Angiogenesis-related protein expression in bevacizumab-treated metastatic colorectal cancer: NOTCH1 detrimental to overall survival.

BACKGROUND: The development of targeted therapies has undoubtedly broadened therapeutic options for patients with colorectal cancer (CRC). The use of bevacizumab to reduce angiogenesis has been associated with improved clinical outcomes. However, an urgent need for prognostic/predictive biomarkers for anti-angiogenic therapies still exists. METHODS: Clinical data of 105 CRC patients treated with bevacizumab in conjunction with chemotherapy were analyzed. The expression of vascular endothelial growth factor (VEGF) receptors, NOTCH1 receptor and its ligand DLL4 were determined by immunohistochemistry. Tumor samples were arranged on a tissue microarray. The association between protein expression and clinicopathological characteristics and outcomes was determined. RESULTS: Bevacizumab was administered as a first-line of treatment in 70.5 % of our cases. The median progression-free survival (PFS) was 10.2 months. The median overall survival (OS) of the total cohort was 24.4 months. Bevacizumab, as the first-line of treatment, and the presence of liver metastasis were independently associated with objective response rate. Membrane VEGFR1 and VEGFR3 expressions were associated with the presence of lung metastasis; interestingly, VEGFR3 was associated with less liver metastasis. NOTCH1 expression was associated with lymph node metastasis. There was a trend toward association between improved PFS and lower NOTCH1 expression (p = 0.06). Improved OS was significantly associated with lower NOTCH1 expression (p = 0.01). In a multivariate analysis, ECOG (Eastern Cooperative Oncology Group) performance status, liver metastasis, histological grade, and NOTCH1 expression were independently associated with OS. CONCLUSION: Our findings illustrated the expression profile of angiogenesis-related proteins and their association with clinicopathological characteristics and outcomes. NOTCH1 expression is a detrimental prognostic factor in metastatic CRC patients treated with chemotherapy plus bevacizumab.

VEGFR1 and VEGFR2 in lip carcinogenesis and its association with microvessel density.

The aim of the present study was to determine the role of vascular endothelial growth factor receptors (VEGFR1 and VEGFR2) in lip carcinogenesis, to investigate correlations between these markers with microvessel density (MVD) and clinicopathological aspects. Medical records from 27 cases of actinic cheilitis (AC) and 46 cases of lower lip squamous cell carcinoma (LLSCC) were analysed and submitted to immunohistochemistry. VEGFR1- and VEGFR2-immunostained sections were analysed based on percentage of positive epithelial and inflammatory cells, while CD31 was submitted to quantitative analysis to determine MVD. Different patterns of VGFR1 and VEGFR2 expression were observed between AC and LLSCC. VEGFR1 expression in epithelial and inflammatory cells and VEGFR2 expression in epithelial cells were higher in AC compared to LLSCC (p < 0.05). VEGFR1 expression in epithelial cells was higher in LLSCC compared to AC (p < 0.001). Expression of both receptors was not associated to MVD or clinicopathological aspects. A direct correlation was found between epithelial VEGFR1 and VEGFR2 expression (p = 0.02) and between VEGFR2 epithelial and inflammatory expression (p < 0.001). Our findings indicate that activation of VEGFR1 and VEGFR2 in epithelial and inflammatory cells appears to be an early event in lip carcinogenesis.

Role of VEGF-A and its receptors in sporadic and MEN2-associated pheochromocytoma.

Pheochromocytoma (PHEO), a rare catecholamine producing tumor arising from the chromaffin cells, may occurs sporadically (76%-80%) or as part of inherited syndromes (20%-24%). Angiogenesis is a fundamental step in tumor proliferation and vascular endothelial growth factor (VEGF-A) is the most well-characterized angiogenic factor. The role of angiogenic markers in PHEO is not fully understood; investigations were therefore made to evaluate the expression of VEGF-A and its receptors in PHEO and correlate to clinical parameters. Twenty-nine samples of PHEO were evaluated for VEGF-A, VEGF receptor-1 (VEGFR-1) VEGFR-2 expression and microvessel density (MVD) by immunohistochemistry. Clinical data were reviewed in medical records. The mean age of patients was 38±14 years, and 69% were woman. VEGF-A, VEGFR-1 and VEGFR-2 staining were detected in nearly all PHEO samples. No significant correlation was observed between VEGF-A, VEGFR-1, VEGFR-2 expression or MVD and age at diagnosis, tumor size or sporadic and hereditary PHEO. However, the levels of expression of these molecules were significantly higher in malignant PHEO samples (p=0.027, p=0.003 and p=0.026, respectively).VEGF-A and its receptors were shown to be up-regulated in malignant PHEO, suggesting that these molecules might be considered as therapeutic targets for unresectable or metastatic tumors.

Vimentin is synthesized by mouse vascular trophoblast giant cells from embryonic day 7.5 onwards and is a characteristic factor of these cells.

