Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by severe itching, erythema and edema resistant to anti-histamine therapy. <em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) is a potent agent that causes hyperpermeability of blood vessels and <em>endothelial</em> cell proliferation, and might be involved in the persisting erythema and edema in AD. In this study, we used extraction of stratum corneum with physiological saline to detect VEGF produced in the lesions of AD. Biological activity of VEGF was assayed by proliferation of cultured human umbilical vein <em>endothelial</em> cells in vitro. As a result, we found that the amount of VEGF produced in lesional scales was approximately 25 times higher than that in normal stratum corneum. Moreover, VEGF <em>121</em> isoform that exclusively induces hyperpermeability of blood vessels was a predominant component in the lesional scales suggesting that this <em>factor</em> plays an important role in the persisting erythema and edema in the AD lesions.

The angiogenic proteins basic fibroblast <em>growth</em> <em>factor</em> (bFGF; FGF-2) and <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> <em>121</em> (VEGF(<em>121</em>)) are each able to enhance the collateral-dependent blood flow after bilateral femoral artery ligation in rats. To study the effect of nitric oxide (NO) synthase (NOS) inhibition on bFGF- or VEGF(<em>121</em>)-induced blood flow expansion, the femoral arteries of male Sprague-Dawley rats were ligated bilaterally, and the animals were given tap water [non-N(G)-nitro-L-arginine methyl ester (L-NAME) group; n = 36] or water that contained L-NAME (L-NAME group; 2 mg/ml, n = 36). Animals from each group were further divided into three subgroups: vehicle (n = 12), bFGF (5 microg x kg(-1) x day(-1), n = 12), or VEGF(<em>121</em>) (10 microg x kg(-1) x day(-1), n = 12). <em>Growth</em> <em>factors</em> were delivered via intra-arterial infusion with osmotic pumps over days 1-14. On day 16, after a 2-day delay to permit clearance of bFGF and VEGF from the circulation, maximal collateral blood flow was determined by (85)Sr- and (141)Ce-labeled microspheres during treadmill running. L-NAME (approximately 137 mg x kg(-1) x day(-1)) for 18 days increased systemic blood pressure (approximately 26%, P<0.001). In the absence of L-NAME, collateral-dependent blood flows to the calf muscles were greater in the VEGF(<em>121</em>)- and bFGF-treated subgroups (85 +/- 4.5 and 80 +/- 2.9 ml x min(-1) x 100 g(-1), respectively) than in the vehicle subgroup (49 +/- 3.0 ml x min(-1) x 100 g(-1), P<0.001). In the presence of NOS inhibition by L-NAME, blood flows to the calf muscles were essentially equivalent among the three subgroups (54 +/- 3.0, 56 +/- 5.1, and 47 +/- 2.0 ml x min(-1) x 100 g(-1) in the bFGF-, VEGF(<em>121</em>)-, and vehicle-treated subgroups, respectively) and were not different from the blood flow in the non-L-NAME vehicle subgroup. Our results therefore indicate that normal NO production is essential for the enhanced <em>vascular</em> remodeling induced by exogenous bFGF or VEGF(<em>121</em>) in this rat model of experimental peripheral arterial insufficiency. These results imply that a blunted <em>endothelial</em> NO production could temper <em>vascular</em> remodeling in response to these angiogenic <em>growth</em> <em>factors</em>.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) is a potent angiogenic <em>factor</em> implicated in many pathological processes. We investigated the regulation of 4 alternatively spliced isoforms (<em>121</em>, 165, 189 and 206 amino acids) by hypoxia, hypoglycemia, acidity, female reproductive hormones and vitamin D in breast carcinoma cell lines representing different tumor phenotypes. There was a 17-fold difference in total VEGF mRNA expression across the cell lines. The isoform expression, <em>121</em>>> 165>> 189, was unchanged by different culture conditions. Hypoxia was the most potent stimulus, and the cell lines demonstrated a 1.4- to 6.9-fold range of VEGF induction, maintained when other hypoxically regulated genes (phosphoglycerate kinase 1 and glucose transporter 1) and a HIF-1-dependent reporter gene were examined. The relative inducibility of the genes was maintained in each cell line, but basal expression was independent of -fold induction. VEGF expression decreased under acidic conditions in 2 cell lines, but the hypoxia stimulus remained effective under acidic conditions. Hypoglycemia, female reproductive hormones and vitamin D exerted no effect on expression, nor did inhibitors of mutant ras. Our results show that VEGF expression varies widely between cell lines and that capacity to respond to hypoxia is also cell specific, relating mostly to the hypoxic sensing of the cell and the signal transduction mechanism. Such characteristics, if maintained in vivo, have implications for the angiogenic potential of different tumor cells under normal and hypoxic conditions.

