FGF2, LIF, and IGF-1 supplementation improves mouse oocyte in vitro maturation via increased glucose metabolism†.

Chemically defined oocyte maturation media supplemented with FGF2, LIF, and IGF-1 (FLI medium) enabled significantly improved oocyte quality in multiple farm animals, yet the molecular mechanisms behind such benefits were poorly defined. Here, we first demonstrated that FLI medium enhanced mouse oocyte quality assessed by blastocyst formation after in vitro fertilization and implantation and fetal development after embryo transfer. We then analyzed the glucose concentrations in the spent media; reactive oxygen species concentrations; mitochondrial membrane potential; spindle morphology in oocytes; and the abundance of transcripts of endothelial growth factor-like factors, cumulus expansion factors, and glucose metabolism-related genes in cumulus cells. We found that FLI medium enabled increased glucose metabolism through glycolysis, pentose phosphate pathway, and hexosamine biosynthetic pathway, as well as more active endothelial growth factor-like factor expressions in cumulus cells, resulting in improved cumulus cell expansion, decreased spindle abnormality, and overall improvement in oocyte quality. In addition, the activities of MAPK1/3, PI3K/AKT, JAK/STAT3, and mTOR signaling pathways in cumulus cells were assessed by the phosphorylation of MAPK1/3, AKT, STAT3, and mTOR downstream target RPS6KB1. We demonstrated that FLI medium promoted activations of all these signaling pathways at multiple different time points during in vitro maturation.

The role of vascular endothelial growth factor as a prognostic and clinicopathological marker in osteosarcoma: a systematic review and meta-analysis.

BACKGROUND: In recent years, numerous investigations have been conducted to determine the clinical significance and critical functions of vascular endothelial growth factor (VEGF) in various malignant cancers. The purpose of this meta-analysis was to comprehensively evaluate the prognostic and clinicopathological value of VEGF in patients with osteosarcoma. METHODS: We performed a systematic literature retrieval of available databases. Odds ratios (ORs) or standard mean difference (SMD) for clinicopathological parameters, hazard ratios (HRs) for overall survival and disease-free survival were calculated to assess the correlation between VEGF expression and prognosis in patients with osteosarcoma. RESULTS: A total of 22 studies with 1144 patients were included in our study. Pooled analyses showed that VEGF overexpression predicted worse overall survival (HR, 2.42; 95% CI, 1.87-3.11, p < 0.001) and disease-free survival (HR, 2.604; 95% CI, 1.698-3.995, p < 0.001), respectively. Furthermore, investigation regarding osteosarcoma clinicopathologic characteristics suggested that high VEGF expression was significantly associated with metastasis (OR, 4.39; 95% CI, 2.77-6.95; p < 0.001), clinical stage (OR, 0.73; 95% CI, 0.62-0.87; p < 0.001), and microvessel density (SMD, 3.33, 95% CI,1.57-5.10, p < 0.001), but not associated with tumor location, gender, age, local recurrence, and chemotherapy response. CONCLUSION: Our meta-analysis findings suggest that elevated VEGF expression may be a predictive biomarker for poor prognosis and adverse clinicopathological characteristics in patients with osteosarcoma.

Hypertonic sodium lactate improves microcirculation, cardiac function, and inflammation in a rat model of sepsis.

