC), high frequency conductance as a parameter for the hydration state of the SC, skin color and casual surface lipid levels, were conducted during late summer and winter months. Skin color was determined with a chromameter according to the L*a*b* CIE 1976 system, where L* is an attribute on the luminance scale, a* that on the red versus green scale and b* that on the yellow versus blue scale. All the measurements were conducted in an environment controlled-chamber (21 ± 1 °C room temperature, and 50 ± 3% relative humidity). Results The barrier function of the SC was found to be significantly impaired in winter on the cheek as compared with that measured in summer, whereas no such seasonal change was apparent both in the hydration state of the SC and sebum levels on the cheek. In the assessment of the skin color on the cheek, a significant increase in a* (redness) and a decrease in b* (yellowness) were observed in winter. In contrast, on the flexor forearm, the values of L* (luminescence) increased in winter, but no seasonal change was noted in the values of a* and b*. In this study, skin changes with aging were also found by the non-invasive bioengineering methods. The value of TEWL on the cheek tended to increase with age, whereas no significant change was observed in the value of TEWL on the forearm. In the assessment of skin color, b* value on the cheek significantly increased with age whereas a* and L* values on the cheek did not show any significant change with age. Summary of this study We think that such an increase in yellowness with aging of the cheek skin is a phenomenon unique to the Japanese (Asians) since an increase in b* value was not observed in Caucasians [2]. The facial skin that is always exposed shows barrier impairment in a dry and cold winter environment and demonstrates increased yellowness in skin color because of a prolonged exposure to the UV light from the sun in the summer season. The non-invasive bioengineering methods are useful to demonstrate even invisible seasonal changes occurring in the same individuals and changes with age occurring in the skin. References 1.,Goh, S.H. The treatment of visible signs of senescence: the Asian experience. Br. J. Dermatol.122, 105,109 (1990). 2.,LeFur, I., Numagami, K., Guinot, C. et al. Age-related reference values of skin color in Caucasian and Japanese healthy women according to skin site. Pigment Cell Res. 7, 67 (1999). 3.,Chung, J.H., Lee, S.H., Youn, C.S. et al. Cutaneous photodamage in Koreans: influence of sex, sun exposure, smoking, and skin color. Arch. Dermatol. 137, 1043,1051 (2001). 4.,Kikuchi, K., Kobayashi, H., Le Fur, I. et al. Winter season affects more severely the facial skin than the forearm skin: comparative biophysical studies conducted in the same Japanese females in later summer and winter. Exog. Dermatol. 1, 32,38 (2002). [source] Neutrophils: key mediators of tumour angiogenesisINTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY, Issue 3 2009Simon Tazzyman Summary It is now well known that most malignant tumours contain a significant amount of leucocytic infiltrates the presence of which has, on many occasions, been linked to poor patient prognosis. These leucocyte populations are recruited to tumours by chemotactic factors released by either viable or necrotic tumour cells, or by cells within the tumour stroma. In recent times, most studies have analysed the role that tumour-associated macrophages (TAM) have on tumour progression. However, there is now increasing evidence to show that neutrophils also actively participate in this process. Whilst there are some data to suggest that neutrophil-derived factors can promote genetic mutations leading to tumourigenesis, or secrete factors that promote tumour cell proliferation; there is now substantial evidence to show that neutrophils, like TAM, significantly affect tumour angiogenesis. In this review, we discuss the likely mechanisms by which neutrophils are recruited into the tumour and then elaborate on how these cells may induce tumour vascularization by the secretion of powerful pro-angiogenic factors. We also discuss possible future chemotherapeutic strategies that are aimed at limiting tumour angiogenesis by inhibiting neutrophil recruitment. [source] On the vascularization and structure of the skin of a Korean bullhead Pseudobagrus brevicorpus (Bagridae, Teleostei) based on its entire body and appendagesJOURNAL OF APPLIED ICHTHYOLOGY, Issue 1 2010J. Y. Park