The human airway epithelium is a pseudostratified heterogenous layer comprised of ciliated, secretory, intermediate, and basal cells. As the stem/progenitor population of the airway epithelium, airway basal cells differentiate into ciliated and secretory cells to replenish the airway epithelium during physiological turnover and repair. Transcriptome analysis of airway basal cells revealed high expression of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> A (VEGFA), a gene not typically associated with the function of this cell type. Using cultures of primary human airway basal cells, we demonstrate that basal cells express all of the three major isoforms of VEGFA (<em>121</em>, 165 and 189) but lack functional expression of the classical VEGFA receptors VEGFR1 and VEGFR2. The VEGFA is actively secreted by basal cells and while it appears to have no direct autocrine function on basal cell <em>growth</em> and proliferation, it functions in a paracrine manner to activate MAPK signaling cascades in endothelium via VEGFR2-dependent signaling pathways. Using a cytokine- and serum-free co-culture system of primary human airway basal cells and human <em>endothelial</em> cells revealed that basal cell-secreted VEGFA activated endothelium to express mediators that, in turn, stimulate and support basal cell proliferation and <em>growth</em>. These data demonstrate novel VEGFA-mediated cross-talk between airway basal cells and endothelium, the purpose of which is to modulate <em>endothelial</em> activation and in turn stimulate and sustain basal cell <em>growth</em>.

BACKGROUND

Vascular endothelial growth factor (VEGF) is a major regulator of angiogenesis and its expression is increased in non-small cell lung cancer (NSCLC). We aimed to determine the expression pattern of VEGF splice variants in NSCLC and its correlation with the clinicopathological characteristics of tumors.

METHODS

We used real-time reverse transcription PCR to quantify the mRNA expression of total VEGF, 4 VEGF splice variants (VEGF(121), VEGF(165), VEGF(183), and VEGF(189)), and 2 VEGF receptors (VEGFR-1 and VEGFR-2) in 27 pairs of cancerous and adjacent noncancerous tissues originating from patients with NSCLC.

RESULTS

Total VEGF, VEGF(121), and VEGF(165) were expressed in all specimens, whereas VEGF(183) and VEGF(189) were present in small amounts in certain samples. Total VEGF, VEGF(121), and VEGF(165) mRNA was upregulated in cancerous compared with healthy tissues, whereas VEGF(183) and VEGF(189) expression tended to be higher in healthy tissues. The expression of VEGFRs was similar between matched specimens. No correlation was found between the expression of total VEGF or VEGF splice variants and the clinicopathological characteristics of tumors. The expression patterns of VEGF splice variants differed between tissue pairs. VEGF(121) was the major variant expressed in all samples; however, its relative expression was higher in cancerous tissues. The relative expression of VEGF(183) and VEGF(189) was upregulated in healthy lung tissues, whereas the ratio of VEGF(165) to total VEGF was similar between matched specimens.

CONCLUSIONS

The expression pattern of certain VEGF splice variants is altered during tumorigenesis. Our data support the hypothesis that during malignant progression an angiogenic switch favoring the shorter diffusible isoforms occurs.

BACKGROUND

Patients with intermittent claudication caused by infrainguinal atherosclerosis have limited pharmacologic options "Therapeutic angiogenesis" is a novel treatment approach that seeks to improve perfusion of ischemic limbs by the induction of collateral vessel formation. This trial is a phase 2 randomized double-blind placebo-controlled proof of concept trial that will use an intramuscular adenoviral gene transfer approach of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em>, <em>121</em> isoform (Ad(GV)VEGF(<em>121</em>.10)) to patients with severe IC caused by infrainguinal disease.

METHODS

This is a phase 2, double-blind, randomized, placebo-controlled, dose-finding, multicenter study. Patients with severe intermittent claudication caused by infrainguinal atherosclerosis predominantly involving the superficial femoral artery confirmed with imaging studies that meet inclusion criteria will be stratified on the basis of the presence or absence of diabetes mellitus and randomized in a 1:1:1 fashion to low dose (4 x 10(9) particle units), high dose (4 x 10(10) particle units), or placebo arms (35-36 patients per group). Subjects are required to have exercise-limiting IC in the index extremity during 2 qualifying exercise treadmill tests, with peak walking times between 1 and 10 minutes. A single dose of Ad(GV)VEGF(<em>121</em>.10) will be administered as 20 intramuscular injections throughout the area of the lower limb requiring collateralization.

RESULTS

The primary efficacy parameter for the Regional Angiogenesis With Vascular Endothelial Growth Factor (RAVE) trial is the change in peak walking time at 12 weeks compared with baseline. The sample size is expected to provide an 80% power to detect a difference of 1.5 minutes between any of the 2 treatment groups and the placebo group. Secondary efficacy parameters include claudication onset time, hemodynamic effects of therapy assessed with ankle-brachial index, assessment of physical impairment, and health-related quality of life as measured with the Walking Impairment Questionnaire and SF-36 Health Survey. All randomized patients will also be evaluated for safety.

