<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) is a mitogen in physiological and pathological angiogenesis. Understanding the expression of different VEGF isoforms might be important for distinguishing angiogenesis in tissue development, <em>vascular</em> remodelling and tumour formation. We examined its expression and noted the presence of the isoforms VEGF(<em>121</em>) and VEGF(165) (<em>121</em> and 165 residues long respectively) in fetal heart, lung, ovary, spleen, placenta and ovarian tumours. Unexpectedly, a 47 kDa species predominated in fetal intestine and muscle. The presumed initiation site in VEGF is an AUG codon (AUG(1039)), 1039 nt from its main transcriptional start site. AUG(1039) is preceded in the 5' untranslated region by an in-frame CUG at nt 499 (CUG(499)), which could produce the 47 kDa form with a 180-residue N-terminal extension. We therefore assessed whether CUG(499) functions as an initiator. CUG(499) initiation produced the 47 kDa VEGF(165) precursor, which was processed at two sites to yield VEGF and three N-terminal fragments. When CTG(499) was mutated to CGC, the precursor and N-terminal fragments were barely detectable. Although the precursor form was predominant in VEGF(165), both CUG(499) and AUG(1039) forms were found in VEGF(<em>121</em>). VEGF precursor induced neither the proliferation of human umbilical vein <em>endothelial</em> cells nor the expression of angiopoietin 2, which can be induced by, and act with, VEGF to induce tumour angiogenesis. The precursor also adheres to the extracellular matrix (ECM), suggesting that it might be a storage form for generating active VEGF in the cell or ECM. Alternate CUG(499) and AUG(1039) initiation and processing of the inactive precursor and its products might be important in regulating angiogenesis.

Chronic mountain sickness (CMS), a maladaptation syndrome to chronic hypoxia, occurs in the Andes. Gene expression differences in Andeans could explain adaptation and maladaptation to hypoxia, both of which are relevant to neurology at sea level. Expression of genes responsive to cellular oxygen concentration, hypoxia-inducible <em>factor</em>-1alpha (HIF-1alpha), three splicing variants of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) and von Hippel-Lindau protein (pVHL) was measured by reverse transcription polymerase chain reaction (RT-PCR) in 12 Cerro de Pasco (CP) (altitude 4338 m) natives and 15 CMS patients in CP. Thirteen high altitude natives living in Lima and five Lima natives were sea level controls. A CMS score (CMS-sc) was assigned clinically. Expression was related to the clinical assessment. High expression of HIF-1alpha and VEGF-<em>121</em> was found in CMS (P<0.001). Samples from CP had higher expression than those from Lima (P<0.001). Expression of HIF-1alpha and VEGF-<em>121</em> was related to age (P<0.001); adjusting for age did not abolish the group effect. Higher CMS-sc was related to expression independent of age (P<0.001). VEGF-165 and -189 were expressed only in CMS. Birth altitude had no effect on gene expression. pVHL was not quantifiable.HIF-1alpha and VEGF-<em>121</em> participate in adaptation to hypoxia. The high levels may explain blood vessel proliferation in Andeans and hold lessons for patients at sea level. VEGF-165 expression suggests that it contributes to preservation of neuronal function in human chronic hypoxia. VHL mutations may mark those destined to develop neural crest tumors which are common in the Andes.

The <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) receptor tyrosine kinase subtype kinase insert domain receptor (KDR) contains seven extracellular Ig-like domains, of which the three most amino-terminal contain the necessary structural features required for VEGF binding. To clarify the functional role of KDR Ig-like domains 4-7, we compared VEGF-induced signaling in human embryonic kidney and porcine aortic <em>endothelial</em> cells expressing native versus mutant receptor proteins in which Ig-like domains 4-7, 4-6, or 7 had been deleted. Western blotting using an anti-receptor antibody indicated equivalent expression levels for each of the recombinant proteins. As expected, VEGF treatment robustly augmented native receptor autophosphorylation. In contrast, receptor autophosphorylation, as well as downstream signaling events, were VEGF-independent for cells expressing mutant receptors. (125)I-VEGF(165) bound with equal or better affinity to mutant versus native receptor, although the number of radioligand binding sites was significantly reduced because a significant percentage of mutant, but not native, receptors were localized to the cell interior. As was the case for native KDR, (125)I-VEGF(165) binding to the mutant receptors was dependent upon cell surface heparan sulfate proteoglycans, and (125)I-VEGF(<em>121</em>) bound with an affinity equal to that of (125)I-VEGF(165) to the native and mutant receptors. It is concluded that KDR Ig-like domains 4-7 contain structural features that inhibit receptor signaling by a mechanism that is independent of neuropilin-1 and heparan sulfate proteoglycans. We speculate that this provides a cellular mechanism for blocking unwanted signaling events in the absence of VEGF.

