OBJECTIVE

To test the influence of vascular endothelial growth factor (VEGF) on normal and ischemic wounds in a noncontractive dermal ulcer standardized model in the rabbit ear and to assay the levels of both VEGF and basic fibroblastic growth factor messenger RNA levels in normal and ischemic wounds at different intervals during the healing process.

METHODS

Dermal ulcers were created in the normal and ischemic ears of 20 anesthetized young female New Zealand white rabbits. Either VEGF 121, VEGF 165 (30 microg per wound), or buffered saline solution alone was applied to each wound and covered. Wounds were harvested at day 7 or 10 and evaluated histologically. Twenty-four similar rabbits were wounded in the same manner and their untreated wounds were harvested at 1, 3, 7, and 10 days after wounding. The wounds were analyzed with reverse transcriptase polymerase chain reaction.

METHODS

Histologic specimens were measured for amount of new epithelium and granulation tissue. Reverse transcriptase polymerase chain reaction was used to determine basic fibroblastic growth factor and VEGF messenger RNA expression.

RESULTS

Both isoforms of VEGF improved granulation tissue formation in both normal and ischemic wounds with a magnitude similar to other vulnerary agents tested in the past. Vascular endothelial growth factor application had no effect on new epithelium formation. In contrast to basic fibroblastic growth factor, VEGF messenger RNA levels were induced 4 fold by ischemia alone and 6 fold by wounding in both ischemic and normal wounds.

CONCLUSIONS

Vascular endothelial growth factor seems to be more important than basic fibroblastic growth factor during ischemic wound healing. Treatment of ischemic wounds with VEGF improves the deficit in wound healing produced by ischemia.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF(165)) is a potent angiogenic mitogen commonly overexpressed in cancerous cells. It contains two main binding domains, the receptor-binding domain (RBD) and the heparin-binding domain (HBD). This study attempted to identify the specific sequences of the VEa5 DNA aptamer that exhibit high binding affinity towards the VEGF(165) protein by truncating the original VEa5 aptamer into different segments. Using surface plasmon resonance (SPR) spectroscopy for binding affinity analysis, one of the truncated aptamers showed a >200-fold increase in the binding affinity for HBD. This truncated aptamer also exhibited high specificity to HBD with negligible binding affinity for VEGF(<em>121</em>), an isoform of VEGF lacking HBD. Exposing colorectal cancer cells to the truncated aptamer sequence further confirmed the binding affinity and specificity of the aptamer to the target VEGF(165) protein. Hence, our approach of aptamer truncation can potentially be useful in identifying high affinity aptamer sequences for the biological molecules and targeting them as antagonist for cancer cell detection.

Much of the morbidity of intracranial meningiomas is related to the degree of tumour <em>vascular</em>ity and the extent of peritumoural vasogenic oedema. Several studies have shown that <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) is up-regulated in meningiomas, although its relationship with tumour vasculature is still unclear. In order to better understand the angiogenic assessment of intracranial meningiomas, we analysed its <em>vascular</em> pattern, both as number and as morphologic configuration of microvessels. Moreover, we investigated the mRNA-VEGF expression, relating this expression to <em>vascular</em> pattern. A total of 40 intracranial meningiomas, classified as benign (31 cases), atypical (7 cases), and anaplastic (2 cases) were analysed. RT-PCR analyses of mRNA-VEGF and competitive-PCR were performed. VEGF expression and microvessel density (MVD) were also immunohistochemically investigated. Grade II-III meningiomas showed numerous small microvessels (mean: 34), while the majority of Grade I showed few larger vessels (mean: 13.09) (P = 0.000003). A microvessel pattern overlapping into atypical subtype was found in eignt of the 31 (25.8%) Grade I meningiomas. A significant association was found between grading and <em>vascular</em> pattern (P = 0.0002), as well as between the MVD and the immunohistochemical expression of VEGF (P = 0.0005). The expression of mRNA agreed with the immunohistochemical expression of the protein (P < 0.0001). A total of 39 cases expressed the <em>121</em> VEGF isoform and, among these, 28 cases also expressed the 165 isoform. Only 9 cases expressed both isoforms 165 and 189. Grade II and III meningiomas showed a preponderant expression of soluble isoforms (<em>121</em> and 165). These results prompt us to speculate that the microvessel pattern could underlie a higher metabolic demand, probably due to a rapid <em>growth</em> with a consequent worse clinical behaviour of the tumour. In this sense, the <em>vascular</em> pattern may be used as a prognostic <em>factor</em>, in order to mostly focus attention on those Grade I meningiomas which have a higher likelihood of either recurrence or development of perilesional oedema. The pattern of vasculature itself seems to be dependent on the types of VEGF isoforms: the Grade II-III meningiomas (that presented numerous microvessels) expressed the soluble isoforms <em>121</em> and 165, while the isoform 189 was more frequently detected in Grade I meningiomas.

