OBJECTIVE

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) exists in three main splice variants, characterized by <em>121</em>, 165 and 189 amino acids (VEGF <em>121</em>, VEGF 165 and VEGF 189) and acts via two specific receptors: VEGF-R1 or Flt-1 and VEGF-R2 or KDR. VEGF plays an important role in the pathogenesis of diabetic retinopathy. This study examined the relationship between VEGF and its isoforms and the severity of diabetic nephropathy in type 2 diabetes.

METHODS

We evaluated the glomerular gene expression of VEGF and its receptors and studied the relationships with renal functional and structural parameters in type 2 diabetic patients.

METHODS

Glomeruli from 17 kidney biopsies were microdissected; 14 out of 17 biopsies were also subjected to electron microscopic morphometric analysis to estimate glomerular structural parameters. VEGF mRNA was studied by comparative kinetic RT-PCR and real-time RT-PCR in order to identify the three different isoforms and to quantify VEGF, VEGF-R1 and VEGF-R2 mRNA levels.

RESULTS

(i) Glomerular VEGF mRNA levels were inversely related to albumin excretion rate (r=-0.66, P=0.004); (ii) both the degree of mesangial and mesangial matrix expansion were inversely related to VEGF 165 mRNA levels (r=-0.73, P=0.005 and r=-0.64, P=0.017), and directly to VEGF <em>121</em> mRNA levels (r=0.74, P=0.003 and r=0.73, P=0.004); and (iii) VEGF and VEGF-R2 mRNA levels were directly related (r=0.62, P=0.033).

CONCLUSIONS

These findings suggested that quantitative and qualitative changes in VEGF expression are present in type 2 diabetic patients with nephropathy and might be involved in the pathogenesis and progression of diabetic glomerulopathy.

BACKGROUND

Vascular endothelial growth factor (VEGF) is an important endothelial cell mitogen associated with increased angiogenesis and aggressive tumor behavior. Its stimulating effect on endothelial cells basically is dependent on the presence of specific VEGF receptors, such as the flk-1(KDR) receptor. This study investigates the roles of VEGF and of a functionally intact angiogenic pathway, "VEGF/flk-1(KDR)," in patients with endometrial carcinoma and their significance in prognosis and therapy.

METHODS

A series of 121 endometrial carcinomas were studied. The expression of VEGF by endometrial tumor cells was assessed using the monoclonal antibody (MoAb) VG1. VEGF/KDR complexes on tumor endothelium or activated microvessel density (aMVD) were identified using the MoAb 11B5. In addition, the standard microvessel density (sMVD) was assessed with anti-CD31. In all tumors, the alkaline phosphatase/antialkaline phosphatase technique was employed. A Fisher exact test or an unpaired, two-tailed t test was used for testing correlations between categoric tumor variables, whereas a log-rank test was used to determine statistical differences between life tables. A Cox proportional hazards model was used to assess the effect of tumor variables on overall survival.

RESULTS

Cytoplasmic VEGF expression in>> 50% of tumor cells was associated significantly with aMVD (P < 0.0001) and with sMVD (P < 0.003). In univariate survival analysis, VEGF (P = 0.0002), aMVD (P = 0.001), and sMVD (P = 0.0009) were significant prognostic variables. Equally important were the histologic parameters tumor type (P = 0.03), tumor grade (P = 0.003), and disease stage (P < 0.0001). In multivariate analysis, disease stage was the most important independent prognostic factor (P < 0.0001), followed by VEGF/KDR (P < 0.01), and VEGF (P < 0.04). Furthermore, VEGF and VEGF/KDR were the only independent prognostic variables for patients with Stage I endometrioid adenocarcinoma.

CONCLUSIONS

sMVD and the angiogenic factor VEGF are important indicators of a poor prognosis in patients with endometrial carcinoma. VEGF/KDR complexes define a subgroup of patients with endometrial carcinoma with an even worse prognosis.