BACKGROUND: Hypertonic sodium lactate (HSL) may be of interest during inflammation. We aimed to evaluate its effects during experimental sepsis in rats (cecal ligation and puncture (CLP)). METHODS: Three groups were analyzed (n = 10/group): sham, CLP-NaCl 0.9%, and CLP-HSL (2.5 mL/kg/h of fluids for 18 h after CLP). Mesenteric microcirculation, echocardiography, cytokines, and biochemical parameters were evaluated. Two additional experiments were performed for capillary leakage (Evans blue, n = 5/group) and cardiac hemodynamics (n = 7/group). RESULTS: HSL improved mesenteric microcirculation (CLP-HSL 736 [407-879] vs. CLP-NaCl 241 [209-391] UI/pixel, p = 0.0006), cardiac output (0.34 [0.28-0.43] vs. 0.14 [0.10-0.18] mL/min/g, p < 0.0001), and left ventricular fractional shortening (55 [46-73] vs. 39 [33-52] %, p = 0.009). HSL also raised dP/dtmax slope (6.3 [3.3-12.1] vs. 2.7 [2.0-3.9] 103 mmHg/s, p = 0.04), lowered left ventricular end-diastolic pressure-volume relation (1.9 [1.1-2.3] vs. 3.0 [2.2-3.7] RVU/mmHg, p = 0.005), and reduced Evans blue diffusion in the gut (37 [31-43] vs. 113 [63-142], p = 0.03), the lung (108 [82-174] vs. 273 [222-445], p = 0.006), and the liver (24 [14-37] vs. 70 [50-89] ng EB/mg, p = 0.04). Lactate and 3-hydroxybutyrate were higher in CLP-HSL (6.03 [3.08-10.30] vs. 3.19 [2.42-5.11] mmol/L, p = 0.04; 400 [174-626] vs. 189 [130-301] µmol/L, p = 0.03). Plasma cytokines were reduced in HSL (IL-1ß, 172 [119-446] vs. 928 [245-1470] pg/mL, p = 0.004; TNFα, 17.9 [12.5-50.3] vs. 53.9 [30.8-85.6] pg/mL, p = 0.005; IL-10, 352 [267-912] vs. 905 [723-1243] pg/mL) as well as plasma VEGF-A (198 [185-250] vs. 261 [250-269] pg/mL, p = 0.009). CONCLUSIONS: Hypertonic sodium lactate fluid protects against cardiac dysfunction, mesenteric microcirculation alteration, and capillary leakage during sepsis and simultaneously reduces inflammation and enhances ketone bodies.

Valproic acid inhibits the angiogenic potential of cervical cancer cells via HIF-1alpha/VEGF signals

Purpose. Cervical cancer is one of the most prevalent malignancies in women worldwide. Therefore, the investigation about the molecular pathogenesis and related therapy targets of cervical cancer is an emergency. The objective of the present study is to investigate the effects of valproic acid (VPA), a histone deacetylase inhibitor, on the angiogenesis of cervical cancer. Methods. The effects and mechanisms of VPA on in vitro angiogenesis and vascular endothelial growth factor (VEGF) expression of human cervical cancer HeLa and SiHa cells were investigated. Results. Our present study reveals that 1 mM VPA can significantly inhibit the in vitro angiogenic potential and VEGF expression of human cervical cancer HeLa and SiHa cells. Further, the transcription and protein levels of hypoxia inducible factor-1α (HIF-1α), and not HIF-1β, are significantly inhibited in VPA-treated cervical cancer cells. Over expression of HIF-1α can obviously reverse VPA-induced VEGF down regulation. VPA-treatment decreases the activation of Akt and ERK1/2 in both HeLa and SiHa cells in a time-dependent manner. The inhibitor of Akt (LY 294002) or ERK1/2 (PD98059) can inhibit VEGF alone and cooperatively reinforce the suppression effects of VPA on HIF-1α and VEGF expression. Conclusion. Collectively, our data reveal that the inhibition of PI3K/Akt and ERK1/2 signals are involved in VPA-induced HIF-1α and VEGF suppression of cervical cancer cells (AU)

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Different expression of VEGF and EGFL7 in human hepatocellular carcinoma.