Autonomic function is altered by altitude in sojourners and natives. We hypothesized that these physiologic responses are modulated by changes in gene expression. We compared gene product levels in 20 natives of Cerro de Pasco (CP), (4338 m), 10 of which had chronic mountain sickness (CMS) established by a CMS-scoring system, with gene products in the same men after 1 h at sea level. We further compared the results with those obtained from 10 US men residing at 1500 m. We measured gene products in white cells by reverse transcription polymerase chain reaction (RT-PCR). We focused on genes important in <em>vascular</em> autonomic physiology, and/or activated by hypoxia; hypoxia inducible <em>factor</em> 1-alpha (HIF 1-alpha), 2 splicing variants of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF); VEGF-<em>121</em>, VEGF-165, and phosphoglycerate kinase 1 (PGK 1). Normal CP natives showed high expression of all genes in CP, compared to US controls. Within 1 h of arrival at sea level, they had comparable levels to US residents. In CMS, the gene products were higher in CP. Although gene products decreased in Lima in this group, they never reached US values. VEGF <em>121</em> and 165 were correlated (P<0.001). VEGF 165 was higher in CMS in CP (P=0.006), and was positively correlated with CMS-score (R=0.86, P<0.001), and negatively correlated with arterial saturation (R=-0.79, P<0.001). Our findings underscore the changes in gene expression levels in intact humans in response to environmental stress. These changes may support the physiologic alterations induced by the ambient hypoxia at altitude and impact organism survival. They also suggest therapeutic strategies for autonomic and neurodegenerative diseases at sea level.

Combination chemotherapy is increasingly practiced for the treatment of malignant prostate cancers. The aim of this study was to evaluate the in vivo efficacy of combined tamoxifen and quercetin in prostate tumor xenografts. Severe combined immune deficient (SCID) mice inoculated with CWR22 prostate tumor cells were treated with either tamoxifen (10 mg/kg/week), quercetin (200 mg/kg/day) or combined tamoxifen-quercetin for 28 days. Tamoxifen or quercetin alone exhibited a moderate antitumor activity. Tamoxifen decreased the Ki-67 index by 52.4%, reduced the <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) <em>121</em> and VEGF165 mRNA by 18.6 and 21.8%, respectively, and suppressed the blood vessel formation, while quercetin modulated the expression and phosphorylation of cdc-2 and cyclin B1, and inhibited the Ki-67 index by 66.0%. Combined tamoxifen-quercetin effectively delayed the appearance of tumors, inhibited the final tumor volume by 73.3% and reduced the endpoint tumor weight by 67.1% (p<0.05). The Ki-67 index, VEGF<em>121</em>, VEGF165 mRNA and microvessel density (MVD) were decreased by 66.9, 22.1, 40.1 and 59.0%, respectively, by the combined treatment. These findings indicate that tamoxifen inhibits CWR22 prostate tumor by modulating the angiogenesis and its antineoplastic effects can be potentiated by combined use with quercetin.

BACKGROUND

Antigen presenting cells, in particular dendritic cells (DCs), are critical elements in antitumor immunity induction. Some of the angiogenic factors released by tumor and stroma cells, including vascular endothelial growth factor (VEGF), are thought to affect DC function.

METHODS

The expression of Vascular Endothelial Growth Factor (VEGF) isoforms VEGF-A (121, 145,165, 189, 206), VEGF-C and VEGF-D were determined by immunohistochemistry, Western blotting and ELISA in 46 patients with Head and Neck Squamous Cell Carcinoma (HNSCC), 30 healthy donors, and two HNSCC tumor cell lines (PCI-1 and PCI-13).

RESULTS

Increased expression of VEGF-A and VEGF-C was found in tumor tissues compared to normal epithelium (P=0.001). However, VEGF-D levels were decreased in patients with cervical nodal metastasis as compared to patients with negative lymph node status. VEGF-A plasma levels were increased in patients with lymph node metastasis (266 pg/ml) compared to patients with negative lymph node status (19.8 pg/ml). Multivariate analysis demonstrated that VEGF-A correlated with microvessel density (P=0.01), disease progression (P=0.038), a reduced number of local and peripheral mature dendritic cells (DC) (P=0.015) and an increased number of peripheral immature DCs (P=0.05). DCs incubated with tumor supernatant or VEGF-A differentiated into immature DCs and did not develop full allostimulatory activity. Allogenic T cells, when co-cultured with these immature DCs, expressed the T regulatory cell marker CD25, CTLA-4, and CD45Ro, and secreted TGF-beta, VEGF-A and IL-10.

