Morphological evaluation of the feline placenta correlates with gene expression of vascular growth factors and receptors†.

Placental angiogenesis is critical for normal development. Angiogenic factors and their receptors are key regulators of this process. Dysregulated placental vascular development is associated with pregnancy complications. Despite their importance, vascular growth factor expression has not been thoroughly correlated with placental morphologic development across gestation in cats. We postulate that changes in placental vessel morphology can be appreciated as consequences of dynamic expression of angiogenic signaling agents. Here, we characterized changes in placental morphology alongside expression analysis of angiogenic factor splice variants and receptors throughout pregnancy in domestic shorthair cats. We observed increased vascular and lamellar density in the lamellar zone during mid-pregnancy. Immunohistochemical analysis localized the vascular endothelial growth factor A (VEGF-A) receptor KDR to endothelial cells of the maternal and fetal microvasculatures. PlGF and its principal receptor Flt-1 were localized to the trophoblasts and fetal vasculature. VEGF-A was found in trophoblast cells and associated with endothelial cells. We detected expression of two Plgf splice variants and four Vegf-a variants. Quantitative real-time polymerase chain reaction analysis showed upregulation of mRNAs encoding pan Vegf-a and all Vegf-a splice forms at gestational days 30-35. Vegf-A showed a marked relative increase in expression during mid-pregnancy, consistent with the pro-angiogenic changes seen in the lamellar zone at days 30-35. Flt-1 was upregulated during late pregnancy. Plgf variants showed stable expression during the first two-thirds of pregnancy, followed by a marked increase toward term. These findings revealed specific spatiotemporal expression patterns of VEGF-A family members consistent with pivotal roles during normal placental development.

Expression of Markers Ki-67, Nestin, VEGF, CD34 and Apoptosis in Relatively Healthy Lung Tissue with Non-Changed and Metaplastic Bronchial Epithelium.

BACKGROUND: Knowledge about the occurrence of processes such as proliferation, apoptosis and angiogenesis in healthy lung tissues with different bronchial epitheliums is limited, and further exploration can contribute to a better understanding of the physiological renewal of lung tissues. The processes mentioned above occur with the help of important tissue factors; therefore, the aim of the study was to determine the expression of markers Ki-67, nestin, CD34 and vascular endothelial growth factor (VEFG) and detect apoptotic cells in relatively healthy lung tissue. METHODS: Samples of relatively healthy lung tissue were obtained from 19 patients and divided into groups of patients with non-changed and patients with metaplastic bronchial epithelium. Tissue samples were examined by hematoxylin and eosin staining. Ki-67, nestin, VEGF and CD34-positive cells were detected by the immunohistochemistry method. Terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay was carried out to detect apoptotic cells. The number of positive structures was counted semi-quantitatively by microscopy. RESULTS: Ki-67-positive cells were detected in only one case. An occasional to moderate number of nestin-positive structures was found in various tissues of relatively healthy lungs with different bronchial epitheliums. No apoptotic cells were seen in non-changed bronchial epithelium, compared with few apoptotic cells in metaplastic bronchial epithelium. Metaplastic bronchial epithelium contained more VEGF-positive cells than non-changed bronchial epithelium. Samples with non-changed, and metaplastic bronchial epithelium both contained a similar number of CD34-positive structures. CONCLUSIONS: Proliferative activity and programmed cell death are not prominent events in normal lung tissue. A moderate number of nestin-positive cells in the alveolar epithelium and cartilage of bronchi with pseudostratified ciliated epithelium suggests a significant role of neuronal origin cells in these structures, to be intensified in metaplastic bronchial epithelium. A practically non-changed number of CD34-positive cells excludes any difference in stimulation of endothelial origin cells between lungs with different types of epithelium, while an increase in VEGF in structures with metaplastic epithelium suggests the presence/influence of tissue ischemia impact on possible development/maintenance of metaplasia.

sRAGE downregulates the VEGF expression in OHSS ovarian granulosa cells.