Stanniocalcin-1 (STC-1) is a glycoprotein hormone originally identified as a regulator of calcium and phosphate homeostasis in bony fish. Up-regulation of the mammalian homolog in numerous gene profiling studies of angiogenesis and <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em>-A (VEGF-A(165))-regulated gene expression, suggests that regulation of this <em>factor</em> may be a key feature of the angiogenic response. Here we investigated the mechanisms mediating VEGF-A(165)-induced STC-1 gene expression in human <em>endothelial</em> cells. VEGF-A(165), acting via VEGFR2/KDR, induced STC-1 through de novo transcription, mediated primarily via intracellular protein kinase C (PKC)- and extracellular signal-regulated protein kinase (ERK)-dependent pathways. VEGF-A(165)-induced STC-1 mRNA expression was synergistically enhanced up to 2-fold by co-treatment with FGF-2, in a mechanism dependent on VEGFR2/KDR and FGFR1. Production of STC-1 protein by <em>endothelial</em> cells was also induced by VEGF-A(165) and synergistically enhanced by co-treatment with FGF-2. Synergism between VEGF-A(165) and FGF-2 was mediated via a novel neuropilin-1 (NP-1)-dependent mechanism, as indicated by the complete inhibition of synergism with either EG3287, a specific neuropilin antagonist, or siRNA-mediated NP-1 knockdown, and by the inability of the VEGF-A(<em>121</em>) isoform to synergise with FGF-2. Surprisingly, we found that NP-1 knockdown also markedly reduced KDR expression in HUVECs, and enhanced the VEGF-A(165)-induced reduction in KDR expression resulting from receptor-mediated endocytosis. These findings support a role for NP-1 in mediating synergistic effects between VEGF-A(165) and FGF-2, which may occur in part through a contribution of NP-1 to KDR stability.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em>-A (VEGF-A) is an important mediator of angiogenesis in normal and neoplastic tissues. Total VEGF-A levels have been associated with melanoma progression, but the relative contributions of each isoform is unknown. To determine whether differences in the production of any or all of the major VEGF-A isoforms are related to stage of progression, we compared message levels for the three major isoforms of VEGF in melanoma specimens from different stages of progression.Primary melanomas (N = 18), primary recurrences (N = 5), regional dermal metastases (N = 11), nodal metastases (N = 12), normal lymph nodes (N = 18), and distant metastases (N = 9) were prospectively collected. Samples from the horizontal and vertical <em>growth</em> phases of primary tumors were also collected from five additional patients. Message levels for the three major VEGF-A isoforms were measured using real-time quantitative reverse-transcriptase polymerase chain reaction and normalized to beta-actin mRNA levels. There was a marked increase in the expression of all three VEGF-A isoforms from the vertical <em>growth</em> phase tissue as compared with the horizontal <em>growth</em> phase tissue. Primary tumors, local recurrences, regional dermal metastases, nodal metastases, and distant metastases all produced more VEGF(<em>121</em>) and VEGF(165) than negative nodes. Nodal metastases produced the highest level of these two isoforms, higher even than distant metastases. There was no significant difference in VEGF(189) message among the groups. Melanomas in the vertical <em>growth</em> phase produce more VEGF-A (all isoforms) than in the horizontal <em>growth</em> phase. Nodal metastases produce the highest levels of VEGF(<em>121</em>) and VEGF(165), but not VEGF(189) as compared with other stages of progression. These data suggest that the soluble forms of VEGF-A might be an important <em>factor</em> in melanoma metastasis to regional lymph nodes.

Cancer, with more than 10 million new cases a year worldwide, is the third leading cause of death in developed countries. One critical requirement during cancer progression is angiogenesis, the formation of new blood vessels. Structural and functional imaging of tumor vasculature has been studied using various imaging modalities such as magnetic resonance imaging (MRI), computed tomography (CT), and ultrasound. Molecular imaging, a key component of the 21st-century cancer-patient management strategy, takes advantage of these traditional imaging techniques and introduces molecular probes to determine the expression of indicative molecular markers at different stages of cancer development. In this chapter, we will focus on two tumor vasculature-related targets: integrin alpha(v)beta(3) and <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> receptor (VEGFR). For imaging of integrin alpha(v)beta(3) on the tumor vasculature, only nanoparticle-based probes will be discussed. VEGFR imaging will be discussed in depth, and we will give a detailed example of positron emission tomography (PET) imaging of VEGFR expression using radio-labeled VEGF(<em>121</em>) protein. Future clinical translation will be critical for maximum patient benefit from these agents. To achieve this goal, multidisciplinary approaches and cooperative efforts from many individuals, institutions, industries, and organizations are needed to quickly translate multimodality tumor vasculature imaging into multiple facets of cancer patient management.

Glomerular podocytes are major determinants of filtration permselectivity in the glomerulus. Although the molecular mechanisms determining the characteristics of the glomerular filtration unit are incompletely understood, <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) has been implicated. To analyze this process in situ, we established a method that allows exploration of in vivo mRNA expression of podocytes using single-cell reverse transcriptase-polymerase chain reaction (RT-PCR). Microdissected mouse glomeruli were held in a patch-clamp apparatus, and single podocytes were harvested by aspiration. After lysis, the cells were reverse transcribed, and PCR was performed (45 cycles). The podocyte nature of the material was confirmed by detection of podocyte-specific mRNA (glomerular epithelial protein 1 and Wilms' tumor protein 1). Using specific oligonucleotide primers, VEGF was detected in mRNA obtained from renal cortex, single microdissected glomeruli, cultured murine podocytes, and single podocytes in situ. All cells examined expressed three VEGF isoforms (<em>121</em>, 165, and 189). These differ in their capacity for binding to extracellular matrix and could have different potencies regulating glomerular <em>endothelial</em> permeability. Our approach should allow a semiquantitative, isoform-specific evaluation of VEGF mRNA expression in podocytes during nephrogenesis and in glomerular disease.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF or <em>vascular</em> permeability <em>factor</em>) is an important angiogenic <em>factor</em> that is up-regulated in numerous benign and malignant disorders, including angiosarcoma, hemangiomas, and solid tumors. To determine the functional role of VEGF in the development of <em>endothelial</em> tumors, we expressed primate VEGF <em>121</em> in an <em>endothelial</em> cell line, MS1, derived from primary murine cells by immortalization with a temperature-sensitive SV40 large T antigen. This cell line expresses the VEGFR-2 (Flk-1/Kdr) receptor for VEGF. Expression of VEGF <em>121</em> led to the development of slowly <em>growing</em> <em>endothelial</em> tumors, which were histologically well-differentiated angiosarcomas. The angiosarcomas generated from MS1 VEGF cells demonstrated up-regulation of the VEGF receptors VEGFR-2 and VEGFR-1 (Flt-1) in vivo compared with benign hemangiomas generated from MS1 cells. Treatment of these cells with the VEGFR-2 tyrosine kinase inhibitor SU 1498 led to decreased expression of ets-1, a transcription <em>factor</em> which has been shown to be stimulated by VEGF. These results suggest that high level expression of VEGF in <em>endothelial</em> cells may result in malignant transformation. This transformation process likely involves both autocrine and paracrine pathways.