We have studied the effect of VEGF(<em>121</em>) homodimer and VEGF(<em>121</em>/165) heterodimer on the chorioallantoic membrane (CAM) of 13-day-old chick embryos. The <em>factors</em> were applied in doses of 2-4 micrograms and the effects were evaluated macroscopically after 2 and 3 days. Histological studies were performed on semi- and ultrathin sections. Proliferation was studied according to the BrdU-anti-BrdU method on whole mounts and sections. The labeling density was quantified in whole mounts. The fractal dimension, D, of the <em>vascular</em> tree was assessed as a value for <em>vascular</em> bifurcation density. Both forms of VEGF induce brush-like vessel formation in the precapillary region. New capillaries are found in the stroma of the CAM, which normally does not contain capillaries. Our results show that VEGF(<em>121</em>) is a specific <em>endothelial</em> cell mitogen. A fourfold increase of BrdU-labeled <em>endothelial</em> cells is found after VEGF(<em>121</em>) application. The fractal dimension of the <em>vascular</em> tree increases from 1.26 in the controls to 1.44 (VEGF(<em>121</em>)) and 1.41 (VEGF(<em>121</em>/165)). The <em>endothelial</em> cells of the newly formed capillaries possess many mitochondria and micropinocytotic vesicles, but no fenestrations. These capillaries are obviously formed by intussusceptive micro<em>vascular</em> <em>growth</em>. Signs of sprouting are almost absent. An effect on the lymphatic vessels of the CAM is not detectable. Compared to VEGF(165) and VEGF(<em>121</em>/165), VEGF(<em>121</em>) diffuses over a slightly greater distance. Using in situ hybridization, VEGF receptor-2 (flk-1/Quek1) and the homologous flt-4 (Quek2) receptor were studied in the CAM of normal quail embryos and after VEGF(<em>121</em>) application on the CAM of 11-day-old quail embryos. During normal development, flk-1 expression becomes restricted to <em>vascular</em> <em>endothelial</em> cells of large vessels in the stroma of the CAM. VEGF(<em>121</em>) application induces expression of flk-1 in capillaries that normally do not express the receptor. In the normal development of the CAM, flt-4 becomes restricted to <em>endothelial</em> cells of vessels that appear to be lymphatic vessels. Application of VEGF(<em>121</em>) does not alter flt-4 expression.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) promotes cartilage-degrading pathways, and there is evidence for the involvement of reactive oxygen species (ROS) in cartilage degeneration. However, a relationship between ROS and VEGF has not been reported. Here, we investigate whether the expression of VEGF is modulated by ROS. Aspirates of synovial fluid from patients with osteoarthritis (OA) were examined for intra-articular VEGF using ELISA. Immortalized C28/I2 chondrocytes and human knee cartilage explants were exposed to phorbol myristate acetate (PMA; 0-20 microg/ml), which is a ROS inducer, or 3-morpholino-sydnonimine hydrochloride (SIN-1; 0-20 microM), which is a ROS donor. The levels of VEGF protein and nitric oxide (NO) production were determined in the medium supernatant, using ELISA and Griess reagent, respectively. Gene expression of VEGF-<em>121</em> and VEGF-165 was determined by splice variant RT-PCR. Expression of VEGF and VEGF receptors (VEGFR-1 and VEGFR-2) was quantified by real-time RT-PCR. Synovial fluid from OA patients revealed markedly elevated levels of VEGF. Common RT-PCR revealed that the splice variants were present in both immortalized chondrocytes and cartilage discs. In immortalized chondrocytes, stimulation with PMA or SIN-1 caused increases in the levels of VEGF, VEGFR-1 and VEGFR-2 mRNA expression. Cartilage explants produced similar results, but VEGFR-1 was only detectable after stimulation with SIN-1. Stimulation with PMA or SIN-1 resulted in a dose-dependent upregulation of the VEGF protein (as determined using ELISA) and an increase in the level of NO in the medium. Our findings indicate ROS-mediated induction of VEGF and VEGF receptors in chondrocytes and cartilage explants. These results demonstrate a relationship between ROS and VEGF as multiplex mediators in articular cartilage degeneration.

Stimulating angiogenesis by gene transfer approaches offers the hope of treating tissue ischemia which is untreatable by currently practiced techniques of vessel grafting and bypass surgery. <em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) and basic fibroblast <em>growth</em> <em>factor</em> (FGF-2) are potent angiogenic molecules, making them ideal candidates for novel gene transfer protocols designed to promote new blood vessel <em>growth</em>. In this study, an ex vivo gene therapy approach utilizing cell encapsulation was employed to deliver VEGF and FGF-2 in a continuous and localized manner. C(2)C(12) myoblasts were genetically engineered to secrete VEGF(<em>121</em>), VEGF(165) and FGF-2. These cell lines were encapsulated in hollow microporous polymer membranes for transplantation in vivo. Therapeutic efficacy was evaluated in a model of acute skin flap ischemia. Capsules were positioned under the distal, ischemic region of the flap. Control flaps showed 50% necrosis at 1 week. Capsules releasing either form of VEGF had no effect on flap survival, but induced a modest increase in distal <em>vascular</em> supply. Delivery of FGF-2 significantly improved flap survival, reducing necrosis to 34.2% (P < 0.001). Flap <em>vascular</em>ization was significantly increased by FGF-2 (P < 0.01), with numerous vessels, many of which had a large lumen diameter, <em>growing</em> in the proximity of the implanted capsules. These results demonstrate that FGF-2, delivered from encapsulated cells, is more efficacious than either VEGF(<em>121</em>) or VEGF(165) in treating acute skin ischemia and improving skin flap survival. Furthermore, these data attest to the applicability of cell encapsulation for the delivery of angiogenic <em>factors</em> for the treatment and prevention of tissue ischemia.