OBJECTIVE: Ovarian hyperstimulation syndrome (OHSS) is mainly caused by human chorionic gonadotropin (hCG) through vasoactive mediators such as vascular endothelial growth factor (VEGF) and various inflammatory factors. Our previous study showed that soluble receptor for advanced glycation end products (sRAGE) played a protective role in PCOS by inhibiting VEGF, so wanted to explore the role of sRAGE in OHSS. METHODS: Two sets of experiments were performed in this study. In part one, sRAGE protein levels in follicular fluid (FF) samples from 60 patients with OHSS and 60 non-OHSS patients were measured by ELISA. In part two, ovarian granulosa cells were isolated from an additional 25 patients with OHSS and cultured. Then, ovarian granulosa cells were treated with different concentrations of sRAGE. Granulosa cells cultured without sRAGE stimulation were used as the control group. The levels of VEGF, amphiregulin (AREG), betacellulin (BTC), and epiregulin (EREG) mRNA were examined by quantitative RT-PCR. The protein levels of VEGF, AREG, BTC, and EREG were measured by ELISA. RESULTS: Compared with non-OHSS patients, patients with OHSS exhibited lower sRAGE levels in both serum and FF (p < .05). Treatment with sRAGE decreased the production of VEGF, and the effects were dependent on the concentration of sRAGE (p < .05). Simultaneously, the expression of the EGF-like growth factors AREG, BTC and EREG was decreased, and their expression was dependent on the concentration of sRAGE (p < .05). CONCLUSIONS: sRAGE downregulate VEGF expression in OHSS ovarian granulosa cells, in which EGF-like growth factor pathway may be involved, and sRAGE may play a potential protective role in OHSS.

Clearance of inflammatory cytokines in patients with septic acute kidney injury during renal replacement therapy using the EMiC2 filter (Clic-AKI study).

BACKGROUND: The EMiC2 membrane is a medium cut-off haemofilter (45 kiloDalton). Little is known regarding its efficacy in eliminating medium-sized cytokines in sepsis. This study aimed to explore the effects of continuous veno-venous haemodialysis (CVVHD) using the EMiC2 filter on cytokine clearance. METHODS: This was a prospective observational study conducted in critically ill patients with sepsis and acute kidney injury requiring kidney replacement therapy. We measured concentrations of 12 cytokines [Interleukin (IL) IL-1ß, IL-1α, IL-2, IL-4, IL-6, IL-8, IL-10, interferon (IFN)-γ, tumour necrosis factor (TNF)-α, vascular endothelial growth factor, monocyte chemoattractant protein (MCP)-1, epidermal growth factor (EGF)] in plasma at baseline (T0) and pre- and post-dialyzer at 1, 6, 24, and 48 h after CVVHD initiation and in the effluent fluid at corresponding time points. Outcomes were the effluent and adsorptive clearance rates, mass balances, and changes in serial serum concentrations. RESULTS: Twelve patients were included in the final analysis. All cytokines except EGF concentrations declined over 48 h (p < 0.001). The effluent clearance rates were variable and ranged from negligible values for IL-2, IFN-γ, IL-1α, IL-1ß, and EGF, to 19.0 ml/min for TNF-α. Negative or minimal adsorption was observed. The effluent and adsorptive clearance rates remained steady over time. The percentage of cytokine removal was low for most cytokines throughout the 48-h period. CONCLUSION: EMiC2-CVVHD achieved modest removal of most cytokines and demonstrated small to no adsorptive capacity despite a decline in plasma cytokine concentrations. This suggests that changes in plasma cytokine concentrations may not be solely influenced by extracorporeal removal. TRIAL REGISTRATION: NCT03231748, registered on 27th July 2017.

A modular 3D printed lab-on-a-chip for early cancer detection.

A functional polymeric 3D device is produced in a single step printing process using a stereolithography based 3D printer. The photocurable formulation is designed for introducing a controlled amount of carboxyl groups (-COOH), in order to perform a covalent immobilization of bioreceptors on the device. The effectiveness of the application is demonstrated by performing an immunoassay for the detection of protein biomarkers involved in angiogenesis, whose role is crucial in the onset of cancer and in the progressive metastatic behavior of tumors. The detection of angiogenesis biomarkers is necessary for an early diagnosis of the pathology, allowing the employment of a less invasive therapy for the patient. In particular, vascular endothelial growth factor and angiopoietin-2 biomarkers are detected with a limit of detection of 11 ng mL-1 and 0.8 ng mL-1, respectively. This study shows how 3D microfabrication techniques, material characterization, and device development could be combined to obtain an engineered polymeric chip with intrinsic tuned functionalities.

High-sensitive immunosensing of protein biomarker based on interfacial recognition-induced homogeneous exponential transcription.