To explore the possibility of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) receptor scintigraphy of primary tumours and their metastases, we analysed the binding properties of (123)I-labelled VEGF(165) ((123)I-VEGF(165)) and (123)I-VEGF(<em>121</em>) to human umbilical vein <em>endothelial</em> cells (HUVECs), several human tumour cell lines (HMC-1, A431, KU812, U937, HEP-1, HEP-G2, HEP-3B and Raji), a variety of primary human tumours (n = 40) and some adjacent non-neoplastic tissues as well as normal human peripheral blood cells in vitro. Two classes of high-affinity (123)I-VEGF(165)-binding site were found on the cell surface of HUVECs. In contrast, one class of high-affinity binding sites for (123)I-VEGF(165) was found on HMC-1, A431, HEP-1, HEP-G2, HEP-3B and U937 cells as well as many primary tumours. For (123)I-VEGF(<em>121</em>), a single class of high-affinity binding site was found on certain cell lines (HUVEC, HEP-1 and HMC-1) and distinct primary tumours (primary melanomas, ductal breast cancers and ovarian carcinomas as well as meningiomas). Tumour cells expressed significantly higher numbers of VEGF receptors compared with normal peripheral blood cells and adjacent non-neoplastic tissues. Immunohistochemical staining revealed that the VEGF receptor Flk-1 is expressed to a much higher extent within malignant tissues compared with neighbouring non-neoplastic cells. We observed significantly greater specific binding of (123)I-VEGF(165) and (123)I-VEGF(<em>121</em>) to a variety of human tumour cells/tissues compared with the corresponding normal tissues or normal peripheral blood cells. In comparison with (123)I-VEGF(<em>121</em>), (123)I-VEGF(165) bound to a higher number of different tumour cell types with a higher capacity. Thus, (123)I-VEGF(165) may be a potentially useful tracer for in vivo imaging of solid tumours.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) mediates increased <em>vascular</em> permeability and <em>endothelial</em> mitogenesis, and may orchestrate normal glomerular permselectivity and proteinuria. Distinct isoforms result from differential gene splicing. VEGF binds to two cell surface tyrosine-kinase receptors, KDR (kinase domain region) and Flt-1 (fms-like tyrosine kinase-1). The latter also exists in a soluble form (sFlt), which is inhibitory. We have studied patterns of VEGF-isoform and VEGF-receptor expression in isolated single normal human glomeruli. mRNA from 190 glomeruli (from 20 individuals) was harvested on to magnetic beads, and nested reverse transcription-PCR was performed using primers for the VEGF isoforms and VEGF receptors. Simultaneous nested reverse transcription-PCR for CD45 was conducted in order to exclude leucocyte contamination. Unexpected products were isolated, cloned and sequenced. Multiple patterns of glomerular VEGF mRNA isoform expression were identified. Most frequently (58%), all three common forms were expressed. VEGF(189) (i.e. 189-amino-acid form of VEGF) was expressed in 63%, VEGF(165) in 85% and VEGF(<em>121</em>) in 84% of glomeruli. Two unexpected PCR products were also identified: 18% of glomeruli expressed VEGF(145), and 27% of glomeruli expressed a new truncated VEGF splice variant, VEGF(148), lacking exon 6, the terminal part of exon 7 and exon 8. Multiple patterns of VEGF-receptor expression were also identified, the most common being expression of all three isoforms (28%). Overall, KDR was seen in 59% of glomeruli, Flt-1 in 45% and sFlt in 57%. Thus the expression of VEGF within normal glomeruli is complex and variable, with inter- and intra-individual variation. Furthermore, sFlt appears to be the co-dominant form of VEGF receptor expressed within glomeruli, suggesting that, in healthy individuals, a degree of VEGF autoregulation is the norm. The physiological importance of VEGF(148) remains to be confirmed.

OBJECTIVE

To investigate the prognostic value of vascular endothelial growth factor messenger RNA (VEGF mRNA) in the peripheral blood (PB) of patients with hepatocellular carcinoma (HCC) undergoing curative resection.

METHODS

Using a reverse-transcription polymerase chain reaction (RT-PCR)-based assay, VEGF mRNA in the PB was determined prospectively in 50 controls and in 50 consecutive patients undergoing curative resection for HCC.