CONCLUSIONS

Taken together, our results identify VEGF-A as a multifunctional factor involved in angiogenesis, tumor progression, immunosuppression and immune tolerance.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) is a key mediator of angiogenesis, whose effect on cancer <em>growth</em> and development is well characterized. Alternative splicing of VEGF leads to several different isoforms, which are differentially expressed in various tumor types and have distinct functions in tumor blood vessel formation. Many cancer therapies aim to inhibit angiogenesis by targeting VEGF and preventing intracellular signaling leading to tumor vascularization; however, the effects of targeting specific VEGF isoforms have received little attention in the clinical setting. In this work, we investigate the effects of selectively targeting a single VEGF isoform, as compared with inhibiting all isoforms. We utilize a molecular-detailed whole-body compartment model of VEGF transport and kinetics in the presence of breast tumor. The model includes two major VEGF isoforms, VEGF(<em>121</em>) and VEGF(165), receptors VEGFR1 and VEGFR2, and co-receptors Neuropilin-1 and Neuropilin-2. We utilize the model to predict the concentrations of free VEGF, the number of VEGF/VEGFR2 complexes (considered to be pro-angiogenic), and the receptor occupancy profiles following inhibition of VEGF using isoform-specific anti-VEGF agents. We predict that targeting VEGF(<em>121</em>) leads to a 54% and 84% reduction in free VEGF in tumors that secrete both VEGF isoforms or tumors that overexpress VEGF(<em>121</em>), respectively. Additionally, 21% of the VEGFR2 molecules in the blood are ligated following inhibition of VEGF(<em>121</em>), compared with 88% when both isoforms are targeted. Targeting VEGF(<em>121</em>) reduces tumor free VEGF and is an effective treatment strategy. Our results provide a basis for clinical investigation of isoform-specific anti-VEGF agents.

To obtain an aptamer with a high affinity for <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF), we focused on the receptor-binding domain (RBD) of VEGF as a target epitope. Three rounds of screening gave Vap7, which bound to the VEGF isoforms VEGF(<em>121</em>) and VEGF(165) with K(D) values of 1.0 nM and 20 nM, respectively. Moreover, Vap7 showed specificity within the VEGF family. Secondary structure predictions and circular dicrhoism suggested that Vap7 folds into a G-quadruplex structure. We obtained a mutant aptamer that contains only this region of the aptamer sequence. This truncated mutant (V7t1) bound to both VEGF(<em>121</em>) and VEGF(165) with K(D) values of 1.1 nM and 1.4 nM, respectively. Its sequence was 5'-TGTGGGGGTGGACGGGCCGGGTAGA-3', and it appeared to form a G-quadruplex structure. We also produced an aptamer heterodimer consisting of our previously derived aptamer (del5-1), which binds to the heparin-binding domain of VEGF, linked to V7t1. The resulting heterodimer bound strongly to VEGF(165) with a K(D) value of 4.7 x 10(2) pM.

Vasculotropin (VAS), also called <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) or <em>vascular</em> permeability <em>factor</em>, is a secreted <em>growth</em> <em>factor</em> whose target cell specificity has been reported as restricted to <em>vascular</em> endothelium. Its effects are mediated by at least two distinct membrane-spanning tyrosine kinase receptors, KDR and flt-1; the expression of which also seems restricted to <em>vascular</em> endothelium. We describe here that cultured human retinal pigment epithelial (HRPE) cells express both KDR and flt-1 receptors, bind VAS/VEGF on two high affinity sites (apparent Kd of 9 and 210 pM corresponding to 940 and 18,800 sites per cell) and proliferate or migrate upon recombinant VAS/VEGF addition. HRPE cells also express the mRNA corresponding to the <em>121</em> and 165 amino acid forms of VAS/VEGF. HRPE cells release in their own culture medium and store in their extracellular matrix self-mitogenic and chemoattractant <em>factors</em> indistinguishable from <em>121</em> and 165 VAS/VEGF isoforms. The autocrine role of VAS/VEGF was confirmed by the inhibition of these bioactivities by neutralizing specific anti-VAS/VEGF antibodies.