A novel and versatile immunosensing strategy was developed for ultrasensitive and specific detection of proteins by organically integrating interfacial specific target recognition and homogeneous transcription amplification. In principle, classic antigen-antibody sandwich structure on the microplate could realize the specific identification of target protein. Biotinylated DNA probe was subsequently introduced by streptavidin-biotin system as a bridge linking interfacial and homogeneous reaction. The biotinylated DNA initiated exponential transcription amplification in the solution, which converted per target recognition event on the interface to numerous single-stranded RNA products in solution for highly sensitive fluorescence immunosensing. The proposed immunoassay based on interfacial recognition-induced homogeneous exponential transcription (IR-HET) for vascular endothelial growth factor (VEGF) detection showed a good linear range from 0.01 to 1000 pg/mL and the limit of detection as low as 1 fg/mL, which was 3 orders lower than traditional ELISA method. The established strategy was also successfully applied to directly detect VEGF from culture supernatants of tumor cells and clinical body fluid samples, proving very high sensitivity, selectivity and low matrix effect. Therefore, IR-HET-based immunosensing strategy might become a potential powerful tool be applied in ultrasensitive detection of low abundance protein biomarker for clinical early diagnosis, treatment and prognosis.

In Vivo Choroidal Neovascularization and Macrophage Studies Provide Further Evidence for a Broad Role of Prostacyclin in Angiogenesis.

PURPOSE: The purpose of these studies was (1) to investigate the ability of human M1 phenotype macrophages to secrete vascular endothelial growth factor (VEGF) and the influence of prostacyclin receptor (IP) stimulation (2) to evaluate the contribution of the proangiogenic prostanoid prostacyclin to experimental choroidal neovascularization Methods: Human macrophages derived from primary blood mononuclear cells were functionally biased toward the M1 phenotype by using tumor necrosis factor α (TNFα). Experimental choroidal neovascularization was produced by laser photocoagulation. Antagonist drugs RO-3244794 (IP antagonist) and GW 627368 (EP4 antagonist) were administered according to an optimal dosing regimen that was predetermined by bioavailability studies. RESULTS: IP receptor stimulation had diametrically opposed effects on VEGF release compared with reported data on cytokine/chemokine secretion from human macrophages. For example, the IP agonist cicaprost stimulated VEGF secretion although it inhibits monocyte chemoattractant protein-1 (MCP-1) secretion: both would favor a proangiogenic effect. The IP receptor antagonist RO-3244794 produced an ∼20% statistically significant reduction in the neovascularized lesion area in the choroidal neovascularization model, which was a similar level to that produced by the EP4 antagonist GW 627368. Combining the 2 drugs produced a statistically significant reduction in neovascularization but only of slightly greater magnitude than that obtained with each antagonist administered alone. CONCLUSIONS: IP receptor stimulation potently and highly efficaciously promoted VEGF release from human M1 macrophages, indicating a possible contribution of the M1 macrophage subtype to VEGF-induced choroidal neovascularization. Studies in living animals suggest that prostacyclin and its target IP receptor contribute to choroidal neovascularization, although to a more modest extent than might have been expected.

Oral implant osseointegration model in C57Bl/6 mice: microtomographic, histological, histomorphometric and molecular characterization.

INTRODUCTION: Despite the successful clinical application of titanium (Ti) as a biomaterial, the exact cellular and molecular mechanisms responsible for Ti osseointegration remains unclear, especially because of the limited methodological tools available in this field. OBJECTIVE: In this study, we present a microscopic and molecular characterization of an oral implant osseointegration model using C57Bl/6 mice. MATERIAL AND METHODS: Forty-eight male wild-type mice received a Ti implant on the edentulous alveolar crest and the peri-implant sites were evaluated through microscopic (µCT, histological and birefringence) and molecular (RealTimePCRarray) analysis in different points in time after surgery (3, 7, 14 and 21 days). RESULTS: The early stages of osseointegration were marked by an increased expression of growth factors and MSC markers. Subsequently, a provisional granulation tissue was formed, with high expression of VEGFb and earlier osteogenic markers (BMPs, ALP and Runx2). The immune/inflammatory phase was evidenced by an increased density of inflammatory cells, and high expression of cytokines (TNF, IL6, IL1) chemokines (CXCL3, CCL2, CCL5 and CXC3CL1) and chemokine receptors (CCR2 and CCR5). Also, iNOS expression remained low, while ARG1 was upregulated, indicating predominance of a M2-type response. At later points in time, the bone matrix density and volume were increased, in agreement with a high expression of Col1a1 and Col21a2. The remodelling process was marked by peaks of MMPs, RANKL and OPG expression at 14 days, and an increased density of osteoclasts. At 21 days, intimate Ti/bone contact was observed, with expression of final osteoblast differentiation markers (PHEX, SOST), as well as red spectrum collagen fibers. CONCLUSIONS: This study demonstrated a unique molecular view of oral osseointegration kinetics in C57Bl/6 mice, evidencing potential elements responsible for orchestrating cell migration, proliferation, ECM deposition and maturation, angiogenesis, bone formation and remodeling at the bone-implant interface in parallel with a novel microscopic analysis.