BACKGROUND

Periodontal regeneration requires a coordinated series of events that includes not only the recruitment of periodontal ligament (PDL)-specific cells, but vascular cells as well. The mechanisms of action of enamel matrix derivative (EMD) are poorly understood, and its effects on vascular cells are unknown. The objective of this study was to examine the extent to which EMD affects angiogenesis and PDL cell recruitment.

METHODS

The effects of EMD on human microvascular endothelial cells (HMVECs) were determined by examining proliferation, chemotaxis, angiogenesis, and migration. Proliferation was determined using water-soluble tetrazolium salt (WST)-1 reagent. Chemotaxis was determined using microporous-culture well inserts. Angiogenesis was assessed on plates containing matrigel. The effects of HMVECs on the migration of PDL cells were assessed by evaluating PDL cell outgrowth from collagen gels cultured in the presence of HMVECs on fibrin matrix and surrounded by fibronectin-containing fibrin clots at 24 hours. Effects of EMD on PDL expression of vascular endothelial cell (VEGF) types (A, B, C, and D) and isoforms were determined using reverse transcription-polymerase chain reaction (RT-PCR). Production of VEGF, platelet-derived growth factor (PDGF)-AA, PDGF-BB, PDGF-AB, and transforming growth factor (TGF)-beta1 by EMD-stimulated PDL cells was assessed quantitatively in conditioned media using specific enzyme-linked immunosorbent assays (ELISAs).

RESULTS

EMD at concentrations <50 microg/ml resulted in significant (P <0.05) stimulation of HMVEC proliferation. Compared to baseline, EMD also stimulated a 100% increase in HMVEC chemotaxis when PDL cells were present (P <0.05). All doses of EMD tested (25, 50, and 100 microg/ml) increased angiogenesis in vitro. HMVECs, in combination with EMD at a concentration of 100 microg/ml, stimulated a 750% increase in migration of PDL cells from collagen gels into fibrin clots compared to controls when neither was present. RT-PCR results indicated that PDL cells expressed VEGF-A, -B, and -C and multiple isoforms of VEGF-A, including VEGF(121), VEGF(165), and VEGF(189), whether or not EMD was present in the culture media. ELISAs determined a 400% increase in VEGF concentration by PDL C cells in EMD-stimulated conditioned media and a similar increase in TGF-beta(1)-stimulated media.

CONCLUSIONS

It is likely that EMD stimulates angiogenesis directly by stimulating endothelial cells and indirectly by stimulating the production of angiogenic factors (VEGF) by PDL cells. Importantly, the data are consistent with the concept that EMD enhances bidirectional communication between HMVEC and PDL cells during angiogenesis associated with healing.

Pheochromocytomas are well-<em>vascular</em>ized tumors, suggesting that a potent angiogenic <em>factor</em> may be involved in the mechanism of their formation. As <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) is a potent mitogen for <em>vascular</em> <em>endothelial</em> cells, here we have investigated the mRNA and protein expression of VEGF and the mRNA expression of its two receptors (Flt-1 and Flk-1/KDR) in pheochromocytomas tissue. An increase in VEGF mRNA (mainly isoforms VEGF(<em>121</em>) and VEGF(165)) and in VEGF protein expression were observed by semi-quantitative RT-PCR and Western blot, respectively, compared to normal adrenomedullary tissue. Flk-1/KDR, and Flt-1 levels of mRNA were also increased markedly in tumors and correlated with levels of VEGF mRNA. Therefore, we speculate that upregulation of VEGF expression and its receptors might be important in the pathogenesis of pheochromocytomas.

BACKGROUND

Vascular endothelial growth factor (VEGF), an angiogenic and chemotactic peptide, is abundantly expressed in normal lung tissue, especially in alveolar and bronchial epithelium, glandular cells of the bronchi, and activated alveolar macrophages.

OBJECTIVE

To investigate the role of VEGF in progressively impaired lung function as the major complication and cause of death in septic patients.

METHODS

We evaluated pulmonary VEGF expression in lung autopsy material from septic patients who had been cared for by intensive care medicine using enzyme-linked immunosorbent assay (ELISA), reverse transcriptase-polymerase chain reaction (RT-PCR), and immunohistochemical methods.