RESULTS

Among the isoforms of VEGF mRNA, VEGF(165) and VEGF(121) were expressed. By multivariate analysis, a higher level of VEGF(165) in preoperative PB correlated with a risk of HCC recurrence with borderline significance (P=0.050) and significantly with recurrence-related mortality (P=0.048); while VEGF(121) did not. Other significant predictors of HCC recurrence included cellular dedifferentiation (P=0.033), an absent or incomplete capsule (P=0.020), vascular permeation (P=0.018), and daughter nodules (P=0.006). The other significant parameter of recurrence related mortality was cellular dedifferentiation (P=0.053). The level of circulating VEGF mRNA, however, did not significantly correlate with tumor size, cellular differentiation, capsule, daughter nodules, vascular permeation, necrosis and hemorrhage of tumors.

CONCLUSIONS

The preoperative level of circulating VEGF mRNA, especially isoform VEGF(165), plays a significant role in the prediction of postoperative recurrence of HCC.

The development of ovarian follicles and subsequent corpus luteum formation is accompanied by very active angiogenesis. Ovarian granulosa cells produce <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF), which is a potent <em>endothelial</em> cell mitogen and an angiogenic agent. The complementary DNAs of two other <em>factors</em> structurally related to VEGF, namely VEGF-B and VEGF-C, were recently cloned, but little is known of their regulation in the ovary. We first studied the expression of the messenger RNAs (mRNAs) of the three VEGF isotypes in freshly isolated human granulosa-luteal (GL) cells obtained at oocyte retrieval for in vitro fertilization. The hormonal regulation of these mRNAs was subsequently studied in primary cultures of human GL cells. Analysis of cultured GL cell RNA by reverse transcription-PCR revealed that these cells express the alternatively spliced transcripts representing <em>121</em>-, 145-, and 165-amino acid VEGF isoforms. Northern blot hybridization analyses indicated that transcripts of 4.5 and 3.7 kilobases for VEGF, and 1.4 and 2.4 kilobases for VEGF-B and VEGF-C, respectively, are expressed in human GL cells. The basal VEGF mRNA levels declined steadily, whereas VEGF-B mRNA levels were rather invariant over a 10-day culture period of GL cells. In contrast, VEGF-C mRNA levels increased toward the end of culture. For studying the hormonal regulation of VEGF isotype mRNAs, GL cells were treated with hCG, recombinant human FSH, PGE2, as well as 8-bromo-cAMP and 12-O-tetradecanoylphorbol 13-acetate, which activate protein kinase A- and protein kinase C-dependent signaling pathways, respectively. All test agents stimulated the expression of VEGF mRNA levels in a concentration-dependent manner. Time-course studies indicated that all treatments induced VEGF mRNA levels as early as incubation for 2 h, and the effect was sustained up to 48 h. VEGF-B mRNA levels were not regulated by any of the test agents. However, we found that hCG and 8-bromo-cAMP decreased VEGF-C mRNA levels with a maximal response observed at 24 and 48 h after cellular treatment. We conclude that the mRNAs of VEGF, VEGF-B, and VEGF-C are expressed in human GL cells and that their mRNA steady state levels are regulated in cultured human GL cells in an isotype-specific manner. The differential regulation of VEGF, VEGF-B, and VEGF-C in human GL cells suggests that distinct VEGF isotypes may play different roles during the <em>vascular</em>ization of the human ovarian follicle and corpus luteum.

Neo<em>vascular</em> eye diseases such as wet age-related macular degeneration and proliferative diabetic retinopathy are two of the most common causes of irreversible visual loss. Although mediated by <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF), the mechanisms of these diseases are not fully understood. Molecular inhibitors of VEGF including pegaptanib, ranibizumab and bevacizumab are used as treatments for these diseases. However, there have been very few direct comparisons between these agents, and as dose and treatment regimes differ their relative efficacies are hard to determine. In vitro comparisons tend to use cells from different sites or species, which show heterogeneity in their responses. The aim of this study was to compare the characteristics of primary cultures of isolated human choroidal <em>endothelial</em> cells (hCEC) and retinal <em>endothelial</em> cells (hREC), and their proliferation responses to stimulation with VEGF <em>121</em> and 165, and to compare the anti-proliferative effects of these three drugs. hCEC and hREC were positive for the cell markers VEGFR1, VEGFR2, CD31, CD34 and von Willebrand's <em>factor</em> (vWF), with greater expression of CD34 on the hREC compared to hCEC. Contrary to previous assumptions VEGF isoforms <em>121</em> and 165 were found to be equally potent in stimulating <em>endothelial</em> cell proliferation. However, hREC exhibited higher proliferation with either VEGF isoform compared to hCEC. The anti-VEGF treatments ranibizumab and bevacizumab were effective in decreasing proliferation of hCEC induced by the two VEGF isoforms, individually and in combination, with ranibizumab being moderately more effective, particularly in hREC. Pegaptanib was effective in controlling the proliferation of hCEC stimulated by VEGF 165, but was ineffective against the stimulatory effect of VEGF <em>121</em>. There were found to be significant differences in micro<em>vascular</em> <em>endothelial</em> cells from the retina and choroid, both in the expression of cell markers and their behaviour in response to <em>growth</em> <em>factors</em> and currently available anti-VEGF agents, highlighting the importance of targeting treatments to specific intraocular <em>vascular</em> beds and/or diseases.