Molecularly Imprinted Nanocavities Capable of Ligand-Binding Domain and Size/Shape Recognition for Selective Discrimination of Vascular Endothelial Growth Factor Isoforms.

Vascular endothelial growth factor 165 (VEGF165) is known to be predominantly expressed in the first stage of vascularization; therefore, the detection of VEGF165 is important in the stage diagnosis of cancers. Molecularly imprinted nanocavities, capable of the selective discrimination of VEGF165 from other VEGF isoforms, were prepared by surface-initiated atom transfer radical polymerization. VEGF165 was immobilized on a gold-coated glass substrate by anchored heparin moieties, where the immobilized heparin was able to capture VEGF165 by binding with the heparin-binding domain (HBD) on VEGF165. Molecular imprinting was conducted on the immobilized VEGF165 by using methacrylic acid (MAA) as a functional monomer to interact with basic amino acids outside of the HBD of VEGF165 by electrostatic interaction. After the removal of VEGF165 from the obtained polymer thin layer (ca. 7 nm), VEGF165-imprinted nanocavities remained, in which the heparin moiety and MAA residues were located in suitable positions for VEGF165 recognition. The molecularly imprinted polymer (MIP) thin layer showed a binding affinity for VEGF165 (dissociation constant: 3.4 nM) that was ten times higher than that of the substrate before polymerization (heparin-immobilized substrate). A much lower binding affinity for VEGF121, which contains no heparin-binding domain, was observed. Moreover, the MIP thin layer distinguished VEGF165 from VEGF189, which possesses a larger molecular size than VEGF165, an amino acid sequence homology of 87%, and contains HBDs, whereas the heparin-immobilized substrate showed almost no selectivity. These results suggested that the heparin moiety within the nanocavity provided HBD selectivity and the polymer matrix composed of the molecularly imprinted nanocavity provided size/shape selectivity, which resulted in the highly selective discrimination of VEGF isoforms.

Oral implant osseointegration model in C57Bl/6 mice: microtomographic, histological, histomorphometric and molecular characterization

Abstract Despite the successful clinical application of titanium (Ti) as a biomaterial, the exact cellular and molecular mechanisms responsible for Ti osseointegration remains unclear, especially because of the limited methodological tools available in this field. Objective: In this study, we present a microscopic and molecular characterization of an oral implant osseointegration model using C57Bl/6 mice. Material and Methods: Forty-eight male wild-type mice received a Ti implant on the edentulous alveolar crest and the peri-implant sites were evaluated through microscopic (μCT, histological and birefringence) and molecular (RealTimePCRarray) analysis in different points in time after surgery (3, 7, 14 and 21 days). Results: The early stages of osseointegration were marked by an increased expression of growth factors and MSC markers. Subsequently, a provisional granulation tissue was formed, with high expression of VEGFb and earlier osteogenic markers (BMPs, ALP and Runx2). The immune/inflammatory phase was evidenced by an increased density of inflammatory cells, and high expression of cytokines (TNF, IL6, IL1) chemokines (CXCL3, CCL2, CCL5 and CXC3CL1) and chemokine receptors (CCR2 and CCR5). Also, iNOS expression remained low, while ARG1 was upregulated, indicating predominance of a M2-type response. At later points in time, the bone matrix density and volume were increased, in agreement with a high expression of Col1a1 and Col21a2. The remodelling process was marked by peaks of MMPs, RANKL and OPG expression at 14 days, and an increased density of osteoclasts. At 21 days, intimate Ti/bone contact was observed, with expression of final osteoblast differentiation markers (PHEX, SOST), as well as red spectrum collagen fibers. Conclusions: This study demonstrated a unique molecular view of oral osseointegration kinetics in C57Bl/6 mice, evidencing potential elements responsible for orchestrating cell migration, proliferation, ECM deposition and maturation, angiogenesis, bone formation and remodeling at the bone-implant interface in parallel with a novel microscopic analysis.