RESULTS

Compared with expression in nonseptic control individuals (n = 10), pulmonary VEGF expression as determined by ELISA was significantly (P <.001) decreased in septic patients (n = 8). As monitored by RT-PCR, mRNA for the 2 splice variants, VEGF(121) and VEGF(165), and for VEGFR-2/KDR were expressed in both groups, the yields being lower in the sepsis group. Samples from septic patients lacked or showed only sparse immunoreaction on bronchial and alveolar epithelium, whereas this reaction was strong in all control samples. However, alveolar macrophages were similarly immunopositive in both groups.

CONCLUSIONS

The precise underlying mechanisms for the distinctly different expression of pulmonary VEGF in septic patients and nonseptic control individuals are not clear at present. Particularly the role of VEGF in the development of sepsis-induced lung injury and acute respiratory distress syndrome in mechanically ventilated patients suffering from severe sepsis remains to be clarified.

BACKGROUND

Angiogenesis is the target of several agents in the treatment of malignancies, including renal cell carcinoma (RCC). There is a real need for surrogate biomarkers that can predict selection of patients who may benefit from antiangiogenic therapies, prediction of disease outcome and which may improve the knowledge regarding mechanism of action of these treatments. Tyrosine kinase inhibitors (TKI) have proven efficacy in metastatic RCC (mRCC). However, the molecular mechanisms underlying the clinical response to these drugs remain unclear.

RESULTS

The present study aimed to identify molecular biomarkers associated with the response to sunitinib, a Tyrosine kinase inhibitor. To evaluate this relationship, primary tumors from 23 metastatic RCC patients treated by sunitinib were analyzed for a panel of 16 biomarkers involved in tumor pathways targeted by sunitinib, using real-time quantitative reverse-transcriptase PCR. Nine of the 23 patients (39%) responded to sunitinib. Among transcripts analyzed, only the levels of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) soluble isoforms (VEGF(<em>121</em>) and VEGF(165)) were associated with the response to sunitinib (P = 0.04 for both). Furthermore, the ratio of VEGF soluble isoforms (VEGF(<em>121</em>)/VEGF(165)) was significantly associated with prognosis (P = 0.02).

CONCLUSIONS

This preliminary study provides a promising tool that might help in the management of metastatic RCC, and could be extended to other tumors treated by TKI.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) is a multifunctional cytokine that exerts in vivo a key role in physiological and pathological neoangiogenesis by stimulating <em>endothelial</em> cell proliferation and vessel hyperpermeability. VEGF exists as one of four different isoforms, respectively, VEGF <em>121</em>, VEGF 165, VEGF 189, VEGF 206, and seems to be a crucial mediator of physiological neoangiogenesis during the embryonic development and the female cycle. VEGF also has a major role in the pathogenesis of many diseases including hypervascularized tumors, rheumatoid arthritis, cutaneous diseases and proliferative retinopathies. Anti-VEGF anti-bodies or VEGF agonists may represent a novel approach in the treatment of these diseases.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) performs as an angiogenic and permeability <em>factor</em> in ovarian cancer, and its overexpression has been associated with poor prognosis. However, models to study its role as a marker of tumor progression are lacking. We generated xenograft variants derived from the A2780 human ovarian carcinoma (1A9), stably transfected with VEGF(<em>121</em>) in sense (1A9-VS-1) and antisense orientation (1A9-VAS-3). 1A9, 1A9-VS-1, and 1A9-VAS-3 disseminated in the peritoneal cavity of nude mice, but only 1A9-VS-1, the VEGF(<em>121</em>)-overexpressing tumor variant, produced ascites. Tumor biopsies from 1A9-VS-1 showed alterations in the <em>vascular</em> pattern and caused an angiogenic response in the chorioallantoic membrane assay. A significant level of soluble VEGF was detectable in the plasma of mice bearing 1A9-VS-1 even at an early stage of tumor <em>growth</em>. Plasma VEGF correlated positively with tumor burden in the peritoneal cavity and ascites accumulation. Cisplatin reduced the tumor burden and ascites in mice bearing 1A9-VS-1; the response was associated with a significant decrease of VEGF in plasma. This 1A9-VS-1 xenograft model reproduces the behavior of human ovarian cancer by <em>growing</em> in the peritoneal cavity, being highly malignant, and producing ascites. Plasma VEGF as a marker of tumor progression offers a valuable means of detecting early tumor response and following up treatments in an animal model.