The <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) has been shown to be strictly related to <em>vascular</em> permeability and <em>endothelial</em> cell <em>growth</em> under physiological and pathological conditions. In tumour development and progression, VEGF plays a pivotal role in the development of the tumoral <em>vascular</em> network, and useful information in the progression of human cancer can be obtained by analysing the <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> expression of the tumours. In this study, we investigated the <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> transcript expression in non-small-cell lung carcinomas to evaluate the significance of this <em>factor</em> in a group of cancers in which the <em>vascular</em> pattern has been shown to significantly affect progression. Surgical samples of 42 patients with NSCLC were studied using reverse transcription polymerase chain reaction (PCR) analysis and in situ hybridization. Thirty-three out of 42 cases (78.6%) showed VEGF transcript expression predominantly as transcripts for the secretory forms of VEGF (isoforms <em>121</em> and 165). In situ hybridization, performed on 24 out of 42 samples, showed that the VEGF transcript expression was in several cases present in the cytoplasm both of neoplastic and normal cells, even if the VEGF mRNA was less expressed in the corresponding non-tumoral part. The VEGF <em>121</em> expression was associated with hilar and/or mediastinal nodal involvement (P = 0.02), and, taken together, the VEGF isoforms were shown to significantly influence overall (P = 0.02) and disease-free survival (P = 0.03). As a regulator of tumour angiogenesis, VEGF may represent a useful indicator of progression and poor prognosis in non-small-cell lung carcinomas.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) is a potent cytokine that binds to specific receptors on the <em>endothelial</em> cells lining blood vessels. The signaling cascade triggered eventually leads to the formation of new capillaries, a process called angiogenesis. Distributions of VEGF receptors and VEGF ligands are therefore crucial determinants of angiogenic events and, to our knowledge, no quantification of abluminal vs. luminal receptors has been performed. We formulate a molecular-based compartment model to investigate the VEGF distribution in blood and tissue in humans and show that such quantification would lead to new insights on angiogenesis and VEGF-dependent diseases. Our multiscale model includes two major isoforms of VEGF (VEGF(<em>121</em>) and VEGF(165)), as well as their receptors (VEGFR1 and VEGFR2) and the non-signaling co-receptor neuropilin-1 (NRP1). VEGF can be transported between tissue and blood via trans<em>endothelial</em> permeability and the lymphatics. VEGF receptors are located on both the luminal and abluminal sides of the <em>endothelial</em> cells. In this study, we analyze the effects of the VEGF receptor localization on the <em>endothelial</em> cells as well as of the lymphatic transport. We show that the VEGF distribution is affected by the luminal receptor density. We predict that the receptor signaling occurs mostly on the abluminal <em>endothelial</em> surface, assuming that VEGF is secreted by parenchymal cells. However, for a low abluminal but high luminal receptor density, VEGF binds predominantly to VEGFR1 on the abluminal surface and VEGFR2 on the luminal surface. Such findings would be pertinent to pathological conditions and therapies related to VEGF receptor imbalance and overexpression on the <em>endothelial</em> cells and will hopefully encourage experimental receptor quantification for both luminal and abluminal surfaces on <em>endothelial</em> cells.

BACKGROUND

Advanced hepatocellular carcinoma (HCC) in humans is characterized by hypervascularity. In the present study, the expressions of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF-2) and endostatin were analyzed in patients with chronic liver disease to clarify the effect of these major angiogenic factors.

METHODS

Serum concentrations of VEGF, FGF-2 and endostatin in 24 patients with HCC, 16 patients with liver cirrhosis (LC) and 13 healthy volunteers were measured by enzyme-linked immunosorbent assay. The expression of VEGF in 21 surgically resected HCC samples was analyzed by immunohistochemistry, and that of VEGF isoforms in 15 HCC samples was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR).

RESULTS

Serum VEGF, FGF-2 and endostatin concentrations were significantly elevated in patients with HCC compared with healthy volunteers; but there was no significant difference between patients with HCC and those with non-HCC liver disease. Immunohistochemical analysis showed that VEGF protein was strongly expressed in both well-differentiated HCC cells and non-cancerous hepatocytes, whereas in moderately and poorly differentiated HCC the expression was stronger in the endothelial cells (EC) lining intratumor vessels than in the cancer cells. On RT-PCR for VEGF isoforms it was found that VEGF-121, VEGF-165 and VEGF-189 were expressed in all but one of the HCC samples and in all corresponding non-HCC samples.