We investigated the effects of histone deacetylase (HDAC) inhibitors such as sodium butyrate (SB) and trichostatin A (TSA) on the expression of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) by human glioblastoma T98G, U251MG, and U87MG cells. The glioblastoma cells secreted three VEGF isoforms, VEGF (189), (165), and (<em>121</em>), although the expression levels of VEGF differed between the cell types. Treatment with either 5mM SB or 100 ng/ml TSA reduced VEGF secretion in conditioned media and reduced VEGF mRNA expression. We also studied the expression of VEGF-B, -C, and -D mRNA in human glioblastoma cells and their modulation by HDAC inhibitors. The PCR products of VEGF-B (357bp), VEGF-C (501bp), and VEGF-D (484bp) were amplified in all glioblastoma cells examined. Treatment with SB reduced the expression of VEGF-D mRNA in U251MG cells and the expression of VEGF-B mRNA in U87MG cells. TSA treatment reduced the expression of VEGF-D in U251MG cells. These results suggest that HDAC inhibitors reduce VEGF secretion and modulate the expression of the other VEGF family members, and therefore may inhibit angiogenesis in glioblastoma tissues.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) is a highly specific <em>factor</em> for <em>vascular</em> <em>endothelial</em> cells. Five VEGF-A isoforms (splice variants <em>121</em>, 145, 165, 189 and 206) are generated as a result of alternative splicing from a single VEGF-A gene. These differ in their molecular weights and in biological properties such as their ability to bind to cell-surface heparan sulfate proteoglycans. Deregulated VEGF-A expression contributes to the development of solid tumors by promoting tumor angiogenesis. More specifically, VEGF-A189 expression is related to angiogenesis and prognosis in certain human solid tumors. VEGF-A189 expression is also related to the xenotransplantability of human cancers into immunodeficient mice in vivo. Consequently, inhibition of VEGF-A or VEGF-A189 signaling regulates the development and metastasis of a variety of tumors. This review focuses on recent studies of the mechanisms by which VEGF-A regulates angiogenesis in the cancer stroma and on our recent findings concerning the potential mechanisms of VEGF-A189 expression on tumor <em>growth</em> and metastasis.

Human cancer cells often produce tumors in animal models that incompletely reproduce the histology of the parental tumor. Kaposi's sarcoma (KS) cells, in particular, have not produced durable angiogenic lesions in animal models that resemble those of KS in humans. We investigated the contribution of transformed KS cells, <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF), and human skin tissue on tumor development in a human skin graft/mouse model. High levels of serum VEGF (322 pg/ml) were seen in HIV-1-infected persons with KS compared with HIV-1-infected persons without KS (115 pg/ml). Human KS lesions expressed VEGF in the spindle cells. Transformed KS cells expressed the mitogenically active <em>121</em>-amino acid and 165-amino acid isoforms of VEGF. Tumors induced by KS cells implanted in the SCID mice grew preferentially in human skin grafts rather than in ungrafted murine skin. Tumors induced in the presence of human skin grafts developed numerous lumens expressing alpha(v)beta(3) integrin. KS cells inoculated with neutralizing anti-VEGF antibody did not form tumors. This study supports an important role for VEGF in tumor development and shows how a human tissue can preferentially promote tumor <em>growth</em>.

BACKGROUND

Myocardial ischemia is the most common cause of congestive heart failure. Angiogenic therapy has recently been demonstrated to enhance myocardial perfusion in the ischemic setting. We therefore hypothesized that administration of adenovirus encoding for vascular endothelial growth factor could be used to enhance myocardial function in a pacing-induced model of heart failure.