The <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF)/VEGF receptor (VEGFR) pathway is considered to be one of the most important regulators of angiogenesis and a key target in anticancer treatment. Imaging VEGFR expression can serve as a new paradigm for assessing the efficacy of antiangiogenic cancer therapy, improving cancer management, and elucidating the role and modulation of VEGF/VEGFR signaling during cancer development and intervention. In this study we developed an Avi-tagged VEGF(<em>121</em>) protein, which is site-specifically biotinylated in the presence of bacterial BirA biotin ligase. BirA biotinylated VEGF(<em>121</em>)-Avi (VEGF(<em>121</em>)-Avib) forms a stable complex with streptavidin-IRDye800 (SA800) that retains high affinity for VEGFR in vitro and allows receptor specific targeting in vivo in a 67NR murine xenograft model. In contrast, chemical coupling of IRDye800 abrogated the VEGFR binding ability of the modified protein both in vitro and in vivo. The VEGF(<em>121</em>)-Avib/SA800 complex (VEGF-Avib/SA800) may be used for quantitative and repetitive near-infrared fluorescence imaging of VEGFR expression and translated into clinic for evaluating cancer and other angiogenesis related diseases.

OBJECTIVE

Esophageal squamous cell carcinoma (ESCC) in the Indian population exhibits insidious symptomatology, late clinical presentation, aggressive behavior, and high propensity for metastasis. Ets-1, a transcription factor, is expressed in esophageal tumors and associated with poor prognosis. The aim of the present study was to determine the relationship between Ets-1 expression, tumor angiogenesis [vascular endothelial growth factor (VEGF) and microvessel density (MVD)] and the biological behavior of ESCCs.

METHODS

In a prospective study the expression of Ets-1, VEGF, and PECAM-1 (CD-31) was determined in 55 ESCCs, by immunohistochemical analysis, correlated with clinicopathological parameters and outcome of the patients.

RESULTS

Overexpression of Ets-1 and VEGF proteins was observed in 44/55 (80%) and 38/55 (69%) of ESCCs, respectively. VEGF immunopositivity was associated with lymph node metastasis ( P=0.002). Analysis of mRNA isoforms using RT-PCR revealed increased expression of VEGF 121 transcripts in ESCCs and MVD was correlated with de-differentiation status of the tumors ( P=0.049). Kaplan-Meier survival analysis showed significant correlation between poor disease-free survival and tumor stage ( P=0.02) and with nodal metastasis ( P=0.05). Concomitant expression of VEGF, Ets-1 proteins, and high MVD was correlated with poor disease-free survival ( P=0.004).

CONCLUSIONS

Significant association of Ets-1 and VEGF proteins with tumor angiogenesis (MVD), lymph node invasion, and poor disease-free survival underscores their relevance regarding aggressive tumor behavior and highlights their potential utility as adverse prognostic factors in esophageal carcinomas.

Critical limb ischemia (CLI) is typified by rest pain and/or tissue necrosis secondary to advanced peripheral arterial disease (PAD) and is characterized by diminution in limb perfusion at rest. We tested the safety of an angiogenic strategy with CI-1023 (Ad(GV)VEGF<em>121</em>.10), a replication-deficient adenovirus encoding human <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> isoform <em>121</em> in patients with CLI as part of a phase I trial. Fifteen subjects >35 years of age with CLI and angiographic disease involving the infra-inguinal vessels underwent intramuscular injection of CI-1023 (4 x 10(8) to 4 x 10(10) particle units, n = 13) or placebo (n = 2). All of the patients tolerated the injection well and there were no serious complications related to the procedure. Transient edema was noted in one patient. A total of 79 adverse events were reported over the course of one year. One death (day 136) and one malignancy (day 332) occurred in the CI-1023 group. CI-1023 appears to be well tolerated and safe for single-dose administration in patients with critical limb ischemia due to PAD. Further studies are needed to determine the efficacy of this form of therapeutic angiogenesis.

BACKGROUND

Vascular endothelial growth factor (VEGF) may contribute to the resolution of myocardial ischemia by stimulating collateral circulation. Morphine analgesia after myocardial ischemia is thought to increase infarct size. We hypothesize that morphine inhibits myocardial VEGF expression by inhibiting hypoxia-induced factor 1alpha (HIF-1alpha) and the signal transduction mechanisms involving Erk-1,2 MAP kinase (p42/p44), and PI3 kinase activity (phospho-Akt).

METHODS

(1) In vitro: primary cultures of rat cardiac myocytes; (2) in vivo: rat coronary ligation model; (3) mRNA measurement: real-time reverse transcriptase-polymerase chain reaction; (4) protein measurements: enzyme-linked immunosorbent assay, Western immunoblot, electromobility shift assay (EMSA), and immunohistochemistry.

RESULTS

Using rat cardiac myocytes in vitro, we show that morphine: (1) decreases hypoxia-induced VEGF(121) and VEGF(165) mRNA expression and VEGF protein concentration through an opioid receptor mechanism; (2) decreases HIF-1alpha protein expression (immunoblot) and nuclear protein binding to the VEGF HIF-1alpha DNA response element (EMSA); and (3) inhibits phospho-Erk-1,2 MAP kinase (immunoblot) and phospho-Akt kinase activity (immunoblot). Using a rat coronary ligation model, we show that morphine treatment: (1) decreases myocardial VEGF protein expression (immunohistochemistry); (2) decreases HIF-1alpha protein expression (immunoblot); and (3) decreases phospho-Erk-1,2 and phospho-Akt expression.