CONCLUSIONS

The results suggest that VEGF, FGF-2, and endostatin concentrations are elevated prior to the emergence of HCC and that the distribution of VEGF changes dynamically during the development of HCC.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) is a proangiogenic <em>factor</em> upregulated in many tumors. The alternative splicing of VEGF mRNA renders 3 major isoforms of <em>121</em>, 165 and 189 amino-acids in humans (1 less amino-acid for each mouse VEGF isoform). We have designed isoform specific real time QRT-PCR assays to quantitate VEGF transcripts in mouse and human normal and malignant prostates. In the human normal prostate, VEGF(165) was the predominant isoform (62.8% +/- 5.2%), followed by VEGF(<em>121</em>) (22.5% +/- 6.3%) and VEGF(189) (p < 0.001) (14.6% +/- 2.1%). Prostate tumors showed a significant increase in the percentage of VEGF(<em>121</em>) and decreases in VEGF(165) (p < 0.01) and VEGF(189) (p < 0.05). However, the amount of total VEGF mRNA was similar between normal and malignant prostates. VEGF(164) was the transcript with the highest expression in the mouse normal prostate. Unlike human prostate cancer, tumors from TRAMP mice demonstrated a significant increase in total VEGF mRNA levels and in each of the VEGF isoforms, without changes in the relative isoform ratios. Morpholino phosphorodiamide antisense oligonucleotide technology was used to increase the relative amount of VEGF(<em>121</em>) while proportionally decreasing VEGF(165) and VEGF(189) levels in human prostate cell lines, through the modification of alternative splicing, without changing transcription levels and total amount of VEGF. The increase in the VEGF(<em>121</em>)/VEGF(165-189) ratio in PC3 cells resulted in a dramatic increase in prostate tumor angiogenesis in vivo. Our results underscore the importance of VEGF(<em>121</em>) in human prostate carcinoma and demonstrate that the relative expression of the different VEGF isoforms has an impact on prostate carcinogenesis.

OBJECTIVE

Angiogenesis is a complex multistep process that involves endothelial cell (EC) migration, proliferation and differentiation into vascular tubes. NO has been reported to be a downstream mediator in the angiogenic response to a variety of growth factors, but the mechanisms by which NO promotes neovessel formation is not clear. We hypothesized that NO directly contributes to EC migration and capillary tube formation.

METHODS

Since previous studies have noted important biological differences between NO produced pharmacologically by NO-donor compounds compared to that from NO synthase (NOS), we used a cell-based gene transfer approach to increase NO production in a co-culture model of in vitro angiogenesis. Rat smooth muscle cells (SMCs) were transfected with plasmids containing VEGF(121), VEGF(165) (SMC(VEGF)), endothelial NOS (SMC(eNOS)) or the empty vector (SMC(Cont)). Expression of the eNOS in SMC(eNOS) was confirmed by Northern analysis, NADPH-diaphorase activity, and nitrite/nitrate levels, whereas VEGF production was confirmed using ELISA. Calf pulmonary artery ECs (CPAECs) were cultured on the fibrin matrix with (co-culture) or without underlying SMCs (monoculture).

RESULTS

Co-culture of ECs with SMC(Cont) had no effect on EC differentiation compared with EC in monoculture (differentiation index, DI=2.8+/-3.4 vs. 2.1+/-2.8, respectively, NS). In contrast, co-culture with SMC(eNOS) resulted in the formation of extensive capillary-like structures within 48 h (DI=17.2+/-5.9, P<0.001 versus SMC(Cont)), which was significantly inhibited using a NOS inhibitor, L-NAME (3 mM) (DI=4.5+/-3.04, P<0.001 versus SMC(eNOS)). Similarly, SMC(VEGF121) induced an angiogenic response (DI=14.2+/-3.8), which was also significantly inhibited by L-NAME (DI=5.9+/-1.8, P<0.05). In using the Boyden chamber model, SMC(eNOS), but not SMC(Cont) increased EC migration to a similar extent as SMC(VEGF121), and both were significantly inhibited with L-NAME.

CONCLUSIONS

These data support an important paracrine role for endogenously produced NO in EC migration and differentiation in vitro, and suggest that the cell-based eNOS gene transfer may be a useful approach to increase new blood vessel formation in vivo.

Recent studies have demonstrated a role for micro<em>vascular</em> and tubulointerstitial injury in some models of salt-sensitive hypertension. We utilized a model of post-cyclosporin A (CsA) nephropathy and hypertension to test the hypothesis that treatment with an angiogenic <em>factor</em> aimed at ameliorating the micro<em>vascular</em> and renal injury would prevent the development of hypertension. CsA was administered with a low-salt diet for 45 days, resulting in a renal lesion characterized by afferent arteriolopathy, focal peritubular capillary loss, and tubulointerstitial fibrosis. Rats were then placed on a high-salt diet and randomized to receive either <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF(<em>121</em>)) or vehicle for 14 days. Placement of rats with established CsA nephropathy on a high-salt diet results in the rapid development of salt-sensitive hypertension. VEGF(<em>121</em>) treatment resulted in lower blood pressure, and this persisted on discontinuing the VEGF. VEGF(<em>121</em>) treatment was also associated with a decrease in osteopontin expression, macrophage infiltration, and collagen III deposition and markedly stimulated resolution of the arteriolopathy (20.9 +/- 7.8 vs. 36.9 +/- 6.1%, VEGF vs. vehicle, P < 0.05). In conclusion, CsA-associated renal micro<em>vascular</em> and tubulointerstitial injury results in the development of salt-sensitive hypertension. Treatment of animals with established CsA nephropathy with VEGF reduces the hypertensive response and accelerates histological recovery. The <em>vascular</em> protective effect of VEGF may be due to the improvement of arteriolopathy. Angiogenic <em>growth</em> <em>factors</em> may represent a novel strategy for treating CsA-associated hypertension and renal disease.