OBJECTIVE

To test the feasibility of myocardial angiogenic gene expression using a novel catheter-based transendocardial injection system.

BACKGROUND

Angiogenesis has been induced by direct injection of growth factors into ischemic myocardium during open-heart surgery. Catheter-based transendocardial injection of angiogenic factors may provide equivalent benefit without need of surgery.

METHODS

A new guidance system for intramyocardial therapy utilizes magnetic fields and catheter-tip sensors to locate a position in space and reconstruct three-dimensional left ventricular (LV) electromechanical maps without using fluoroscopy. A retractable 27G needle was coupled with the guidance system for LV transendocardial injection. In 12 pigs, the catheter was used to inject 0.1 ml of methylene-blue (MB) dye and 8 pigs had myocardial injections of adenoviral vector (1 x 10(10) particles per site) containing the LacZ transgene. Ten pigs underwent catheter-based transendocardial injection and six pigs were injected using transepicardial approach with the gene encoding adenovirus <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em>-<em>121</em> (Ad.VEGF<em>121</em>; 1 x 10(10) viral particles x 6 sites) and sacrificed at 24 h. Injection sites were identified with ultraviolet light by coinjection of fluorescent beads.

RESULTS

Overall, 138 of 152 attempted injection MB tracks (91%) were found after sacrifice. Tissue staining was 7.1+/-2.1 mm in depth and 2.3+/-1.8 mm in width. No animal had pericardial effusion or tamponade. In Ad.LacZ injected animals, gross pathology showed positive staining in injected zones, and histology confirmed positive myocyte staining. Adenovirus <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em>-<em>121</em> injected sites showed high levels of VEGF<em>121</em> production that was of similar magnitude whether injected using the transendocardial (880.4+/-412.2 pg VEGF<em>121</em>/mg protein) or transepicardial (838.3+/-270 pg VEGF<em>121</em>/mg protein) delivery approach (p = 0.62).

CONCLUSIONS

Using this magnetic guidance catheter-based navigational system, transgenes can effectively be transfected into designated myocardial sites. Thus, if it is determined that direct intramyocardial injection of angiogenic factors enhances collateral function in patients, this less invasive catheter-based system offers a similar gene delivery efficiency and, thus, may have clear advantages compared with the surgically-based transepicardial injection approach.

<AbstractText>To assess change in retinal nonperfusion (NP) after anti-<em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) therapy for diabetic macular edema (DME) using 2 different imaging modalities: swept-source widefield (SS-WF) OCT angiography (OCTA) and ultra-widefield (UWF) fluorescein angiography (FA).</AbstractText><AbstractText>Observational case series.</AbstractText><AbstractText>Ten eyes of 9 patients with severe nonproliferative diabetic retinopathy (NPDR) or proliferative DR (PDR) initiating 3 monthly anti-VEGF intravitreal injections for DME.</AbstractText><AbstractText>All eyes were imaged with UWF color fundus photographs (CFPs), UWF FA, and SS-WF OCTA at baseline (M0) and 1 month after the third anti-VEGF injection (M3). All images were aligned and divided into 16 boxes for analysis of NP areas by 2 blinded retina specialists.</AbstractText><AbstractText>The number of discrepancies between SS-WF OCTA and UWF FA regarding the detection of NP areas and small vessels passing through NP areas; assessment of DR severity on UWF CFP; and change in each NP area between M0 and M3: number of boxes/eye with presence of at least 1 NP area, number of arterioles or venules that disappeared or reappeared, and number of NP areas in which capillaries disappeared or reappeared.</AbstractText><AbstractText>The diabetic retinopathy (DR) severity score improved by at least 1 stage in 8 of 10 eyes, with a significant decrease in the mean number of microaneurysms and retinal hemorrhages on UWF CFP at M3 versus M0 (n = 40±28 vs. <em>121</em>±57; P = 0.0020) and regression of fundus neo<em>vascular</em>ization when present. All NP areas detected on FA were seen on SS-WF OCTA, but additional NP areas were detected on SS-WF OCTA at M0 in 29% (46/160) of boxes. No reperfusion of arterioles or venules was observed at M3 on FA or SS-WF OCTA. Retinal capillaries were only visible on OCTA, and no reperfusion in NP areas was observed even when a reduction in dark areas was visible on FA.</AbstractText><AbstractText>No reperfusion of vessels or capillary network was detected in NP areas using 2 imaging techniques, UWF FA and SS-WF OCTA, in eyes with DR after 3 anti-VEGF injections. The detection rate of NP areas was higher with SS-WF OCTA than with UWF FA.</AbstractText>

OBJECTIVE

To compare the expression of nerve growth factor (NGF) and its high-affinity receptor trkA in normal ovaries and in epithelial ovarian carcinomas. Given NGF acts as an angiogenic factor through a vascular endothelial growth factor (VEGF)-mediated mechanism in several types of tissues, we examined whether NGF regulates the expression of VEGF isoforms in epithelial ovarian cancer (EOC).