[Role of immunological factors in the development of myopic choroidal neovascularization]. / Vliyanie immunologicheskikh faktorov na mekhanizmy formirovaniya miopicheskoi khorioidal'noi neovaskulyarizatsii.

AIM: To study the concentration of cytokines in the aqueous humor of the anterior chamber in patients with myopic choroidal neovascularization (mCNV) and to compare the results to their ophthalmic status. MATERIAL AND METHODS: A total of 19 patients (19 eyes) with mCNV treated with intravitreal ranibizumab were included in the study. The control group consisted of 15 patients (15 eyes) with myopia who had cataract surgery. Age, sex, and refractive error distribution were similar to that in the study group. All patients underwent a detailed ophthalmic examination as well as immunological study of the aqueous humor for cytokines concentrations using flow fluorometry (Bio-Plex Pro Human Cytokine Panel, 27-Plex, Bio-Rad Laboratories, USA). RESULTS: Significant differences in concentrations of 10 cytokines were found between the mCNV and study groups. Vascular endothelial growth factor (VEGF) level was twice as low in patients with mCNV as that in the controls (191.15±142.3 pg/ml and 320.06±170.05 pg/ml, respectively) (p<0.05). The other 9 cytokines were higher in mCNV, namely, platelet-derived growth factor (PDGF-BB), inflammatory and anti-inflammatory cytokines (IL-2, IL-15, IL-17А and IL-5, IL-13, respectively), tumor necrosis factor (TNFα), and chemokines (IL-8, RANTES). The degree of myopia as well as morphological and functional changes in the macular zone were shown to be in close correlation with cytokines involved in inflammation and VEGF. VEGF level appeared to be negatively related to axial eye length, refractive error, and three cytokines: IL-13, INF-γ, and RANTES. At the same time, numerous (6, 8 and more) close correlations were established between inflammatory and anti-inflammatory cytokines, chemokines, and growth factors. CONCLUSION: Patients with mCNV have been found to have higher than usual levels of inflammatory and anti-inflammatory cytokines, chemokines, and growth factors as well as a significantly decreased VEGF concentration. Immunological status of these patients differs from that in other ocular neovascular diseases suggesting possible involvement of alternative pathogenetic mechanisms.

Detection of angiogenic factors in midtrimester amniotic fluid and the prediction of preterm birth.

OBJECTIVE: We investigated whether the level of vascular endothelial growth factor (VEGF), placental growth factor (PlGF), and soluble VEGF receptor-1 (sFlt-1) in midtrimester amniotic fluid of preterm birth have different values compared with term delivery. MATERIALS AND METHODS: Our participants were 86 pregnant women who had undergone amniocentesis from 16 to 19 weeks of gestation. Forty-three cases were women with preterm delivery, and the other 43 cases were matched women with full-term delivery. Stored amniotic fluid was investigated after the delivery. The levels of VEGF, PlGF, and sFlt-1 were measured by enzyme-linked immunosorbent assay and Western blot. RESULTS: The levels of VEGF and PlGF in the preterm group were significantly higher than in the control group (30.48 ± 8.57 pg/mL vs. 26.06 ± 8.24 pg/mL and 28.83 ± 7.83 pg/mL vs. 25.35 ± 8.26 pg/mL, respectively) (p = 0.017 and 0.048, respectively). In terms of sFlt-1, the levels were decreased in the preterm group (10,478.51 ± 4012.56 pg/mL vs. 12,544.05 ± 4140.96 pg/mL) (p = 0.021). CONCLUSION: This study explains that elevated levels of VEGF and PlGF, suggestive of angiogenesis and tendency of inflammation at midtrimester, are predictive of preterm delivery, and their availability is maximized by downregulation of sFlt-1.

Aqueous levels of VEGF correlate with retinal non-perfusion areas in patients with diabetic macular edema and macular edema secondary to central retinal vein occlusion.