METHODS

Yorkshire swine underwent a left thoracotomy with placement of a ventricular epicardial pacing system. Animals received adenovirus coding either for the 121-amino-acid isoform of vascular endothelial growth factor (Ad(CU)VEGF121.1 group, n = 8) or a null vector coding for no genes (AdNull group, n = 8). The adenovirus was administered in the left ventricular free wall as 10 transepicardial injections of 100 microL each (total dose of 10(11) particle units). After a 1-week recovery period, animals were paced at a rate of 230 beats/min for 7 days to induce heart failure. Transthoracic echocardiographic and sonomicrometric measurements were performed before pacing (baseline), on termination of pacing (day 0), and then weekly for 3 weeks.

RESULTS

The fractional area change was significantly decreased in AdNull animals at day 0 after pacing compared with the Ad(CU)VEGF121.1 animals (29% +/- 14% vs 46% +/- 8%, P =.02). The fractional area change recovered to baseline values within 7 days in the Ad(CU)VEGF121.1 animals (62% +/- 7%) but remained significantly impaired in the AdNull group compared with that in the Ad(CU)VEGF121.1 animals up to day 21 (P =.04). Similarly, fractional wall thickening demonstrated a decrease at day 0 after pacing that was greater (P <.05) in the AdNull group compared with that in the Ad(CU)VEGF121.1 group in 5 of 6 segments. Fractional wall thickening returned to levels approximating prepacing values in all segments within 7 days in the Ad(CU)VEGF121.1 group but remained significantly impaired compared with prepacing fractional wall thickening (P <.05) in the AdNull group in 5 of 6 segments up to day 21 after pacing. Segmental shortening, as measured by sonomicrometry, also was significantly decreased at day 7 in the AdNull group compared with that in the Ad(CU)VEGF121.1 group (10% +/- 4% vs 16% +/- 3%, P =.004) and remained significantly impaired (P <.05) in the AdNull group at day 14 and 21 when compared with baseline values.

CONCLUSIONS

Preservation of cardiac performance and a more rapid recovery of myocardial function can be achieved in a model of pacing-induced cardiomyopathy with adenovirus-mediated administration of vascular endothelial growth factor compared with that seen in a null virus control group. These data suggest that angiogenic therapy may be useful clinically in treating cardiomyopathy.

<em>Vascular</em> <em>endothelial</em> <em>growth</em>/permeability <em>factor</em> (VEG/PF) has an established role in angiogenesis, however, the regulation of placental VEG/PF expression during primate pregnancy is incompletely understood. A temporal study was conducted in baboons to determine the effect of acute administration of estradiol on the expression of VEG/PF by cells of the villous placenta. VEG/PF mRNA levels were determined by reverse transcription-polymerase chain reaction in isolated placental cell fractions of baboons after acute i.v. and i.m. administration of estradiol. Within 2 h of estradiol treatment, VEG/PF mRNA (attomoles/ micrograms total RNA) increased within villous cytotrophoblasts to a level (mean +/- SEM, 12,612 +/- 2419) that was almost 2-fold greater (P < 0.05) than in untreated controls (6810 +/- 1368). Cytotrophoblast VEG/PF mRNA levels remained elevated (P < 0.01) 6 h after estradiol treatment (15,006 +/- 506), but were not different from controls 18 h after estradiol administration. VEG/ PF mRNA levels in whole villous tissue also were greater 6 h (12,667 +/- 2284, P < 0.05) and 18 h (16,080 +/- 3816, P < 0.01) after estradiol treatment than in untreated animals (3380 +/- 594). In contrast, VEG/PF mRNA levels in cells of the inner villous core were not altered by estradiol treatment. Expression of both the VEG/PF(<em>121</em>) and VEG/PF(165) mRNA species appeared to increase in the placenta 6 h after estradiol treatment of baboons. We propose that estrogen regulates VEG/PF expression within the placenta in a cell-specific manner, providing a paracrine system to promote vascularization of the villous placenta during the first half of primate pregnancy.

BACKGROUND

Vascular endothelial growth factor (VEGF)-A and angiopoietin (Ang)-1 and Ang-2 are key factors in angiogenic signaling. In this study the expression of these factors was identified in cartilage tumors. As interleukin (IL)-1beta has been found to be an indispensable factor in angiogenic signaling, we further analyzed the effect of IL-1beta on the expression of VEGF-A, Ang-1, and Ang-2 using a previously established cell culture model.