METHODS

The expression and localization of NGF and tyrosine kinase receptor A (trkA) in normal ovarian samples and in ovarian cancer samples were analyzed by RT-PCR and immunohistochemistry. NGF regulates the expression of three VEGF isoforms (VEGF(121), VEGF(165) and VEGF(189)); these were examined using RT-PCR in explants of EOC and ELISA in culture media.

RESULTS

TrkA mRNA levels were over-expressed in ovarian cancer compared to normal ovarian samples, whereas NGF mRNA levels remained unchanged. NGF and trkA proteins were absent or found in very low levels in normal ovarian surface epithelium (OSE), whereas they were highly expressed in epithelial cells of EOC. Additionally, NGF stimulated the expression of VEGF isoforms in cancer explants. The effect was dose-dependent and inhibited by a NGF antibody and by K(252a), a trk receptor inhibitor.

CONCLUSIONS

The abundance of NGF and trkA receptors in epithelial cells of EOC, together with the ability of NGF to increase VEGF expression strongly suggests an autocrine role of NGF in EOC. These findings suggest that blocking neurotrophin action could be a therapeutic target in treating ovarian cancer.

The acute increase in <em>vascular</em> permeability produced by <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF-A(165)) requires activation of <em>endothelial</em> Flk-1 receptors (VEGFR-2) and stimulation of platelet-activating <em>factor</em> (PAF) synthesis. Like PAF, VEGF-A(165) promotes translocation of P-selectin to the <em>endothelial</em> cell (EC) surface. However, the mechanisms involved remain unknown. By treating human umbilical vein <em>endothelial</em> cells (HUVECs) with VEGF analogs, we show that activation of VEGFR-1 or VEGFR-2 or both induced a rapid and transient translocation of <em>endothelial</em> P-selectin and neutrophil adhesion to activated ECs. The effects mediated by VEGF-A(165) and VEGF-A(<em>121</em>) (VEGFR-1/VEGFR-2 agonists) were blocked by a selective VEGFR-2 inhibitor, SU1498. VEGF-A(165) was twice as potent as VEGF-A(<em>121</em>), which can be explained by the binding capacity of VEGF-A(165) to its coreceptor neuropilin-1 (NRP-1). Indeed, treatment with NRP-1 antagonist (GST-Ex7) reduced the effect of VEGF-A(165) to the levels observed upon stimulation with VEGF-A(<em>121</em>). Finally, the use of selective PAF receptor antagonists reduced VEGF-A(165)-mediated P-selectin translocation. Together, these data show that maximal P-selectin translocation and subsequent neutrophil adhesion was mediated by VEGF-A(165) on the activation of VEGFR-2/NRP-1 complex and required PAF synthesis.

BACKGROUND

Administration of angiogenic <em>factors</em> stimulates neo<em>vascular</em>ization in ischemic tissues. However, there is no evidence that angiogenesis can be induced in normoperfused skeletal muscles. We tested the hypothesis that adenovirus-mediated intramuscular (IM) gene transfer of the <em>121</em>-amino-acid form of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (AdCMV.VEGF(<em>121</em>)) could stimulate neo<em>vascular</em>ization in nonischemic skeletal muscle and consequently attenuate the hemodynamic deficit secondary to surgically induced ischemia.

RESULTS

Rabbits and rats received IM injections of AdCMV.VEGF(<em>121</em>), AdCMV.Null, or saline in the thigh, 4 weeks (rabbits) or 2 weeks (rats) before femoral artery removal in the injected limb. In unoperated rats, at the site of injection of AdCMV.VEGF(<em>121</em>), we found 96% and 29% increases in length density of arterioles and capillaries, respectively. Increased tissue perfusion (TP) to the ischemic limb in the AdCMV.VEGF(<em>121</em>) group was documented, as early as day 1 after surgery, by improved blood flow to the ischemic gastrocnemius muscle measured by radioactive microspheres (AdCMV.VEGF(<em>121</em>)=5.69+/-0.40, AdCMV.Null=2.97+/-0.50, and saline=2.78+/-0.43 mL x min(-1) x 100 g(-1), P<0.001), more angiographically recognizable collateral vessels (angioscore) (AdCMV. VEGF(<em>121</em>)=50.58+/-1.48, AdCMV.Null=29.08+/-4.22, saline=11.83+/-1.90, P<0.0001), and improvement of the bioenergetic reserve of the gastrocnemius muscle as assessed by (31)P NMR spectroscopy. Follow-up studies showed that superior TP to the ischemic limb in the AdCMV.VEGF(<em>121</em>) group persisted until it was equalized by spontaneous collateral vessel development in untreated animals.

CONCLUSIONS

IM administration of AdCMV.VEGF(<em>121</em>) stimulates angiogenesis in normoperfused skeletal muscles, and the newly formed vessels preserve TP after induction of ischemia.