Early cancer diagnosis is very important for the prevention or mitigation of metastasis. However, effective and efficient methods are needed to improve the diagnosis and assessment of cancer. Here, we report a single-step detection method using a nanoplasmonic aptamer sensor (aptasensor), targeting a <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em>-165 (VEGF(165)), a predominant biomarker of cancer angiogenesis. Our single-step detection is accomplished by (1) specific target recognition by an aptamer-target molecule interaction and (2) direct readouts of the target recognition. The readout is achieved by inactivation of surface plasmon enhancement of fluorescent probes preattached to the aptamers. Our aptasensor provides the appropriate sensitivity for clinical diagnostics with a wide range of linear detection from 25 pg/mL to 25 μg/mL (=from 1.25 pM to 1.25 μM), high specificity for VEGF(165) against PDGF-BB, osteopontin (OPN), VEGF(<em>121</em>), NaCl, and temporal/thermal/biological stability. In experiments with 100% serum and saliva from clinical samples, readouts of the aptasensor and an ELISA for VEGF(165) show good agreement within the limit of the ELISA kit. We envision that our developed aptasensor holds utilities for point-of-care cancer prognostics by incorporating simplicity in detection, low-cost for test, and required small sample volumes.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) is an angiogenic <em>factor</em> secreted by various tumors, including epithelial tumors of the ovary, and is involved in tumor progression and maintenance. The significance and function of other members of the VEGF family in the ovary has not yet been elucidated. In the present study, we have defined the expression of mRNA encoding VEGF-B, VEGF-C, and placenta <em>growth</em> <em>factor</em> (PIGF), compared with that of VEGF mRNA, in normal ovary and a range of ovarian epithelial tumors. Analysis by reverse transcription-PCR indicated that mRNA encoding VEGF (isoforms <em>121</em> and 165), VEGF-B (isoforms 167 and 186), and VEGF-C, but not PIGF, were present in all ovarian tissues examined. By in situ hybridization, neither VEGF-C nor PIGF transcripts were detected in any of the samples. The expression pattern of VEGF-B mRNA was generally similar to that of VEGF mRNA, in that transcripts were readily detected in the epithelial cells of all histologic types of ovarian carcinoma, but could not be detected in normal or benign tumor epithelium. Specific differences in the expression of the two genes were noted in areas of tumor necrosis, in which the expression of VEGF mRNA, but not VEGF-B mRNA, was further enhanced, and in a sample in which VEGF-B mRNA was strongly expressed in tumor-associated macrophages that did not hybridize with the riboprobe to VEGF mRNA. These results imply that a second member of the VEGF family, VEGF-B, may play a significant role in the angiogenesis, progression, and maintenance of ovarian carcinomas.

An imbalance in circulating proangiogenic and antiangiogenic <em>factors</em> is postulated to play a causal role in preeclampsia (PE). We have described an inbred mouse strain, BPH/5, which spontaneously develops a PE-like syndrome including late-gestational hypertension, proteinuria, and poor feto-placental outcomes. Here we tested the hypothesis that an angiogenic imbalance during pregnancy in BPH/5 mice leads to the development of PE-like phenotypes in this model. Similar to clinical findings, plasma from pregnant BPH/5 showed reduced levels of free <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) and placental <em>growth</em> <em>factor</em> (PGF) compared to C57BL/6 controls. This was paralleled by a marked decrease in VEGF protein and Pgf mRNA in BPH/5 placentae. Surprisingly, antagonism by the soluble form of the FLT1 receptor (sFLT1) did not appear to be the cause of this reduction, as sFLT1 levels were unchanged or even reduced in BPH/5 compared to controls. Adenoviral-mediated delivery of VEGF(<em>121</em>) (Ad-VEGF) via tail vein at embryonic day 7.5 normalized both the plasma-free VEGF levels in BPH/5 and restored the in vitro angiogenic capacity of serum from these mice. Ad-VEGF also reduced the incidence of fetal resorptions and prevented the late-gestational spike in blood pressure and proteinuria observed in BPH/5. These data underscore the importance of dysregulation of angiogenic <em>factors</em> in the pathogenesis of PE and suggest the potential utility of early proangiogenic therapies in treating this disease.