METHODS

Surgical specimens of enchondromas, conventional chondrosarcomas, and dedifferentiated chondrosarcomas were obtained from 72 patients. VEGF-A, Ang-1, and Ang-2 mRNA expression was detected by conventional and quantitative reverse transcription polymerase chain reaction (PCR). VEGF-A expression was also detected by immunohistochemistry or Western blot.

RESULTS

Differential expression of VEGF-A, Ang-1, and Ang-2 was clearly demonstrated in cartilage tumors. VEGF-A expression was positively correlated with the tumor type. Higher VEGF-A expression levels were detected in conventional chondrosarcomas Grades II and III (using a 3-tier grading system) than in dedifferentiated chondrosarcomas (P < .05). A typical pattern of VEGF-A isoforms was identified, including VEGF(121), VEGF(145), VEGF(165), and VEGF(189). Ang-1 presented as a low-level transcript with slightly elevated levels in chondrosarcomas (P < .05). Highly variable Ang-2 expression levels were detected in solitary cases of conventional chondrosarcomas. IL-1beta regulated VEGF-A and Ang-1 expressions in a dose-dependent manner. Whereas low IL-1beta concentrations increased VEGF-A and Ang-1 transcription, high IL-1beta concentrations had the opposite effect. IL-1beta did not activate Ang-2 expression.

CONCLUSIONS

Angiogenic signaling in cartilage tumors is variable and at least partly regulable by IL-1beta. The findings are of therapeutic relevance, either as a desired effect or a side effect in medical treatment.

It has been documented that hypoxia enhances coronary vasculogenesis and angiogenesis in cultured embryonic quail hearts via the upregulation of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF). In this study, we compared the functions of two VEGF splice variants. Ventricles from 6-day-old embryonic quail hearts were cultured on three-dimensional collagen gels. Recombinant human VEGF(<em>121</em>) or VEGF(165) were added to the culture medium for 48 h, and <em>vascular</em> <em>growth</em> was visualized by immunostaining with a quail-specific <em>endothelial</em> cell (EC) marker, QH1. VEGF(165) enhanced <em>vascular</em> <em>growth</em> in a dose-dependent manner: 5 ng/ml of VEGF(165) slightly increased the number of ECs, 10 ng/ml of VEGF(165) increased the incorporation of ECs into tubular structures, and at 20 ng/ml of VEGF(165) wider tubes were formed. This pattern plateaued at the 50 ng/ml dose. In contrast, VEGF(<em>121</em>) did not enhance either the number of ECs or tube formation at these or higher dosages. Combined effects of hypoxia and exogenous VEGF(165) were then compared. Tube formation from the heart explants treated with both hypoxia and 50 ng/ml of VEGF(165) had a morphology intermediate to those treated with hypoxia or VEGF(165) alone. Immunocytochemistry study revealed EC lumenization under all culture conditions. However, the addition of VEGF(165) stimulated the coalescence of ECs to form larger vessels. We conclude the following: 1) VEGF(<em>121</em>) and VEGF(165) induced by hypoxia have different functions on coronary <em>vascular</em> <em>growth</em>, 2) unknown <em>factors</em> induced by hypoxia can modify the effect of VEGF(165), and 3) EC lumenization observed in the heart explant culture closely mimics in vivo coronary vasculogenesis.

OBJECTIVE

To examine the effect of loss of cell-cell contacts on the gene expression of vascular endothelial growth factor (VEGF) and other factors in primary culture of human retinal pigment epithelial (RPE) cells with real-time reverse transcription-PCR.

METHODS

The dissociation of postconfluent RPE cells was induced by calcium chelation, low-calcium medium, anti-E-cadherin, and anti-N-cadherin antibodies. Total RNA was isolated from the cultured RPE cells and reverse transcribed to cDNA. VEGF was quantified by real-time PCR with a fluorescence detector. VEGF isoforms were differentially measured by specific exon-spanning primers. Besides VEGF, the gene expression levels of some other growth factors were also examined in calcium-mediated dissociation.