Neuropilin 1 (NRP1) is a co-receptor for <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF(165)), an inducer of <em>vascular</em> permeability and angiogenesis. Numerous physiological <em>factors</em> enhance VEGF expression and function but only a few have been shown to be negative regulators. Previously, we have shown that the naturally occurring soluble form of NRP1 (sNRP1) inhibits binding of VEGF(165) to <em>endothelial</em> cells in vitro and impairs tumor <em>growth</em> in vivo. To investigate the role of sNRP1 in the regulation of <em>vascular</em> development and function, sNRP1 expression was targeted to the skin, where it is not normally expressed, using a keratin 14 (K14) promoter expression construct. K14-sNRP1 transgenic mice displayed normal skin architecture with a subtle abnormal <em>vascular</em> phenotype. While the overall number of skin blood vessels remained unchanged, the lumen size of smooth muscle-associated dermal vessels was reduced. K14-sNRP1 mice had reduced <em>vascular</em> permeability in response to VEGF(165), but also to VEGF(<em>121</em>) and platelet activating <em>factor</em>, suggesting that the lack of permeability was not solely due to the sequestration of VEGF. sNRP1 also reversed the increase in inflammation and edema induced by transgenic VEGF overexpression in cutaneous delayed-type hypersensitivity reactions. In summary, sNRP1 appears to primarily regulate vessel permeability while its effect on physiological angiogenesis is less evident in this model.

The aim of this study was to raise and characterize a monoclonal antibody reactive with VEGF (<em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em>) in routinely fixed specimens and to use it to investigate its tissue distribution in normal and pathological specimens. Recombinant VEGF 189 protein was used to raise a monoclonal antibody. The specificity of the antibody was confirmed using COS cells transfected with cDNA coding for VEGF <em>121</em>, 165 and 189 protein and by western blotting studies. The resulting antibody VG1 was shown to react with the <em>121</em>, 165 and 189 isoforms of VEGF protein in routinely processed material. In normal tissues, there was strong staining of endometrial and salivary glands and of the mucosa of the gastro-intestinal tract. In tumours, a proportion of the neoplastic cells in lung and breast cancer and in melanoma were labelled. In all tissues, whether normal or malignant, striking VEGF positivity was seen in plasma in vessels and stroma. This study has shown that antibody VG1 detects the <em>121</em>, 165 and 189 VEGF isoforms in routinely fixed specimens. The results of the normal tissue and tumour labelling are in agreement with other studies using alternative methods of detection. This should be a useful and reliable reagent for studies of VEGF and angiogenesis in human pathological material.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) is an angiogenic mitogen, specific for <em>endothelial</em> cells. Hypoxia-induced VEGF in <em>endothelial</em> cells and cardiomyocytes leads to autocrine and paracrine stimulation, respectively. During myocardial ischemia, VEGF is upregulated in the endothelium and myocardium, and may mediate angiogenesis. Morphine sulfate is commonly used in pain relief for patients with acute myocardial infarction. We investigated the effect of morphine sulfate on VEGF expression in cultured <em>endothelial</em> cells and cardiac myocytes subjected to hypoxia. Enzyme-linked immunosorbent assays showed that morphine sulfate significantly inhibited hypoxia-induced VEGF expression in mouse heart microvascular <em>endothelial</em> cells (SMHEC4), primary cultures of human umbilical vein <em>endothelial</em> cells (HUVECs) and in primary cultures of rat cardiac myocytes (P<0.05). Real time reverse transcriptase polymerase chain reaction showed that morphine treatment (100 ng/ml) of hypoxic HUVECs resulted in a significant reduction in mRNA levels of VEGF(<em>121</em>) and VEGF(165) isoforms. Transfection of HUVECs with a human VEGF promoter-luciferase construct showed that hypoxia-induced transcriptional activation of VEGF was markedly inhibited by morphine sulfate (P<0.05). Phosphatidyl inositol-3 kinase and protein kinase C-mediated activation of the VEGF promoter was also inhibited by morphine. The opioid antagonist naloxone significantly reversed the inhibitory effects of morphine in <em>endothelial</em> cells suggesting the involvement of opioid receptors. Our results show that the inhibitory effects of morphine on hypoxia-induced VEGF expression in <em>endothelial</em> cells and cardiac myocytes can lead to a decrease in the autocrine and paracrine stimulation and hence limit neovascularization of the ischemic myocardium.

BACKGROUND

Angiogenesis is essential for tumor <em>growth</em> or metastasis. A method involving noninvasive detection of angiogenic activity in vivo would provide diagnostic information regarding antiangiogenic therapy targeting <em>vascular</em> <em>endothelial</em> cells as well as important insight into the role of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) and its receptor (flt-1 and KDR) system in tumor biology. We evaluated radioiodinated VEGF(<em>121</em>), which displays high binding affinity for KDR, and VEGF(165), which possesses high binding affinity for flt-1 and low affinity for KDR, as angiogenesis imaging agents using the LS180 tumor xenograft model.

METHODS

VEGF(<em>121</em>) and VEGF(165) were labeled with (125)I by the chloramine-T method. Biodistribution was observed in an LS180 human colon cancer xenograft model. Additionally, autoradiographic imaging and immunohistochemical staining of tumors were performed with (125)I-VEGF(<em>121</em>).