AIM: To evaluate the association between the level of vascular endothelial growth factor in the aqueous humor and the size of capillary non-perfusion areas in patients with macular edema secondary to retinal vein occlusion and diabetic retinopathy. MATERIAL AND METHODS: The study group consisted of 24 patients (24 eyes) at the age of 55-78 years, with diffuse macular edema secondary to retinal vein occlusion and diabetic retinopathy. The control group consisted of 26 subjects aged 55-87 years who were admitted for scheduled cataract surgery. The VEGF aqueous humor levels, retinal thickness using optical coherence tomography, as well as the size of non-perfusion areas measured on fluorescein angiography images were evaluated in each enrolled subject. RESULTS: The vascular endothelial growth factor aqueous humor levels were found to be significantly higher in patients with macular edema as compared to controls (p = 0.0002). In the diabetic macular edema and retinal vein occlusion group, the con- centration of vascular endothelial growth factor in aqueous humor positively correlated with the extent of non-perfusion areas measured on fluorescein angiograms (Rs = + 0.45, p = 0.02;). Multivariate analysis of patients and controls performed using the general linear model, adjusted for age, sex, intraocular pressure and the presence of diabetes, revealed that macular edema was an independent factor associated with higher aqueous VEGF concentrations (ß = +0.74, p = 0.0012). CONCLUSIONS: Macular edema secondary to either retinal vein occlusion or diabetic retinopathy is associated with the increased levels of vascular endothelial growth factor in the aqueous humor. Therefore, the management of patients with macular edema secondary to retinal vein occlusion or diabetic retinopathy should aim at reducing the ocular vascular endothelial growth factor concentrations, especially in the presence of capillary non-perfusion areas.

Gingival crevicular fluid vascular endothelial cell growth factor and platelet-derived growth factor-BB release profile following the use of perforated barrier membranes during treatment of intrabony defects: a randomized clinical trial.

BACKGROUND AND OBJECTIVE: Perforated barrier membranes open channels between the suprabony and intrabony compartments of the defect, which could allow for more physiologic cellular interactions between different components of the periodontium during guided tissue regeneration surgery. To test this assumption, this study was designed to evaluate levels of vascular endothelial cell growth factor (VEGF) and platelet-derived growth factor (PDGF)-BB in gingival crevicular fluid during the early stages of healing of localized intrabony defects treated with perforated membranes (PMs) or non-PMs, as compared with open flap debridement. MATERIAL AND METHODS: Thirty non-smoking patients with severe chronic periodontitis participated in this prospective, randomized and single blinded trial. Each patient contributed one interproximal defect that was randomly assigned to the PM group (n = 10), occlusive membrane (OM) group (n = 10) or open flap debridement (OFD) group (n = 10). Plaque index, gingival index, probing depth, clinical attachment level and the intrabony depth of the defect were measured at baseline and reassessed at 6 and 9 mo after therapy. Gingival crevicular fluid samples were collected on days 1, 3, 7, 14, 21 and 30 d after therapy for the changes in VEGF and PDGF-BB levels. RESULTS: During the early stages of healing (1, 3 and 7 d), the mean VEGF and PDGF-BB concentrations at sites treated with PMs and OFD peaked with a statistically significant difference as compared with the OM-treated group. VEGF and PDGF-BB levels at sites treated with PMs and OFD were not statistically different. Growth factor levels decreased sharply in the samples obtained at days 21 and 30 with non-significant differences between the three groups. Nine months after therapy, the PM-treated group showed a statistically significant improvement in probing depth, clinical attachment level and intrabony defect compared to the OM and OFD groups. CONCLUSIONS: Within the limits of the present study, one can conclude that PM coverage of periodontal defects is associated with initial gingival crevicular fluid growth factor upregulation that could improve the clinical outcomes of guided tissue regeneration surgery.

Expression of lymphatic markers and lymphatic growth factors in psoriasis before and after anti-TNF treatment

BACKGROUND: Angiogenesis is an early stage of psoriatic lesion development, but less is known about lymphagiogenesis and its role in the development of psoriasis. OBJECTIVE: To examine the expression of specific lymphatic markers and lymphatic growth factors in untreated psoriatic skin, in the unaffected skin of patients and skin of healthy volunteers, as well as their alteration after treatment with an anti-TNF agent. METHODS: Immunohistochemistry for the lymphatic markers D2-40 and LYVE-1, in addition to the VEGF-C and VEGF-D growth factors, was performed in the skin biopsies of psoriatic lesions and adjacent non-psoriatic skin of 19 patients before and after treatment with etanercept, as well as in the skin biopsies of 10 healthy volunteers. RESULTS: The expressions of D2-40, VEGF-C and VEGF-D on lymphatic vessels underwent statistically significant increases in untreated psoriatic skin compared with non-lesional skin, in contrast to LYVE-1, which did not involve significant increase in expression in psoriatic skin. VEGF-C expression on lymphatic vessels diminished after treatment with etanercept. Moreover VEGF-C and VEGF-D staining on fibroblasts presented with higher expression in lesional skin than in non-lesional adjacent skin. CONCLUSION: Remodeling of lymphatic vessels possibly occurs during psoriatic lesion development, parallel to blood vessel formation. The exact role of this alteration is not yet clear and more studies are necessary to confirm these results. .