BACKGROUND: Vascular endothelial growth factor (VEGF) is one of several angiogenic factors expressed in cirrhosis and during progression to malignancy, that seem to play a major role in hepatocellular carcinoma development. Lately, another angiogenic factor, epidermal growth factor-like domain multiple 7 (EGFL7), has attracted interest due to its possible relationship with hepatocellular carcinoma metastasis. AIMS: To evaluate expression of VEGF and EGFL7 in human hepatocellular carcinoma, compared to corresponding cirrhotic surrounding tissue. METHODS: Tumoural and cirrhotic tissue was harvested from 12 consecutive patients undergoing surgical resection. VEGF and EGFL7 were assessed by immunofluorescence and quantitative reverse transcriptase-polymerase chain reaction, compared with normal controls. RESULTS: Both angiogenic factors were over-expressed in cirrhotic livers compared to normal controls. VEGF and EGFL7 expressions did not differ according to disease aetiology, nodule size or other clinical variables. While VEGF expression was constant, regardless of tumour differentiation stage and unchanged compared to surrounding cirrhotic tissue, EGFL7 expression increased in less differentiated hepatocellular carcinoma. CONCLUSIONS: The preferential expression of EGFL7 in less differentiated hepatocellular carcinoma compared to VEGF, suggests a possible important role of this angiogenic factor in a later oncogenic and infiltrative/metastatic phase.

Prognostic significance of epidermal growth factor-like domain 7 in pancreatic cancer.

BACKGROUND: Recent studies have shown the clinical significance of epidermal growth factor-like domain 7 (EGFL7) in a variety of cancers. However, the relationship between EGFL7 and the prognosis of pancreatic cancer (PC) remains unclear. The present study was undertaken to investigate the role of EGFL7 in the prognosis of PC. METHODS: The expression of EGFL7 in nine PC cell lines was first determined by Western blotting analysis. Tissue microarray-based immunohistochemical staining was performed in paired formalin-fixed paraffin-embedded tumor and non-tumor samples from 83 patients with PC. Finally, correlations between EGFL7 expression and clinicopathological variables as well as overall survival were evaluated. RESULTS: EGFL7 was widely expressed in all PC cell lines tested. EGFL7 expression in tumor tissues was significantly higher than that in non-tumor tissues (P=0.040). In addition, univariate analysis revealed that high EGFL7 expression in tumor tissues was significantly associated with poor overall survival, accompanied by several conventional clinicopathological variables, such as gender, histological grade and lymph node metastasis. In a multivariate Cox regression test, EGFL7 expression was identified as an independent marker for long-term outcome of PC. CONCLUSION: Our data showed that EGFL7 is extensively expressed in PC and that EGFL7 is associated with poor prognosis.

Real-time immuno-polymerase chain reaction in a 384-well format: detection of vascular endothelial growth factor and epidermal growth factor-like domain 7.

Immuno-polymerase chain reaction (immuno-PCR) combines the specificity of antibodies with the amplification power of PCR to detect low levels of proteins. Here, we describe the development of a 384-well immuno-PCR method that uses streptavidin coated on a PCR plate to capture complexes of biotinylated capture antibody, antigen, and DNA-labeled detection antibody. Unbound molecules are removed by a wash step using a standard plate washer. Antibody-DNA molecules in bound complexes are then detected directly on the plate using real-time PCR. Circulating human vascular endothelial growth factor concentrations measured by this method correlated with measurements obtained from enzyme-linked immunosorbent assay (ELISA). Using this method, we developed an assay for human epidermal growth factor-like domain 7 (EGFL7), an extracellular matrix-bound angiogenic factor. EGFL7 is expressed at a higher level in certain cancers, although endogenous EGFL7 concentrations have not been reported. Our 384-well EGFL7 immuno-PCR assay can detect 0.51pM EGFL7 in plasma, approximately 16-fold more sensitive than the ELISA, utilizing the same antibodies. This assay detected EGFL7 in lysates of non-small-cell lung cancer and hepatocellular carcinoma cell lines and also hepatocellular carcinoma, breast cancer, and ovarian cancer tissues. This 384-well immuno-PCR method can be used to develop high-throughput biomarker assays.

Plasma rich in growth factors promotes bone tissue regeneration by stimulating proliferation, migration, and autocrine secretion in primary human osteoblasts.