Summary To investigate the vascularization and structure of the skin and its relationship to cutaneous respiration in Pseudobagrus brevicorpus, a histological study by light microscopy was carried out on 15 regions of the skin, including eight body regions, six fins and the barbel. The skin consisted of the epidermis, dermis and subcutis in all regions, except for the barbel that had a relatively thin dermis and subcutis. The epidermis was composed of the outermost layer, the middle layer and the stratum germinativum. There were two kinds of gland cells: the unicellular mucus cells and large club cells. The middle layer had a small number of fine blood capillaries accompanied by dermal collagen in all regions; the mean number of blood capillaries ranged from 0.9 to 5.9. The mean diffusion distance between the capillary endothelial cells and the surface of the epidermis ranged from 50.6 to 126.8 ,m. Based on these intra-epithelial blood capillaries, the relative surface area of the respiratory epithelium ranged from 0.1 to a maximum value of 1.2%. The dermis lacking scales had collagen bundles arranged parallel to each other, but vertical fiber bundles around the dorso-lateral regions were seen at intervals. Sensory organs such as taste buds, pit organs and lateral canals were found whereby the taste buds in particular were more abundant in the epidermis of the barbel. The vascularization of the skin may be closely related to an additional respiratory system used to deal with an extreme hypoxic condition during dry seasons. [source] Secretion of SDF-1, by bone marrow-derived stromal cells enhances skin wound healing of C57BL/6 mice exposed to ionizing radiationJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 6b 2010Yannick Landry Abstract Patients treated for cancer therapy using ionizing radiation (IR) have delayed tissue repair and regeneration. The mechanisms mediating these defects remain largely unknown at present, thus limiting the development of therapeutic approaches. Using a wound healing model, we here investigate the mechanisms by which IR exposure limits skin regeneration. Our data show that induction of the stromal cell-derived growth factor 1, (SDF-1,) is severely impaired in the wounded skin of irradiated, compared to non-irradiated, mice. Hence, we evaluated the potential of bone marrow-derived multipotent stromal cells (MSCs), which secrete high levels of SDF-1,, to improve skin regeneration in irradiated mice. Injection of MSCs into the wound margin led to remarkable enhancement of skin healing in mice exposed to IR. Injection of irradiated MSCs into the wound periphery of non-irradiated mice delayed wound closure, also suggesting an important role for the stromal microenvironment in skin repair. The beneficial actions of MSCs were mainly paracrine, as the cells did not differentiate into keratinocytes. Specific knockdown of SDF-1, expression led to drastically reduced efficiency of MSCs in improving wound closure, indicating that SDF-1, secretion by MSCs is largely responsible for their beneficial action. We also found that one mechanism by which SDF-1, enhances wound closure likely involves increased skin vascularization. Our findings collectively indicate that SDF-1, is an important deregulated cytokine in irradiated wounded skin, and that the decline in tissue regeneration potential following IR can be reversed, given adequate microenvironmental support [source] Spatial and temporal patterns of bone formation in ectopically pre-fabricated, autologous cell-based engineered bone flaps in rabbitsJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 4 2008Oliver Scheufler Abstract Biological substitutes for autologous bone flaps could be generated by combining flap pre-fabrication and bone tissue engineering concepts. Here, we investigated the pattern of neotissue formation within large pre-fabricated engineered bone flaps in rabbits. Bone marrow stromal cells from 12 New Zealand White rabbits were expanded and uniformly seeded in porous hydroxyapatite scaffolds (tapered cylinders, 10,20 mm diameter, 30 mm height) using a perfusion bioreactor. Autologous cell-scaffold constructs were wrapped in a panniculus carnosus flap, covered by a semipermeable membrane and ectopically implanted. Histological analysis, substantiated by magnetic resonance imaging (MRI) and micro-computerized tomography scans, indicated three distinct zones: an outer one, including bone tissue; a