Expression of lymphatic markers and lymphatic growth factors in psoriasis before and after anti-TNF treatment.

BACKGROUND: Angiogenesis is an early stage of psoriatic lesion development, but less is known about lymphagiogenesis and its role in the development of psoriasis. OBJECTIVE: To examine the expression of specific lymphatic markers and lymphatic growth factors in untreated psoriatic skin, in the unaffected skin of patients and skin of healthy volunteers, as well as their alteration after treatment with an anti-TNF agent. METHODS: Immunohistochemistry for the lymphatic markers D2-40 and LYVE-1, in addition to the VEGF-C and VEGF-D growth factors, was performed in the skin biopsies of psoriatic lesions and adjacent non-psoriatic skin of 19 patients before and after treatment with etanercept, as well as in the skin biopsies of 10 healthy volunteers. RESULTS: The expressions of D2-40, VEGF-C and VEGF-D on lymphatic vessels underwent statistically significant increases in untreated psoriatic skin compared with non-lesional skin, in contrast to LYVE-1, which did not involve significant increase in expression in psoriatic skin. VEGF-C expression on lymphatic vessels diminished after treatment with etanercept. Moreover VEGF-C and VEGF-D staining on fibroblasts presented with higher expression in lesional skin than in non-lesional adjacent skin. CONCLUSION: Remodeling of lymphatic vessels possibly occurs during psoriatic lesion development, parallel to blood vessel formation. The exact role of this alteration is not yet clear and more studies are necessary to confirm these results.

Effects of botulinum toxin A on patient-specific keloid fibroblasts in vitro.

OBJECTIVES/HYPOTHESIS: To test whether therapeutic effects of botulinum toxin A on patient- specific keloid tissue can be reproduced on the cellular level. Specifically, effects on cell proliferation and expression of growth factors and cytokines relevant for wound healing were to be tested. STUDY DESIGN: Experimental study including patient specific cell cultures of keloids. METHODS: Patient-specific keloid tissue was tested in a cell culture model for effects of botulinum toxin incubation on cell proliferation and expression of the following cytokines and growth factors: IL-6 (interleukin-6), VEGF (vascular endothelial growth factor), TGF-ß (transforming growth factor-ß). RESULTS: None of the tested parameters of human keloid tissue were affected by botulinum toxin A incubation. CONCLUSION: The present study does not add evidence to suggest a significant therapeutic role of botulinum toxin injections for the treatment of keloids. LEVEL OF EVIDENCE: N/A.

Study on vascular endothelial growth factor and its receptor in the vitreous of diabetic rats / Estudio del factor de crecimiento endotelial vascular y su receptor en el humor vítreo de ratas diabéticas

OBJECTIVE: To investigate the vitreous level of vascular endothelial growth factor (VEGF) and kinase insert domain-containing receptor (KDR) in diabetic rats, and to explore the role of VEGF and KDR in diabetic retinopathy. METHODS: Eighty-four adult Wistar rats were randomly divided into two groups. Fifty-eight rats in group A were injected intraperitoneally with streptozotocin to induce diabetes and 20 rats in group B were injected with physiological saline. Blood glucose meter was used to detect the blood glucose level at 72 hours after injection; blood glucose level >16.67 mmol/L was considered to be successful modelling. Blood glucose level was assayed and body mass was measured on the same modelling day, one week, two weeks and four weeks after modelling. Four weeks after modelling, the vitreous was taken and the VEGF and KDR levels were detected by enzyme-linked immunosorbent assay (ELISA). The eyeballs were fixed with paraform and embedded by petrolin for haematoxylin and eosin (H & E) staining. RESULTS: Forty-two rats survived and 16 rats died in group A. No rats died in group B. The blood glucose at one week, two weeks and four weeks between the two groups had statistical differences (p < 0.05). The weight at one week and two weeks between the two groups was not different but there was statistical difference at four weeks between the two groups (p < 0.01). The ELISA results showed that the VEGF and KDR levels were 0.276 ± 0.026 ng/mL and 2.936 ± 0.295 ng/mL in group A, 0.231 ± 0.021 ng/mL and 2.394 ± 0.227 ng/mL in group B, respectively. The VEGF and KDR levels of group A were higher than those of group B (p < 0.05). CONCLUSIONS: The changes of VEGF and KDR levels in the vitreous of diabetic rats were related to the early retinopathy induced by diabetes.