Although peritoneal metastasis is an important <em>factor</em> determining the prognosis of patients with gastrointestinal cancer, the mechanisms have not yet been clearly defined. Human peritoneal mesothelial cells (HPMC) are the first line against disseminated tumor cells. Recent reports have shown that mesothelial cells are capable of secreting various cytokines and <em>growth</em> <em>factors</em>. In this study, we isolated human mesothelial cells from surgically resected omental tissue and examined the production and interaction of two major angiogenic <em>factors</em>, <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) and basic fibroblast <em>growth</em> <em>factor</em> (FGF-2). Quiescent HPMC produced a considerable amount of VEGF at almost the same level as tumor cells. Interestingly, addition of FGF-2 to the culture significantly increased the mRNA synthesis and protein secretion of VEGF in a dose-dependent manner, as determined by Northern blot and ELISA. The addition of 0.5 ng/mL FGF-2 was enough to stimulate VEGF production, and the effect reached a plateau at 5 ng/mL. Reverse-transcribed polymerase chain reaction (RT-PCR) method clarified that the HPMC-derived VEGF consisted mostly of VEGF(<em>121</em>) and VEGF(165), which are both predominantly soluble forms. These data suggest that HPMC contribute to the development of metastases and the accumulation of malignant ascites due to the production of VEGF, especially in cancers that do not express enough amount of VEGF.

Direct radiolabeling of proteins can result in the loss of targeting activity, requires highly customized procedures, and yields heterogeneous products. Here we describe a novel imaging complex comprised of a standardized (99m)Tc-radiolabeled adapter protein noncovalently bound to a "Docking tag" fused to a "Targeting protein". The assembly of this complex is based on interactions between human 109-amino acid (HuS) and 15-amino acid (Hu-tag) fragments of ribonuclease I, which serve as an "Adapter protein" and a Docking tag, respectively.

METHODS

HuS modified with hydrazinonicotinamide (HYNIC) was radiolabeled using (99m)Tc-tricine to a specific activity of 3.4-7.4 MBq/microg. Protein complexes were then formed by mixing (99m)Tc-HuS with equimolar amounts of either Hu-tagged VEGF(<em>121</em>) (Hu-VEGF [<em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em>]) or Hu-tagged anti-VEGFR-2 single-chain antibody (Hu-P4G7) and incubating on ice for 15 min. 4T1 luc/gfp luciferase-expressing murine mammary adenocarcinoma cells (1 x 10(4)) were implanted subcutaneously or injected intravenously into BALB/c mice. Bioluminescent imaging (BLI) was performed 10 d later. Immediately after BLI visualization of tumor, 18.5-37 MBq of tracer (5-10 microg of protein) were injected via tail vein. One hour later planar or SPECT images were obtained, followed by killing the mice.

RESULTS

There was significantly (P = 0.0128) increased uptake of (99m)Tc-HuS/Hu-VEGF (n = 10) within subcutaneous tumor as compared with (99m)Tc-HuS/Hu-P4G7 (n = 5) at biodistribution assay (2.68 +/- 0.75 vs. 1.8 +/- 0.21; tumor-to-subcutaneous tissue [ratio of specific activities], respectively), despite similar molecular weights. The focal (99m)Tc-HuS/Hu-VEGF uptake seen on planar images (3.44 +/- 1.16 [tumor to soft-tissue background]) corresponded directly to the locations of tumor observed by BLI. Region of interest analyses of SPECT images revealed a significant increase of (99m)Tc-HuS/Hu-VEGF (n = 5) within the lungs with BLI-detectable pulmonary tumor nodules as compared with controls (n = 4) (right: 4.47 +/- 2.07 vs. 1.79 +/- 0.56; left: 3.66 +/- 1.65 vs. 1.62 +/- 0.45, tumor lung [counts/pixel]/normal lung [counts/pixel], respectively).

CONCLUSIONS

(99m)Tc-HuS/Hu-VEGF complex is stable for at least 1 h in vivo and can be effectively used to image mouse tumor neovasculature in lesions as small as several millimeters in soft tissue. We expect that a similar approach can be adapted for in vivo delivery of other targeting proteins of interest without affecting their bioactivity.

Evidence from pathophysiological studies support the concept that embryonic development, tumor progression, and hormonally-regulated tissue masses such as adult prostate and corpus luteum are angiogenesis-dependent. We examined if the prostatic expression of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF), the major regulator of normal and pathological angiogenesis, was regulated by testosterone. Northern blot of VEGF messenger ribonucleic acid (mRNA) extracted from a human immortalized epithelial prostatic cell line (PNT1) showed that dihydrotestosterone (DHT) up-regulated VEGF mRNA at a level comparable to that observed upon exposure to <em>growth</em> <em>factors</em>. Polymerase chain reaction of reverse transcribed mRNA demonstrated that the ratio of the two splice variants encoding the <em>121</em> and 165 isoforms of VEGF were not affected by DHT. VEGF biological activity, measured in the conditioned medium by radio receptor assay, was increased by DHT. Injection of testosterone in adult rats induced a transient increase of the ventral lobe weight and the specific activity of prostatic VEGF, leading to a 7-fold increase in the prostate content of VEGF.