RESULTS

Disruption of cell-cell contacts of RPE cells was induced by calcium chelation and low-calcium medium, but not by anti-E-cadherin and anti-N-cadherin antibodies. Calcium-mediated dissociation of RPE cells significantly increased the gene expression levels of VEGF. The mRNA levels of VEGF increased by 6.3-fold on treatment with EGTA and by 4.7-fold in the low-calcium medium at 6 hours. Splice variants of VEGF showed the differential pattern of gene expression. Whereas the expression of VEGF(121) and VEGF(165) was upregulated on calcium-induced dissociation of RPE cells, that of VEGF(145) and VEGF(189) was unchanged. VEGF(206) was not detected. On calcium-induced dissociation, bFGF, IL-6, matrix metalloproteinase (MMP)-1, and placental growth factor (PlGF) were upregulated, whereas acidic (a)FGF and pigment epithelium-derived factor (PEDF) were both downregulated.

CONCLUSIONS

The results show that loss of intercellular contacts promotes increased gene expression of VEGF and other angiogenic factors in human RPE cells.

<em>Vascular</em> <em>endothelial</em> cell <em>growth</em> <em>factor</em> (VEGF) is a potent mitogen and permogen that increases in the plasma and decreases in the alveolar space in respiratory diseases such as acute respiratory distress syndrome (ARDS). This observation has led to controversy over the role of this potent molecule in lung physiology and disease. We hypothesized that some of the VEGF previously detected in normal lung may be of the anti-angiogenic family (VEGF(xxx)b) with significant potential effects on VEGF bioactivity. VEGF(xxx)b protein expression was assessed by indirect immunohistochemistry in normal and ARDS tissue. Expression of VEGF(xxx)b was also detected by immunoblotting in normal lung tissue, primary human alveolar type II (ATII) cells, and bronchoalveolar lavage (BAL) fluid in normal subjects and by ELISA in normal, "at risk," and ARDS subjects. The effect of VEGF(165) and VEGF(165)b on both human primary <em>endothelial</em> cells and alveolar epithelial cell proliferation was assessed by [(3)H]thymidine uptake. We found that VEGF(165)b was widely expressed in normal healthy lung tissue but is reduced in ARDS lung. VEGF(<em>121</em>)b and VEGF(165)b were present in whole lung, BAL, and ATII lysate. The proliferative effect of VEGF(165) on both human primary <em>endothelial</em> cells and human alveolar epithelial cells was significantly inhibited by VEGF(165)b (P < 0.01). These data demonstrate that the novel VEGF(xxx)b family members are expressed in normal lung and are reduced in ARDS. A specific functional effect on primary human <em>endothelial</em> and alveolar epithelial cells has also been shown. These data suggest that the VEGF(xxx)b family may have a role in repair after lung injury.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) is implicated in numerous pathologies through complex relationships with cellular adhesion molecules (CAMs) and inflammation markers. These have not been assessed in non-pathological conditions. Our aim was the evaluation of associations between VEGF and CAM/inflammation molecules in a healthy population, and of possible genomic interplays in order to better apprehend the underlying mechanisms leading to the pathology. We examined the associations between VEGF and ICAM-1, VCAM-1, E-, L-, P-selectins, TNF-α, CRP and IL-6 plasma levels in 403 healthy individuals. Gene expression of CAM/inflammation molecules and VEGF isoforms (<em>121</em>, 145, 165, and 189) were quantified in peripheral blood mononuclear cells (PBMCs). The effect of four genetic variants (explaining ≈ 50% of the heritability of circulating VEGF levels) and of their interactions on plasma and mRNA levels of CAM/inflammation molecules was examined. VEGF was associated with ICAM-1 and E-selectin in plasma. In PBMCs, VEGF(145) mRNA was associated with ICAM-1, L-selectin and TNF-α expression. Interactions of the genetic variants were shown to affect ICAM-1, E-selectin, IL-6 and TNF-α plasma levels, while rs4416670 was associated with L-selectin expression. These findings propose a biological connection between VEGF and CAM/inflammation markers. Common genetic and transcriptional mechanisms may link these molecules and control their effect in healthy conditions.

BACKGROUND

Overexpression of vascular endothelial growth factor (VEGF) is associated with increased angiogenesis, growth and invasion in solid tumors, and hematologic malignancies. The expression of isoforms of VEGF, which mediate different effects, can be discriminated by splice-variant-specific quantitative reverse transcription-PCR (RT-PCR), but current methods have only modest sensitivity and precision and suffer from heteroduplex formation.