BACKGROUND: Alveolar bone loss can be a major clinical concern affecting both functionality and esthetics. Osteoblasts are the main cells charged with the repair and regeneration of missing bone tissue. Plasma rich in growth factors (PRGF) allows delivery of a cocktail of proteins and growth factors that promote wound healing and tissue regeneration to the site of injury. This study tests the effect of this endogenous regenerative technology to stimulate alveolar osteoblast bone-forming potential. METHODS: Primary human osteoblasts were retrieved from alveolar bone of patients undergoing oral surgery. Cell proliferation was evaluated, and culture inserts and permeable transwell supports were used to assess cell migration and chemotaxis. The expression of differentiation markers was quantified by enzyme-linked immunosorbent assay. RESULTS: PRGF succeeded in increasing proliferation, migration, and chemotaxis of osteoblasts. Also, PRGF significantly enhanced the autocrine expression of two relevant proangiogenic factors, vascular endothelial growth factor and hepatocyte growth factor, and three markers of osteoblastic activity, procollagen I, osteocalcin, and alkaline phosphatase. CONCLUSION: The results indicate that PRGF can stimulate some of the biologic processes of the main cells responsible for bone regeneration and help support the positive clinical outcomes that have been reported with this technology.

Novel role for epidermal growth factor-like domain 7 in metastasis of human hepatocellular carcinoma.

UNLABELLED: Epidermal growth factor-like domain 7 (Egfl7) is a recently identified secreted protein that is believed to be primarily expressed in endothelial cells (ECs). Although its expression was reported elevated during tumorigenesis, whether and how Egfl7 contributes to human malignancies remains unknown. In the present study overexpression of Egfl7 was found predominantly in hepatocellular carcinoma (HCC) cells in HCC tissues and closely correlated with poor prognosis of HCC. The expression of Egfl7 in cancer cells was further verified with HCC cell lines including HepG2, MHCC97-L, and HCCLM3, and the Egfl7 expression levels positively correlated with metastatic potential of HCC cell lines was tested. To functionally characterize Egfl7 in HCC, we depleted its expression in HCCLM3 cells by using small interfering RNA. Interestingly, reduction of Egfl7 expression resulted in significant inhibition of migration but not growth of HCCLM3 cells. Biochemical analysis indicated that Egfl7 could facilitate the phosphorylation of focal adhesion kinase (FAK) and therefore promote the migration of HCCLM3 cells. In addition, this effect was almost completely blocked by inhibition of epidermal growth factor receptor (EGFR), indicating that the activation of FAK by Egfl7 is mediated through EGFR. Finally, we used a mouse model to demonstrate that down-regulation of Egfl7 was associated with suppression of intrahepatic and pulmonary metastases of HCC. Collectively, our study shows for the first time that overexpression of Egfl7 in HCC and Egfl7 promotes metastasis of HCC by enhancing cell motility through EGFR-dependent FAK phosphorylation. CONCLUSION: Our study suggests Egfl7 as a novel prognostic marker for metastasis of HCC and a potential therapeutic target.

Effect of low shear stress on permeability and occludin expression in porcine artery endothelial cells.

INTRODUCTION: Although both fluid shear stress and mass transport of atherogenic substances into the vascular wall are known to be important factors in atherogenesis, there has been little research on the effect of shear stress on vascular permeability. Therefore, the effects of shear stress on the permeability of arteries and the expression of the endothelial cell tight junction molecule occludin, an important regulator of vascular permeability, were investigated. METHODS: Porcine carotid arteries were perfusion cultured ex vivo with low (1.5 dyne/cm(2)) or physiologic (15 dyne/cm(2)) shear stress and 100 mmHg pressure for 24 hours. Subsequently, 20 nm gold particles in solution were infused into the lumen of vessels at 100 mmHg for 30 minutes. Frozen sections were then cut and stained for gold particles. Image analysis was used to determine the density of the particles in the vessel walls. The expression of endothelial cell occludin mRNA and protein were determined using reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, respectively. RESULTS: Permeability results showed a 2.8-fold increase in the apparent permeability of vessels cultured with low versus physiologic shear stress. RT-PCR and Western blotting results showed significant decreases in occludin mRNA and protein expression at 12 and 24 hours in vessels cultured with low versus physiologic shear stress. CONCLUSIONS: These results demonstrate that low shear stress increases vascular permeability in porcine carotid arteries, possibly owing to decreased occludin expression. These results may have implications in the preferential formation of atherosclerotic vascular disease adjacent to branches and bifurcations where low mean shear stresses may occur.