Hair follicle <em>vascular</em>ization appears to be closely related to the processes involved in hair cycle regulation, in which <em>growth</em> <em>factors</em>, cytokines and other bioactive molecules are involved. In particular, <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF), essential for angiogenesis and <em>vascular</em> permeability, may be responsible for maintaining proper vasculature around the hair follicle during the anagen <em>growth</em> phase. The aim of our study was to compare the in vitro angiogenic capacity, i.e. the steady-state expression of the VEGF gene, of different cultured cell types derived from normal human hair follicles, corresponding to different follicular compartments. Human dermal papilla cells (DPC), fibrous sheath fibroblasts, dermal fibroblasts, and follicular and interfollicular keratinocytes were cultured and studied in vitro for VEGF expression at the mRNA level using RT-PCR, and for VEGF protein synthesis by radioimmunoprecipitation and immunocytochemistry. In vivo examination for VEGF expression in human terminal hair follicles was performed using immunohistochemical methods. In the present report the expression of four different VEGF molecular isoforms, differing in their angiogenic capacity, are described in different cultured follicular cell types for the first time. Cultured follicular cells strongly expressed mRNA of four VEGF molecular species identified as the <em>121</em>-, 145-, 165- and 189-amino acid splice variants, the most prominent being the <em>121</em>-amino acid molecule. DPC, and also other mesenchymal cells such as fibrous sheath fibroblasts and dermal fibroblasts, in vivo and in vitro strongly expressed VEGF mRNA and synthesized a 46-kDa VEGF protein, whereas follicular and interfollicular keratinocytes in vitro expressed lower levels of VEGF mRNA and proteins than mesenchymal cells. As the highest expression of VEGF was found in DPC, we suggest that DPC are mainly responsible for angiogenic processes possibly related to the human hair cycle.

The <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) is a critical <em>factor</em> for development of the <em>vascular</em> system in physiological and pathological angiogenesis. This <em>growth</em> <em>factor</em> exists under at least three isoforms, VEGF120/<em>121</em>, VEGF164/165 and VEGF188/189 which are generated by alternative splicing. VEGF isoforms have different affinities for heparan sulphate as well as for VEGF receptors, and may play distinct roles in <em>vascular</em> development. The role of VEGF189 as an <em>endothelial</em> mitogen, however, remains controversial. VEGF189 is almost entirely bound to the cell surface or extracellular matrix, and is considered active after its cleavage and release from its extracellular binding site. In the present study, we demonstrate that VEGF189 induces <em>endothelial</em> cell proliferation and migration in vitro. The 30-60% increase observed with VEGF189 (10 ng/ml) in HUVEC proliferation was similar to that observed with VEGF165. However, the proliferative effect observed with VEGF189 appeared dependent on the origin of the <em>endothelial</em> cell, since the proliferation was clearly observed with HUVEC but not with BAEC or capillary <em>endothelial</em> cells from dermis (HMEC). The effect of VEGF189 on <em>endothelial</em> cell migration was also analyzed using the wound healing and the Boyden chamber assays. The migration effect was observed with BAEC which do not proliferate with VEGF189, suggesting that different mechanisms are involved in proliferation and migration. In addition, VEGF189 as well as VEGF165 induced a 2-fold increase of Flk-1/KDR expression in HUVEC, the receptor involved in proliferation and migration of <em>endothelial</em> cells. In the Matrigel plug assay in vivo, both VEGF189 and 165 (100 ng/ml) increased the infiltration of <em>endothelial</em> cells. These data suggest that VEGF189 induced <em>endothelial</em> cell migration and proliferation under certain circumstances.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> is a powerful mitogen for <em>endothelial</em> cells, recently reported to be produced by keratinocytes. In the present work, we examined human keratinocytes in primary culture for the splice variants of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em>. In situ hybridization revealed that 100% of cultured human keratinocytes expressed mRNA for this cytokine, and analysis by reverse transcriptase-polymerase chain reaction indicated that three species of mRNA were produced. Southern hybridization and size calculations of PCR products revealed mRNA species corresponding to <em>121</em>, 165, and 189 amino-acid forms of this cytokine. Using a rabbit anti-<em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> antiserum, we radioimmunoprecipitated two molecular weight forms (approximately 45 and 58 kDa, non-reducing conditions) from keratinocyte culture supernatants. Under reducing conditions, three bands of approximately 15, 20, and 24 kDa appeared, corresponding with the predominant forms of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> described. We propose that secretion of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> by human keratinocytes in vivo sustains angiogenesis during physiologic tissue repair and in pathologic states accompanied by neo<em>vascular</em>ization.