METHODS

We used a real-time RT-PCR assay on the LightCycler system. Applicability for detection of different VEGF mRNAs and total VEGF message was tested on seven healthy tissues (each pooled from healthy donors) and seven correlated malignant tissues. Results were normalized to beta(2)-microglobulin mRNA. Amplification of VEGF splice variants was performed exclusively with variant-specific reverse primers, whereas forward primer and fluorescent probe were common to obtain similar RT-PCR kinetics.

RESULTS

Highly specific detection of VEGF splice variants was achieved with minor intra- and interassay variation (<0.22 threshold cycle). Total VEGF expression was higher in malignant tissues. In healthy tissues, the mRNA encoding diffusible variants VEGF(121) and VEGF(165) constituted on average 78% (SD = 9.3%) of the total VEGF message, and the cell-adherent variant VEGF(189) constituted on average 22% (SD = 5.4%). In contrast, in malignant tissues VEGF(121) and VEGF(165) accounted for 94% (SD = 7.6%) and VEGF(189) only 6% (SD = 3.7%).

CONCLUSIONS

Because of the ability for quantification of VEGF splice variants with high specificity, sensitivity, and reproducibility, this new LightCycler assay is superior to conventional semiquantitative competitive RT-PCR and immunological assays and may contribute to better understanding of VEGF-mediated angiogenesis.

<em>Endothelial</em> cells lining vessels of endocrine tissues are fenestrated. Interactions with the local environment via either soluble <em>factors</em> or cell-cell interactions appear to govern this terminal <em>endothelial</em> differentiation. Adrenocorticotropin (ACTH) has previously been reported to modulate <em>endothelial</em> fenestration in the rat adrenal cortex. Since <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) has been characterized as a potent inducer of <em>endothelial</em> fenestration, we aimed to characterize the status of VEGF expression in the bovine adult adrenal cortex and asked whether ACTH may regulate VEGF expression. By immunohistochemical analysis, we observed VEGF expression in steroidogenic cells from both zona glomerulosa and zona fasciculata of the bovine adrenal cortex. Double-labeling experiments performed on isolated cells in primary culture revealed VEGF immunoreactivity, essentially colocalized with the Golgi apparatus. The expression of two predominant VEGF isoforms, VEGF(<em>121</em>) and VEGF(165), was observed by RT-PCR analysis. ACTH (10 nM) was found to rapidly (within 2-4 h) increase the abundance of these VEGF transcripts, as assessed by both RT-PCR and Northern blot analysis. In parallel, ACTH significantly induced VEGF secretion into the medium of fasciculata cells in primary culture. Thus, our data are consistent with the involvement of ACTH, through its regulation of VEGF expression, in the maintenance of the adult adrenal cortex endothelium.

Micro<em>vascular</em> surgery has emerged as an attractive area for recent advances in the field of gene therapy. The present study investigated the survival of ischemic, experimental skin flaps after treatment with the gene encoding <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF). In 30 Sprague-Dawley rats, anterior abdominal skin flaps supplied by the epigastric artery and vein were created. Ten animals were treated with a mixture of liposomes and the cDNA encoding the <em>121</em>-amino acid isoform of VEGF. Another 10 animals were treated with control plasmid DNA and liposome transfection medium; a third group of 10 animals was given physiologic saline. Each solution was injected directly into the femoral artery distal to the origin of the epigastric pedicle supplying the flap. Four days after injection, the pedicle was ligated and blood flow in the flap was approximated using dye fluorescence. Seven days later, the amount of viable tissue within the flap was measured by planimetry. After the animals were killed, specimens from both the operated and nonoperated sides of the abdomen were harvested for immunohistologic evidence of VEGF protein expression. Average dye fluorescence indices of the three groups (VEGF cDNA, control plasmid, and saline) 2 hours after pedicle ligation were 35.9, 23.9, and 53.9 percent, respectively (p < 0.05). Compared with the two control groups, flaps receiving VEGF cDNA had significantly greater tissue viability at the end of 7 days: 93.9 versus 28.1 percent for the control plasmid DNA group and 31.9 percent for the saline group (p < 0.05). Immunohistochemical staining documented increased deposition of VEGF protein in flaps that were infused with the VEGF cDNA versus saline alone (p < 0.05). The results indicated that the survival of ischemic tissues can be enhanced by administration of a cDNA encoding VEGF, a protein known to be important in the process of angiogenesis and wound healing.