¿Qué utilidad tiene en la práctica clínica medir la función endotelial? / What is the utility of measuring endothelial function in clinical practice?

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Cell signalling: growth factors and tyrosine kinase receptors

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Results of initial operations in non-small cell lung cancer patients with single-level N2 disease.

BACKGROUND: There is still debate regarding the use of surgery in the management of non-small cell lung cancer patients with N2 disease. The primary aim of the present study was analysis of the results of initial operations in non-small cell lung cancer patients with single-level N2 disease. METHODS: Ninety-four patients with the disease who underwent initial surgery consisting of complete resection of the primary site plus systematic lymphadenectomy were examined. We also immunohistochemically examined lymphatic vessel density and vascular endothelial growth factor-C expression. RESULTS: The overall 5- and 10-year survival rates for the 94 patients were 27.1% and 12.1%, respectively, with a median survival of 22 months. When stratified by skipping status, the 5-year survival rates for the patients in skip-N2 and non-skip-N2 groups were 33.4% and 19.8%, respectively (p = 0.0189). Skip metastasis, T factor, subcarinal lymph node metastasis, and adjuvant chemotherapy were recognized as independent prognostic indicators. In both skip-N2 and non-skip-N2 groups, distant relapse was the dominant pattern of recurrence. Although the peritumoral lymphatic vessel density was associated with vascular endothelial growth factor-C expression in tumors, the lymphangiogenic profile appeared to be different between skip-N2 and non-skip-N2 tumors, suggesting different nodal metastatic process. CONCLUSIONS: Lung cancer patients with single-level N2 disease are an oncologically heterogeneous cohort. Although further studies involving randomized comparisons are required, the poor outcomes found here indicate that the initial operation has yet to be validated for patients with this disease.

Erupción acneiforme por inhibidores del receptor de crecimiento epidérmico / Acneiform eruption from epidermal growth factor receptor inhibitors

Uno de los objetivos fundamentales de la investigación oncológica es la búsqueda de moléculas con mayor eficacia antitumoral y menor toxicidad que los agentes quimioterápicos habituales. Los inhibidores del receptor de crecimiento epidérmico son un nuevo grupo de fármacos que, por su efecto más específico frente a las células neoplásicas, parecen cumplir estas características. Entre los efectos adversos asociados a su uso, destacan por su frecuencia las erupciones cutáneas, secundarias a un efecto inhibitorio directo del fármaco sobre la homeostasis de la epidermis y del folículo pilosebáceo 1,2. Recientemente se han publicado varios casos de toxicidad cutánea en pacientes tratados con inhibidores del receptor de crecimiento epidérmico. Presentamos 3 casos de erupciones acneiformes atribuibles a diferentes fármacos de esta familia (cetuximab, gefitinib y erlotinib)

One of the fundamental aims of oncological research is the search for molecules with greater efficacy against tumors and less toxicity than the usual chemotherapeutic agents. Epidermal growth factor receptor inhibitors are a new group of drugs which, because of their more specific effect against neoplastic cells, seem to meet these characteristics. Skin eruptions are one of the most frequent adverse effects associated with their use, secondary to the drug's direct inhibitory effect on homeostasis of the epidermis and of the pilosebaceous follicle. Several cases of cutaneous toxicity in patients treated with epidermal growth factor receptor inhibitors have recently been published. We present three cases of acneiform eruptions attributable to different drugs in this family (cetuximab, gefitinib and erlotinib)

Significance of serum vascular endothelial growth factor, insulin-like growth factor-I levels and nitric oxide activity in breast cancer patients.