INTRODUCTION: A few days after implantation, the embryo grows intensely and trophoblast giant cells (TGC) undergo cell rearrangement, especially of their cytoskeleton. Although we previously showed vimentin in mouse antimesometrial TGC at embryonic days (E) 8.5-10.5, by immunostaining, we did not demonstrate what is the first embryonic day that TGC synthesize vimentin and whether mouse chorioallantoic placental TGC express vimentin. This is of particular interest because cytokeratin is a marker for TGC in the placenta. METHODS: We performed in situ hybridization and immunolocalization, combined with histological and stereological techniques, to study vimentin expression between E6.5 and E12.5 and we investigated Vegf and Flt1/Vegfr1 expression in TGC. RESULTS: Analyses of morphologic parameters of TGC showed that the highest expansion of nuclear and cytoplasmic volumes (p ≤ 0.05) occurred at E7.5. We detected vimentin expression in TGC from E7.5 onwards; vimentin disappeared as TGC degeneration advanced. Primary and secondary TGC showed intense positive immunostaining for vimentin, Vegf and Flt1/Vegfr1 at E7.5. In the chorioallantoic placenta, parietal TGC (zone of giant cells), spiral artery-associated TGC, maternal blood canal-associated TGC and TGC within the sinusoidal spaces of the labyrinth exhibited an intense immunopositive-reaction for vimentin. DISCUSSION: At E7.5 TGC acquire vimentin, Vegf and Flt1/Vegfr1; at the same time, blood begins to drain from maternal vessels. Vimentin synthesis initiates during a differentiation process of TGC and continues throughout the stage of vascular TGC. CONCLUSIONS: We propose that vimentin is a characteristic factor of specialized (vascular) TGC, being a valuable tool for studying pathological pregnancies associated with defects in vascular trophoblasts in mice.

Upregulation of the vascular endothelial growth factor, Flt-1, in rat hippocampal neurons after envenoming by Phoneutria nigriventer; age-related modulation.

This study characterizes the distribution and quantifies the expression of the tyrosine kinase receptor for the vascular endothelial growth factor (VEGF), Flt-1, in the rat hippocampus following intra-peritoneal injection of Phoneutria nigriventer venom (PNV). Post-natal day 14 (P14) and 8-10 weeks (adult) old rats were used and analyses were done at 1, 2, 5 and 24 h after venom exposure and compared with saline-injected counterparts. PNV-injected animals showed hippocampal venules with perivascular edema indicating blood-brain barrier (BBB) dysfunction. This was accompanied by significant overexpression of Flt-1 which though was not the same for CA1, CA2, CA3 and dentate gyrus (DG) hippocampal regions, neither for P14 and adult rats. Regional analysis using GIMP methodology showed that Flt-1 was constitutively distributed more densely in neurons of DG, followed by CA1/CA2 and CA3 of both control P14 and adult animals, without variation over time, but significantly more expressed in P14 than in adults. A time-course analysis showed that Flt-1 upregulation was progressive and that neurons VEGFR1/Flt-1+ of PNV-exposed animals are timely and regionally modulated depending on the hippocampal region, being CA2 the least responsive region regardless animal's age, whilst DG was the most susceptible with adult animals having higher upregulation than neonates. Since VEGF has been reported to confer protection in several pathological processes we suggest that VEGF may be involved in hippocampal neurons response via Flt-1 mediation following PNV envenoming; its higher upregulation in adult envenomed rats may be an indication that Flt-1 neuroprotective mediation is more efficient with age. The Flt-1 upregulation and the incidence of perivascular edema in young animals may indicate a pro-inflammatory role of the receptor.

Vascular endothelial growth factor (VEGF) and VEGF-receptor expression in placenta of hyperglycemic pregnant women.

Hyperglycemia occurs in a variety of conditions such as overt diabetes, gestational diabetes and mild hyperglycemia, all of which are generally defined based on the oral glucose tolerance test and glucose profiles. Whereas diabetes has received considerable attention in recent decades, few studies have examined the mechanisms of mild hyperglycemia and its associated disturbances. Mild gestational hyperglycemia is associated with macrosomia and a high risk of perinatal mortality. Morphologically, the placenta of these women is characterized by an increase in the number of terminal villi and capillaries, presumably as part of a compensatory mechanism to maintain homeostasis at the maternal-fetal interface. In this study, we analised the expression of VEGF and its receptors VEGFR-1 (Flt-1) and VEGFR-2 (KDR) in placentas from mildly hyperglycemic women. This expression was compared with that of normoglycemic women and women with gestational and overt diabetes. Immunohistochemistry revealed strong staining for VEGF and VEGFR-2 in vascular and trophoblastic cells of mildly hyperglycemic women, whereas the staining for VEGFR-1 was discrete and limited to the trophoblast. The pattern of VEGF and VEGF-receptor reactivity in placentas from women with overt diabetes was similar to that of normoglycemic women. In women with gestational diabetes, strong staining for VEGFR-1 was observed in vascular and trophoblastic cells whereas VEGF and VEGFR-2 were detected only in the trophoblast. The expression of these proteins was confirmed by western blotting, which revealed the presence of an additional band of 75 kDa. In the decidual compartment, only extravillous trophoblast reacted with all antibodies. Morphological analysis revealed collagen deposition around large arteries in all groups with altered glycemia. These findings indicate a placental response to altered glycemia that could have important consequences for the fetus. The change in the placental VEGF/VEGFR expression ratio in mild hyperglycemia may favor angiogenesis in placental tissue and could explain the hypercapillarization of villi seen in this gestational disturbance.