Cerebro<em>vascular</em> angiopathy affects late-onset Alzheimer's disease (LOAD) brains by possibly increasing <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF). A expression, thereby stimulating <em>endothelial</em> cell proliferation and migration. Indeed, VEGF-A gene upregulation, with increased VEGF-A protein content of reactive astrocytes and microglia, occurs in LOAD brains, and neo<em>vascular</em>ization was observed one week after injecting amyloid-β (Aβ)(1-42) into rat hippocampus. We have now found, with cultured 'normoxic' normal adult human astrocytes (NAHAs), that fibrillar Aβ(25-35) (an active Aβ(1-42) fragment) or a cytokine mixture (the (CM)-trio (interleukin [IL]-1β+interferon [IFN]-γ+tumor necrosis <em>factor</em> [TNF]-α), or pair (IFN-γ+TNF-α) like those produced in LOAD brains) stimulates the nuclear translocation of stabilized hypoxia-inducible <em>factor</em> (HIF)-1α protein and its binding to VEGF-A hypoxia-response elements; the mRNA synthesis for three VEGF-A splice variants (<em>121</em>, 165, 189); and the secretion of VEGF-A165. The CM-trio was the most powerful stimulus, IFN-γ+TNF-α was less potent, and other cytokine pairs or single cytokines or Aβ(35-25) were ineffective. While Aβ(25-35) did not change HIF-1β protein levels, the CM-trio increased both HIF-1α and HIF-1β protein levels, thereby giving an earlier and stronger stimulus to VEGF-A secretion by NAHAs. Thus, increased VEGF-A secretion from astrocytes stimulated by Aβ(1-42) and by microglia-released cytokines might restore angiogenesis and Aβ(1-42) <em>vascular</em> clearance.

Extracellular matrix (ECM) molecules, such as fibronectin (FN), regulate fibroblast sensitivity to soluble <em>growth</em> <em>factors</em>, in part, by controlling cellular levels of phosphatidylinositol bis-phosphate (PIP2), the substrate for phospholipase C-gamma (McNamee et al., 1993, J. Cell Biol. <em>121</em>, 673-678). In the present study, we extended these investigations by exploring whether cells of the <em>vascular</em> wall also exhibit this response and analyzing the mechanism by which adhesion to ECM regulates intracellular PIP2 mass. Capillary <em>endothelial</em> cells, pulmonary <em>vascular</em> smooth muscle cells, and C3H 101/2 fibroblasts were all found to exhibit a similar two- to threefold increase in PIP2 mass within 3 h after binding to dishes coated with FN. Furthermore, similar effects were observed using dishes coated with a variety of different ECM molecules, including collagen types I and IV as well as a synthetic RGD-containing peptide. An increase in PIP2 mass also was produced when suspended cells bound to microbeads (4.5 micron diameter; coated with RGD-peptide or anti-integrin beta 1 antibody) that induce local integrin clustering and focal adhesion formation, independently of cell spreading. In contrast, neither binding of soluble FN nor binding of microbeads coated with ligands for other transmembrane surface receptors (e.g., acetylated low-density lipoprotein, antibodies against heparan sulfate) had any effect on PIP2 mass. While these results suggest that integrin clustering stimulates PIP2 synthesis, no change in total cellular or cytoskeletal-associated phosphatidylinositol-4-phosphate kinase (PIP kinase) activity could be detected when cells bound to immobilized integrin ligands. However, when focal adhesion complexes were isolated from these cells using a magnetic procedure (G. Plopper and D. E. Ingber, 1993, Biochem. Biophys. Res. Commun. 193, 571-578), this subfraction of the cytoskeleton was found to be enriched for PIP kinase activity by more than twofold relative to the whole cytoskeleton. These data suggest that ECM binding may increase PIP2 mass in <em>vascular</em> cells by clustering cell surface integrin receptors and activating cytoskeletal-associated PIP kinases locally within the focal adhesion complex.

BACKGROUND

The experience with direct myocardial injection of adenovirus encoding angiogenic <em>growth</em> <em>factor</em> is limited to invasive surgical approach. Accordingly, we sought to evaluate, for the first time, in a randomized, double-blind, placebo-controlled, phase I pilot study the safety and feasibility of percutaneous catheter-based intramyocardial delivery of a replication-deficient adenovector encoding the <em>121</em>-amino-acid isoform of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (AdVEGF<em>121</em>).