middle zone, formed by fibrous connective tissue; and a central zone, essentially necrotic. The depths of connective tissue and of bone ingrowth were consistent at different construct diameters and significantly increased from respectively 3.1 ± 0.7 mm and 1.0 ± 0.4 mm at 8 weeks to 3.7± 0.6 mm and 1.4 ± 0.6 mm at 12 weeks. Bone formation was found at a maximum depth of 1.8 mm after 12 weeks. Our findings indicate the feasibility of ectopic pre-fabrication of large cell-based engineered bone flaps and prompt for the implementation of strategies to improve construct vascularization, in order to possibly accelerate bone formation towards the core of the grafts. [source] Evaluation of processed bovine cancellous bone matrix seeded with syngenic osteoblasts in a critical size calvarial defect rat modelJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 3 2006U. Kneser Abstract Introduction: Biologic bone substitutes may offer alternatives to bone grafting procedures. The aim of this study was to evaluate a preformed bone substitute based on processed bovine cancellous bone (PBCB) with or without osteogenic cells in a critical size calvarial defect rat model. Methods: Discs of PBCB (Tutobone®) were seeded with second passage fibrin gel-immobilized syngenic osteoblasts (group A, n = 40). Cell-free matrices (group B, n = 28) and untreated defects (group C; n=28) served as controls. Specimens were explanted between day 0 and 4 months after implantation and were subjected to histological and morphometric evaluation. Results: At 1 month, bone formation was limited to small peripheral areas. At 2 and 4 months, significant bone formation, matrix resorption as well as integration of the implants was evident in groups A and B. In group C no significant regeneration of the defects was observed. Morphometric analysis did not disclose differences in bone formation in matrices from groups A and B. Carboxyfluorescine-Diacetate-Succinimidylester (CFDA) labeling demonstrated low survival rates of transplanted cells. Discussion: Osteoblasts seeded into PBCB matrix display a differentiated phenotype following a 14 days cell culture period. Lack of initial vascularization may explain the absence of added osteogenicity in constructs from group A in comparison to group B. PBCB is well integrated and represents even without osteogenic cells a promising biomaterial for reconstruction of critical size calvarial bone defects. [source] Modifications of the fibroblast growth factor-2 gene led to a marked enhancement in secretion and stability of the recombinant fibroblast growth factor-2 proteinJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2007Shin-Tai Chen Abstract Progress in FGF-2 gene therapy has been hampered by the difficulty in achieving therapeutic levels of FGF-2 secretion. This study tested whether the addition of BMP2/4 hybrid secretion signal to the FGF-2 gene and mutation of cys-70 and cys-88 to serine and asparagine, respectively, would increase the stability and secretion of active FGF-2 protein in mammalian cells using MLV-based vectors. Single or double mutations of cys-70 and cys-88 to ser-70 and asp-88, respectively, markedly increased the amounts of FGF-2 protein in conditioned media and cell lysates, which may be due to glycosylation, particularly at the mutated asp-88 residue. Addition of BMP2/4 secretion signal increased FGF-2 secretion, but also suppressed FGF-2 biosynthesis. The combination of BMP2/4 secretion signal and double cys-70 and cys-88 mutations increased the total amount of secreted FGF-2 protein >60-fold. The modifications did not alter its ability to stimulate cell proliferation and Erk1/2 phosphorylation in marrow stromal cells or its ability to bind heparin in vitro, suggesting that the modified FGF-2 protein was functionally as effective as the unmodified FGF-2. An ex vivo application of rat skin fibroblasts (RSF) transduced with the modified FGF-2 vector in a subcutaneous implant model showed that rats with implants containing cells transduced with the modified FGF-2 vector increased serum FGF-2 level >15-fold, increased growth of the implant, and increased vascularization within the implant, compared to rats that received implants containing ,-galactosidase- or wild-type FGF-2-transduced control cells. This modified vector may be useful in FGF-2 gene therapy investigations. J. Cell. Biochem. 100: 1493,1508, 2007. © 2007 Wiley-Liss, Inc. [source] | ||||||||||||||||||||||||||||||||||