Increased expression of vascular endothelial growth factor and its receptors, VEGFR-1 and VEGFR-2, in medullary thyroid carcinoma.

BACKGROUND: Vascular endothelial growth factor (VEGF-A) expression is upregulated in the majority of human tumors, where it stimulates proliferation, migration, and survival of endothelial cells. Studies have suggested that VEGF inhibitors can be used as an alternative therapy in medullary thyroid carcinoma (MTC), but data about expression of VEGF-A and its receptor in this tumor are scarce. The aims of this study were to evaluate VEGF-A, VEGF receptor (VEGFR)-1, VEGFR-2, and microvessel density (MVD) expression in MTC samples and correlate it with clinical parameters. METHODS: Paraffin-embedded samples from 38 MTC patients were evaluated for VEGF-A, VEGFR-1, VEGFR-2, and MVD expression by immunohistochemistry. Clinical data were retrospectively reviewed in medical records. RESULTS: Thirty-eight patients aged 31.8 +/- 17.1 years were enrolled. Twenty-seven patients had hereditary disease (71.1%). Twenty-five of them were found to have multiple endocrine neoplasia (MEN) 2A and two were found to have MEN 2B. VEGF-A immunohistochemical staining was detected in 95% (36/38), VEGFR-1 in 96% (36/37), and VEGFR-2 in 91% (31/34) of MTC samples. Age at surgery was positively correlated with VEGFR-2 (p = 0.003). There was no correlation between VEGF-A, VEGFR-2, and tumor stage (tumor node metastasis). Nevertheless, VEGFR-1 was found to be inversely correlated with tumor node metastasis (p = 0.034). We also observed a trend toward an association between VEGFR-1 signal intensity and cure of disease, although this did not reach statistical significance (p = 0.054). Neither VEGF-A nor VEGFR-2 was associated with disease outcome after a median follow-up period of 5 years (p = 0.882 and p = 0.236, respectively). As expected, MVD was correlated with age at surgery (p = 0.005) and tumor size (p = 0.03). Patients with the hereditary form of the disease had a stronger intensity for VEGFR-1 (p = 0.039), whereas patients with sporadic disease displayed higher MVD counts (44 [27-63] vs. 21 [9-49], p = 0.018). CONCLUSION: The VEGF-A, VEGFR-1, and VEGFR-2 immunoreactive proteins are overexpressed in MTC lesions and might be implicated in tumor progression. It is not clear, however, if expression of these molecules provides prognostic information regarding the spread or outcome of MTC.

Multiparity increases trophoblast invasion and vascular endothelial growth factor expression at the maternal-fetal interface in mice.

To analyze immunomodulating effects related to parity status, we studied trophoblast invasion grade, placental expression and systemic concentration of VEGF and its receptor Flt-1 in normal fertile (CBA/JxBALB/c) mice and abortion-prone (CBA/JxDBA/2) H-2(d)xH-2(k) mice. BALB/c or DBA/2 mated CBA/J females were, respectively, divided into the following groups: primiparous young (3.0+/-0.5 months old); primiparous old (8.5+/-0.5 months old) and multiparous old (8.5+/-0.5 months old, with 4 pregnancies). Immunohistochemical analysis of term placentae from both multiparous groups revealed various layers of invasive trophoblast tissue, identified as cytokeratin+/vimentin- cells, in contrast to the single layer detected in the placentae of primiparous animals, indicating that multiparity increases trophoblast invasion regardless of the success of the pregnancy outcome. Invasive trophoblast tissue from primiparous CBA/JxDBA/2 placentae showed diminished VEGF expression in comparison with the normal fertile group, while both multiparous groups demonstrated high expression of VEGF in the invasive trophoblast tissue. Placental expression of Flt-1 was similar in all groups. However, the primiparous CBA/JxBALB/c group showed the highest plasma concentration of sFlt-1 at term, while both multiparous groups demonstrated low circulating levels. No differences in circulating VEGF levels were observed among the groups. These results demonstrate an increase in trophoblast invasion tissue and expression of VEGF in the maternal-fetal interface in multiparous mice compared to primiparous mice. Moreover, the placenta appears to be able to regulate the circulating levels of VEGF by releasing sFlt-1.