Vascular endothelial growth factor (VEGF) is used to evaluate the angiogenic activity in breast carcinoma. Nitric oxide (NO) and insulin-like growth factor-I (IGF-I) are also implicated in breast tumorigenesis, including angiogenesis. We measured serum VEGF, IGF-I and nitrate+nitrite levels in 38 patients with metastatic and 23 with nonmetastatic breast cancer and in 16 controls. Serum VEGF and IGF-I levels were higher in patients with metastatic disease than in those with nonmetastatic disease or in controls (P<0.001). Serum nitrate+nitrite levels were higher in patients with metastatic and nonmetastatic disease than in controls (P<0.001). Patients with visceral metastasis and local metastasis had higher serum VEGF and nitrate+nitrite levels than patients with bone metastasis (P<0.05). In the metastatic disease group, there was a positive correlation between serum VEGF levels and nitrate+nitrite levels (r=0.436, P<0.05). Within the group with nonmetastatic disease, premenopausal patients had higher serum IGF-I levels than did postmenopausal patients (P<0.001). NO may involve an angiogenic process that is stimulated by VEGF in breast carcinoma. Larger studies are required to clarify these suggestions.

Endometriosis-associated ovarian carcinoma: differential expression of vascular endothelial growth factor and estrogen/progesterone receptors.

BACKGROUND: Multiple epidemiologic and histologic studies have suggested that ovarian endometriosis can give rise to malignant ovarian tumors, primarily those of epithelial origin. The progression of endometriosis to endometriosis-associated ovarian carcinoma (EAOC) has not been investigated thoroughly and is poorly understood at best. Using immunohistochemical methods, we compared the differential expression patterns of various cytokines and growth factors in atypical endometriosis (AE) and EAOC. METHODS: Using the Johns Hopkins Pathology Data Bank, tissue blocks from patients diagnosed with EAOC or AE were identified. Tissue blocks were stained for 4 markers: vascular endothelial growth factor (VEGF), Ki-67, estrogen receptor (ER), and progesterone receptor (PR). RESULTS: Seventeen cases of EAOC and 8 cases of AE were identified. Staining for VEGF was documented in 16 of 17 (94%) EAOC tissue blocks and in only 1 of 8 (12.5%) AE tissue blocks (P < 0.0001). Only 4 of the 17 (23%) EAOC tissue blocks exhibited positive staining for ER, compared with 8 of 8 (100%) AE tissue blocks (P = 0.0005). Positive staining for PR was noted in only 6 of 17 (35%) EAOC samples but was present in 8 of 8 (100%) AE samples (P = 0.003). Seventy percent of EAOC samples exhibited positive staining for Ki-67, compared with 37.5% of AE samples (P = 0.19). CONCLUSIONS: EAOC appears to be associated with overexpression of VEGF and reduced expression of both ER and PR. Variations in VEGF expression may be associated with the malignant transformation of endometriosis and may present both diagnostic and therapeutic options for the treatment of ovarian malignancies.

Vascular endothelial growth factor (VEGF) expression and the effect of exogenous VEGF on survival of a random flap in the rat.

The induction of endogenous vascular endothelial growth factor (VEGF) production in the skin flap with ischemic injury and the effect of exogenous VEGF on survival of the ischemic skin flap were studied in rats. A dorsal flap model (3x10 cm(2)) was used in this study. In Part I, biopsies were taken from the flap at 2.5, 5.5, and 8.5 cm distances from the distal edge at 0, 6, 12, and 24 h after the flaps were sutured. Malonyldialdehyde (MDA) and VEGF(165) protein level were measured. In Part II, exogenous VEGF (1 microg/ml) was injected subdermally into the flaps in 14 rats before the flaps were replaced. Flaps that received a saline injection were used as the controls. The skin paddle survival was measured on postoperative day five. The results showed that the MDA level in the distal part of the flap significantly increased at 24 h postoperatively when compared to MDA in other parts of the flap. However, VEGF levels in the distal part of the flap significantly decreased when compared to the middle part of the flap. Subdermal injection of exogenous VEGF to the distal area of the flap could significantly improve survival of the distal flap (89% of total skin paddle) when compared to the control, which had a 64% mean percent survival. We conclude that production of endogenous VEGF protein is significantly increased in the skin flap with mild ischemia, but decreased in the flap with severe ischemia. Administration of exogenous VEGF could significantly enhance survival of ischemic flaps.