RESULTS

(125)I-VEGF(<em>121</em>) and (125)I-VEGF(165) exhibited strong, continuous uptake by tumors and the uterus, an organ characterized by angiogenesis. (125)I-VEGF(<em>121</em>) uptake in tumors was twofold higher than that of (125)I-VEGF(165) (9.12+/-98 and 4.79+/-1.08 %ID/g at 2 h, respectively). (125)I-VEGF(<em>121</em>) displayed higher tumor to nontumor (T/N) ratios in most normal organs in comparison with (125)I-VEGF(165). (125)I-VEGF(<em>121</em>) accumulation in tumors decreased with increasing tumor volume. Autoradiographic and immunohistochemical analyses confirmed that the difference in (125)I-VEGF(<em>121</em>) tumor accumulation correlated with degree of tumor <em>vascular</em>ity.

CONCLUSIONS

Radioiodinated VEGF(<em>121</em>) is a promising tracer for noninvasive delineation of angiogenesis in vivo.

In this review we bring forward what is currently known about the role of type I insulin-like <em>growth</em> <em>factor</em> receptor (IGF-1R) in mediating breast cancer invasion and metastasis. We begin by addressing how activated IGF-1R could allow pre-cancerous cells to become invasive. To this effect, we discuss clinical reports suggesting that activation of IGF-1R could stimulate ductal carcinoma in situs to become invasive. In the same light, we review basic research from our laboratory showing that IGF-1R differentially regulates the expression of breast cancer progression genes when pre-malignant breast epithelial cells were stimulated with insulin-like <em>growth</em> <em>factor</em>-I (IGF-I) over time. The discussion then turns toward the ability of IGF-1R to stimulate invasion of breast cancer cells that have acquired a malignant phenotype. At this stage of breast cancer, it appears that IGF-I stimulates cells to invade in part by inducing urokinase plasminogen activator. Finally, we consider the potential role of IGF-1R in regulating breast cancer metastases by facilitating angiogenesis and lymphangiogenesis. In support of this idea, there is evidence for IGF-1R in both of these processes through the induction of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factors</em> (VEGF(165) and VEGF(<em>121</em>)). Thus, IGF-1R affords breast cancer cells many opportunities to become invasive and eventually metastatic. We conclude that disrupting IGF-1R signaling has many important implications in the treatment and management of breast cancer.

<em>Growth</em> of trophoblast tissue in early pregnancy is rapid and accomplished in an unusually hypoxic environment. Hypoxia has been reported to upregulate mRNA production of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF), and VEGF receptors have been found on trophoblast cells. These observations suggest that VEGF may have an important role in early placentation. This study examines the influence of hypoxia on both the production of the VEGF message and protein and on the production of human chorionic gonadotrophin (hCG) protein by the cell lines JEG, JAr and BeWo. Cells were grown under normoxic and hypoxic conditions for 72 h. The average oxygen tension in the culture media of the hypoxic cultures (6-7 kPa) was significantly less than in the normoxic cultures (19-21 kPa). RNA was extracted and message for VEGF(<em>121</em>), VEGF(165) and VEGF(189) found in all cell lines by reverse transcription and the polymerase chain reaction (RT-PCR). These messages were upregulated by hypoxia; findings confirmed by competitive PCR for VEGF and expression of the house keeping gene GAPDH. hCG and VEGF were measured by immunoassay. Hypoxia resulted in an increase in VEGF production (P<0.05) but had inconsistent effects on hCG production. In some experiments the absolute concentrations of hCG and VEGF in the culture media were noted to be significantly correlated (r>0.5, P<0.05). In addition to its role in angiogenesis, VEGF may have direct effects on trophoblast cells encouraging proliferation and invasion. These effects may be regulated in part through oxygen supply and hCG.

The presence of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) in the ovary has been reported in a number of species. The objective of the present study was to demonstrate the expression of VEGF, VEGF receptor (R)-1, and VEGFR-2 in detail by different methodological approaches in bovine corpora lutea (CL) obtained from different stages of the estrous cycle and during pregnancy. VEGF and VEGF receptor transcripts were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and ribonuclease protection assay. All components of the VEGF system were found in the bovine CL during the estrous cycle and pregnancy. Analysis of VEGF transcript by RT-PCR shows that CL tissues expressed predominantly the smallest isoforms (VEGF(<em>121</em>) and VEGF(165)). The highest mRNA expression for VEGF and VEGFR-2 mRNA was detected during the early luteal phase, followed by a significant decrease of expression during the mid and late luteal phase and a further decrease of VEGF mRNA after regression. During pregnancy, high levels of expression were always present. In contrast, no significant change in VEGFR-1 mRNA expression during the estrous cycle and pregnancy was found. The VEGF protein concentration in CL tissue was significantly higher (20.9-23.4 ng/g wet weight) during the early luteal phase (Days 1-7), followed by a decrease at the late luteal phase (14.3-18.7 ng/g wet weight) and, especially, after CL regression (2.8 ng/g wet weight). However, relatively high levels were found during pregnancy (10.1 ng/g wet weight). As achieved by immunohistochemistry, VEGF protein was localized predominantly in luteal cells. High VEGF protein and transcript concentrations and increased VEGFR-2 expression during the early luteal phase coincided with luteal <em>vascular</em>ization. These results suggest an important role of VEGF in angiogenesis of the newly formed CL. The high VEGF mRNA expression and protein levels during matured vasculature in the mid-stage CL and pregnancy also suggest also a survival function for <em>endothelial</em> cells.