CONCLUSIONS

(1) Morphine inhibits hypoxia-induced VEGF transcription, in part, through an HIF-1alpha-mediated mechanism and (2) morphine inhibition of hypoxia-induced HIF-1alpha may be mediated by inhibition of ERK 1,2 MAP kinase activity and PI3 kinase activity.

The hematopoietic <em>growth</em> <em>factor</em> erythropoietin (Epo) circulates in plasma and controls the oxygen carrying capacity of the blood (Fisher. Exp Biol Med (Maywood) 228:1-14, 2003). Epo is produced primarily in the adult kidney and fetal liver and was originally believed to play a role restricted to stimulation of early erythroid precursor proliferation, inhibition of apoptosis, and differentiation of the erythroid lineage. Early studies showed that mice with targeted deletion of Epo or the Epo receptor (EpoR) show impaired erythropoiesis, lack mature erythrocytes, and die in utero around embryonic day 13.5 (Wu et al. Cell 83:59-67, 1995; Lin et al. Genes Dev. 10:154-164, 1996). These animals also exhibited heart defects, abnormal <em>vascular</em> development as well as increased apoptosis in the brain suggesting additional functions for Epo signaling in normal development of the central nervous system and heart. Now, in addition to its well-known role in erythropoiesis, a diverse array of cells have been identified that produce Epo and/or express the Epo-R including <em>endothelial</em> cells, smooth muscle cells, and cells of the central nervous system (Masuda et al. J Biol Chem. 269:19488-19493, 1994; Marti et al. Eur J Neurosci. 8:666-676, 1996; Bernaudin et al. J Cereb Blood Flow Metab. 19:643-651, 1999; Li et al. Neurochem Res. 32:2132-2141, 2007). Endogenously produced Epo and/or expression of the EpoR gives rise to autocrine and paracrine signaling in different organs particularly during hypoxia, toxicity, and injury conditions. Epo has been shown to regulate a variety of cell functions such as calcium flux (Korbel et al. J Comp Physiol B. 174:<em>121</em>-128, 2004) neurotransmitter synthesis and cell survival (Velly et al. Pharmacol Ther. 128:445-459, 2010; Vogel et al. Blood. 102:2278-2284, 2003). Furthermore Epo has neurotrophic effects (Grimm et al. Nat Med. 8:718-724, 2002; Junk et al. Proc Natl Acad Sci U S A. 99:10659-10664, 2002), can induce an angiogenic phenotype in cultured <em>endothelial</em> cells and is a potent angiogenic <em>factor</em> in vivo (Ribatti et al. Eur J Clin Invest. 33:891-896, 2003) and might enhance ventilation in hypoxic conditions (Soliz et al. J Physiol. 568:559-571, 2005; Soliz et al. J Physiol. 583, 329-336, 2007). Thus multiple functions have been identified breathing new life and exciting possibilities into what is really an old <em>growth</em> <em>factor</em>.This review will address the function of Epo in non-hematopoietic tissues with significant emphasis on the brain and heart.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) promotes neo<em>vascular</em>ization, micro<em>vascular</em> permeability, and <em>endothelial</em> proliferation. We described previously VEGF mRNA and protein induction by estradiol (E2) in human endometrial fibroblasts. We report here E2 induction of VEGF expression in human venous muscle cells [smooth muscle cells (SMC) from human saphenous veins; HSVSMC] expressing both ER-alpha and ER-beta estrogen receptors. E2 at 10(-9) to 10(-8) M increases VEGF mRNA in HSVSMC in a time-dependent manner (3-fold at 24 h), as analyzed by semiquantitative RT-PCR. This level of induction is comparable with E2 endometrial induction of VEGF mRNA. Tamoxifen and hypoxia also increase HSVSMC VEGF mRNA expression over control values. Immunocytochemistry of saphenous veins and isolated SMC confirms translation of VEGF mRNA into protein. Immunoblot analysis of HSVSMC-conditioned medium detects three bands of 18, 23, and 28 kDa, corresponding to VEGF isoforms of <em>121</em>, 165, and 189 amino acids. Radioreceptor assay of the conditioned medium produced by E2-stimulated HSVSMC reveals an increased VEGF secretion. Our data indicate that VEGF is E2, tamoxifen, and hypoxia inducible in cultured HSVSMC and E2 inducible in aortic SMC, suggesting E2 modulation of VEGF effects in angiogenesis, <em>vascular</em> permeability, and integrity.