OBJETIVO: Investigar el nivel vítreo del factor de crecimiento endotelial vascular (FCEV) y receptor con dominio inserto-quinasa (KDR) en ratas diabéticas, y explorar el papel de FCEV y KDR en la retinopatía diabética. MÉTODOS: Ochenta y cuatro ratas adultas Wistar fueron divididas aleatoriamente en dos grupos. A cincuenta y ocho ratas en el grupo A se les inyectó estreptozotocinapor vía intraperitonealpara inducir diabetes, mientras que a 20 ratas en el grupo B se les inyectó una solución salina fisiológica. Se usó un medidor de glucosa en sangre para detectar el nivel de glucosa en sangre a las 72 horas después de la inyección. Un nivel de glucosa en sangre > 16.67 mmol/L se consideró como un modelo exitoso. Se analizó el nivel de glucosa en sangre, y se midió la masa corporal en el mismo día del modelado, y una semana, dos semanas, y cuatro semanas después del modelado. Cuatro semanas después del modelado, se tomó el humor vítreo, y los niveles de FCEV y KDR fueron detectados mediante ensayo por inmunoabsorción ligado a enzimas (ELISA). Los globos oculares fueron fijados con para formaldehido e incrustados por petrolin para tinción (H & E) hematoxilina-eosina. RESULTADOS: Cuarenta y dos ratas sobrevivieron y 16 ratas murieron en el grupo A. Ninguna de las ratas en el grupo B murió. La glucosa en la sangre a la semana, las dos semanas, y las cuatro semanas entre los dos grupos tuvo diferencias estadísticas (p < 0.05). El peso a la semana y a las dos semanas entre los dos grupos no fue diferente, pero hubo diferencia estadística a las cuatro semanas entre los dos grupos (p < 0.01). Los resultados de ELISA mostraron que los niveles de FCEV y KDR fueron 0.276 ± 0.026 ng/mLy 2.936 ± 0.295 ng/mL en el grupo A, 0.231 ± 0.021 ng/mL y 2.394 ± 0.227 ng/mL en el grupo B, respectivamente. Los niveles de FCEV y KDR del grupo A fueron superiores a los del grupo B (p < 0.05). CONCLUSIONES: Los cambios de nivel FCEV y KDR en el humor vítreo de las ratas diabéticas estaban asociados con la retinopatía temprana inducida por diabetes.

Prognostic significance of vascular endothelial growth factor expression in esophageal carcinoma: a meta-analysis.

PURPOSE: The purpose of this study was to comprehensively and quantitatively review eligible published studies to explore the prognostic significance of vascular endothelial growth factor (VEGF) expression in patients with esophageal carcinoma (EC). METHODS: PubMed and Embase databases were searched until September 11, 2011. A meta-analysis was performed to demonstrate any relationship between VEGF and 5-year overall survival (OS) in EC patients. RESULTS: The final analysis included 1453 patients from 19 studies. The studies were grouped by patient source, histology, VEGF isoform and cutoff value. The estimated risk of death suggested that VEGF positivity had negative impact on prognosis of patients with EC, esophageal squamous cell carcinoma (ESCC) and Asian patients. The risk ratios (RR) and 95% confidence interval (95% CI) were 1.26 (1.16-1.37) in EC patients, 1.28 (1.16-1.40) in ESCC patients and 1.35 (1.24-1.48) in Asian patients. Furthermore, when the cutoff value was set at 10% in 6 studies, the RR (95% CI) was 1.48 in the VEGF positive group (1.27-1.73). In addition, VEGFC was also correlated with patient poor prognosis with a RR (95% CI) of 1.30 (1.15-1.48). However, EC patients from non-Asian countries and cutoff value at 30% showed no significant correlation with survival. Data were not sufficient to determine the prognostic value of VEGF expression in esophageal adenocarcinoma (EA) patients and VEGFD expression. CONCLUSIONS: VEGF positivity indicated poor prognosis in patients with EC, ESCC and of Asian origin. Cutoff value at 10% may be a more appropriate standard to define VEGF positivity. VEGFC also correlated with poor prognosis in EC patients.