Expression of VEGF and its receptors by myeloma cells.

Angiogenesis or new vessel formation is an essential component in the growth and progression of neoplasms and there is growing evidence of its importance in hematological malignancies including multiple myeloma (MM). Vascular endothelial growth factor (VEGF) is believed to play a role in tumor angiogenesis. We studied the expression of VEGF and its receptors (VEGFR1 or Flt-1 and VEGFR2 or Flk-1/KDR) by myeloma cell lines and plasma cells isolated from patients, using different methods. VEGF expression by the plasma cells was demonstrated by immunohistochemistry in 18 of 20 patients with MM. Enzyme-linked immunosorbent assay demonstrated VEGF secretion in all six different myeloma cell lines studied. Five patient marrow samples and seven different myeloma cell lines were then studied for VEGF mRNA expression by reverse-transcriptase polymerase chain reaction (RT-PCR), which was positive in all. We further evaluated the expression of both VEGFR1 and VEGFR2 in different myeloma cell lines and five sorted myeloma bone marrow samples by RT-PCR. All the myeloma cell lines expressed VEGFR1 and three of the cell lines expressed VEGFR2. VEGFR1 expression was detected in all and VEGFR2 in all but one of the sorted marrow samples. Increased expression of VEGF by the myeloma cells taken in the context of the suspected prognostic value of marrow angiogenesis suggests a pathogenetic role for this cytokine and presence of its receptors on myeloma cells points toward an autocrine mechanism. Demonstration of the presence of VEGFR2 in our study provides a potential biological explanation for the preclinical activity observed with VEGFR2 inhibitors.

Expression of pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor (VEGF) in sickle cell retina and choroid.

Pigment epithelium-derived factor (PEDF) has been shown to be an inhibitor of angiogenesis as well as a multipotent neurotrophic factor in the mammalian eye. Changes in PEDF levels have been correlated with development of retinal neovascularization in oxygen-induced retinopathy. The purpose of this study was to determine the localization and relative level of PEDF in human retinas and choroids using immunohistochemistry and evaluate the changes in PEDF and vascular endothelial growth factor (VEGF) localization and their relation to the progression of proliferative sickle cell retinopathy. Cryopreserved tissues from eyes of normal subjects and subjects with non-proliferative or proliferative sickle cell retinopathy were used with streptavidin peroxidase immunohistochemistry. A rabbit polyclonal antibody was made against recombinant human PEDF. Binding of the antibody was blocked by preincubation of the antibody with excess human recombinant PEDF. Relative levels of immunoreactivity were scored with a seven-point grading system and by microdensitometric analysis.The most prominent sites of PEDF localization in the normal eye were the vitreous condensed at the internal limiting membrane and RPE-Bruch's membrane-choriocapillaris complex. PEDF was also prominent in choroidal stroma. There was limited immunoreactivity in some cells of the neural retinas, in blood vessels and in the interphotoreceptor matrix (IPM). There was no difference in ratio (1.47 vs. 1.44) of PEDF/VEGF or the relative levels of either growth factor in the retinal vasculatures of the control subjects and perfused area of non-proliferative sickle cell retinas. The ratio was increased in the non-perfused area of the non-proliferative sickle cell retinas (2.24). In eyes with proliferative sickle cell retinopathy, elevated PEDF and VEGF immunostaining was present in viable vessels of sea fan neovascular formations as well as feeder vessels of sea fans. The PEDF/VEGF ratio in sea fans was 1.0. Immunoreactivity for PEDF was prominent in retinal vessels in non-perfused regions and in atrophic sea fans, while VEGF immunoreactivity was weak or absent in these structures. In conclusion, PEDF and VEGF were both significantly elevated in viable sea fan formations in sickle cell disease (p<0.05) but only PEDF was present in non-viable sea fans. The highest levels of PEDF in all eyes were associated with extracellular matrices (vitreous, choroidal stroma, IPM, and walls of blood vessels). PEDF might play an important role in inhibiting angiogenesis and inducing the regression of sea fans. Progression of angiogenesis may be dependent on the ratio of PEDF/VEGF.