In traction tendons, whose line of action corresponds to that of the muscle, few blood vessels are uniformly distributed within the tendon tissue. In gliding tendons, which change their direction of pull, an a<em>vascular</em> zone is normally found in the region where the tendon wraps around the pulley. This a<em>vascular</em> fibrocartilaginous gliding zone is predisposed for degenerative changes and spontaneous rupture. Since <em>factors</em> regulating angiogenesis in tendons are largely unknown, we analyzed the expression of the <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) and its receptors VEGFR-1 (flt-1) and VEGFR-2 (KDR) in human fetal and adult tendon tissue by immunohistochemical, biochemical, and molecular biology methods. In order to elucidate whether mechanical stress might influence VEGF expression in tendon tissue we loaded primary cultures of rat tenocytes with intermittent hydrostatic pressure in a special cell culture chamber (amplitude: 0.2 Mpa, frequency: 0.1 Hz, time period: 5 h/day) and measured VEGF expression using ELISA. In fetal tendons high VEGF levels could be quantified by ELISA, whereas negligible ones were found in adult tissue. VEGF could be immunostained in tenocytes and <em>endothelial</em> cells. In the tibialis posterior tendon - as an example for a gliding tendon - VEGF immunostaining decreased in the gliding zone adjacent to the bony hypomochlion between week 20 and week 24 after gestation. This region remained largely a<em>vascular</em> during the fetal period. In the peritendineum and in regions proximally and distally of the gliding zone immunostaining for VEGF was positive and <em>factor</em> VIII-positive microvessels could be detected. In these vessels, the VEGFR-1 (flt-1) and the VGEFR-2 could also be visualized. Reverse transcription-polymerase chain reaction (RT-PCR) confirmed the results regarding VEGF expression and showed further that the splice variants VEGF(<em>121</em>) and VEGF(165) are expressed exclusively during angiogenesis in fetal tendons. Monolayer cultures of tendon cells released measurable amounts of VEGF. Application of intermittent hydrostatic pressure decreased VEGF expression significantly. Thus, the angiogenic peptide VEGF is present in human fetal tendons, which are exposed to traction, but not in the a<em>vascular</em> zone of gliding tendons, which are predominantly exposed to compressive and shearing forces. These findings support the view that the development of a<em>vascular</em> zones in tendons might be caused by a mechanically induced downreglation of VEGF expression.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) is being investigated as a potential interventional therapy for spinal cord injury (SCI). In the current study, we examined SCI-induced changes in VEGF protein levels using Western blot analysis around the epicenter of injury. Our results indicate a significant decrease in the levels of VEGF(165) and other VEGF isoforms at the lesion epicenter 1 day after injury, which was maintained up to 1 month after injury. We also examined if robust VEGF(165) decrease in injured spinal cords affects neuronal survival, given that a number of reported studies show neuroprotective effect of this VEGF isoform. However, exogenously administered VEGF(165) at the time of injury did not affect the number of sparred neurons. In contrast, exogenous administration of VEGF antibody that inhibits actions of not only VEGF(165) but also of several other VEGF isoforms, significantly decreased number of sparred neurons after SCI. Together these results indicate a general reduction of VEGF isoforms following SCI and that isoforms other than VEGF(165) (e.g., VEGF(<em>121</em>) and/or VEGF(189)) provide neuroprotection, suggesting that VEGF(165) isoform is likely involved in other pathophysiological process after SCI.

Administration of adenovirus (Ad) vectors to animals induces innate immune responses, typified by elevated interleukin-6 (IL-6). To assess innate responses to Ad vectors in humans, we evaluated serum IL-6 following administration of E1(-) E3(-) Ad vectors to different human hosts and the relationship among peak IL-6 and peak anti-Ad neutralizing antibodies. We administered: 1) Ad(GV)CFTR.10, a vector carrying the normal human CFTR cDNA (3 x 10(7) to 2 x 10(10) particle units (pu)) to airways of individuals with cystic fibrosis (CF); 2) Ad(GV)VEGF<em>121</em>.10, a vector carrying the normal human <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF)<em>121</em> cDNA, to the myocardium (4 x 10(8) to 4 x 10(10) pu) of individuals with coronary artery disease (CAD) and to lower extremity muscles (4 x 10(8) to 4 x 10(9.5) pu) of individuals with peripheral <em>vascular</em> disease (PVD); and 3) Ad(GV)CD.10, a vector carrying the Escherichia coli cytosine deaminase gene to skin (7 x 10(7) to 7 x 10(9) pu) and airways (7 x 10(8) to 7 x 10(10) pu) of normal individuals and to liver metastasis (4 x 10(8) to 4 x 10(9) pu) of individuals with colon carcinoma. IL-6 increased mildly (up to 220 pg/ml) following vector administration to skin and lung airways of normal individuals and of individuals with CF, and to muscle and liver metastasis of individuals with PVD and colon cancer, respectively. IL-6 responses were higher (up to 1100 pg/ml) following myocardial administration. Control individuals who had chest surgery and bronchoscopy, but no vector administration, had comparable IL-6 increases. Thus, both administration of Ad vectors of humans up to 10(10) pu and the procedures used to administer the vectors elicit systemic IL-6 responses. There was no correlation among peak IL-6 and peak anti-Ad antibodies. These observations indicate that the innate host responses following administration of Ad vectors to humans may result from the procedures used to administer the vector, and from the vector per se.