METHODS

Ten "no-option" patients with severe coronary artery disease were randomized (2:1) to receive AdVEGF<em>121</em> (4 x 10(10) pu) or placebo as fifteen 100 microL, evenly distributed, endomyocardial injections using a nonflouroscopic, 3-dimensional mapping and injection (NOGA) catheter-based system.

RESULTS

Injection procedure was successfully completed in all cases and was associated with no major adverse events. AdVEGF<em>121</em> was considered potentially associated with a single serious adverse event of transient moderate fever. Elevated postprocedure CK and CK-MB fraction levels were recorded in two placebo-treated and three AdVEGF<em>121</em>-treated patients; all CK measured values were <1.5 times upper limit of normal. All adenoviral cultures (urine and throat swab) were negative 24-hr after dosing, and no significant changes in serial plasma VEGF levels were noted over time. At 12 months follow-up, no cancers, proliferative retinal changes, or significant abnormalities in hepatic, renal or hematological indices were observed.

CONCLUSIONS

Percutaneous, catheter-based AdVEGF<em>121</em> intramyocardial injection is a practical, feasible, and potentially safe approach for intramyocardial gene transfer. A larger randomized, phase II efficacy study is warranted.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) is thought to promote tumor <em>growth</em> and angiogenesis. Whereas VEGF is up-regulated in only a portion of anaplastic astrocytoma (AA), it is overexpressed in most glioblastoma multiforme (GBM), and the level of expression is correlated with grade of glioma. To explore the possibility that VEGF may act as a driving force in the progression of AA to GBM, the VEGF isoforms VEGF(<em>121</em>) and VEGF(165) were overexpressed in genetically modified, mutant H-Ras-transformed human astrocytes that on intracranial implantation form AA-like tumors. The ability of the VEGF isoforms to stimulate <em>growth</em>, angiogenesis, oxygenation, and the formation of necrotic GBM-like tumors was then monitored. The parental mutant H-Ras-modified astrocytes expressed four times more endogenous VEGF than normal human astrocytes, but on intracranial implantation formed hypo<em>vascular</em>, hypoxic, small AA-like tumors. Whereas these modest levels of VEGF overexpression were insufficient to drive oxygenation and GBM formation, an additional 8-fold increase in VEGF expression mediated by retroviral infection with constructs encoding either VEGF (<em>121</em>) or VEGF (165) resulted in cells which, after intracranial implantation, formed tumors that were larger, more <em>vascular</em>, and better oxygenated than those formed by the mutant H-ras parental cells. However, the tumors formed by the cells expressing exogenous VEGF (<em>121</em>) or VEGF (165) retained the phenotype of AA, lacking areas of necrosis that are the hallmark of the GBM phenotype. These results suggest that whereas the VEGF(<em>121</em>) and VEGF(165) isoforms can contribute to glioma <em>vascular</em>ization, oxygenation, and <em>growth</em>, they do not in and of themselves drive the formation of the GBM phenotype.

The purpose of this study was to examine the expression of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) during osteoblastic differentiation of cultured human periosteal-derived cells. Periosteal tissues were obtained from mandible during surgical extraction of lower impacted third molars. Periosteal-derived cells were introduced into cell culture. After passage 3, the periosteal-derived cells were further cultured for 42 days in an osteogenic-inductive culture medium containing dexamethasone, ascorbic acid, and beta-glycerophosphate. The alkaline phosphatase activity in the cultured periosteal-derived cells increased rapidly up to day 14, followed by decrease in activity. The Runx2 protein was expressed at day 7 and day 14, and its expression was not observed thereafter. Both VEGF(165) and VEGF(<em>121</em>) were expressed strongly at days 35 and 42 of culture, particularly during the later stages of differentiation. Alizarin red S-positive nodules were first observed on day 14 and then increased in number during the entire culture period. Osteocalcin and VEGF were first detected in the culture medium on day 14, and their levels increased thereafter in a time-dependent manner. These results suggest that VEGF secretion from cultured human periosteal-derived cells increases along with the mineralization process of the extracellular matrix.

OBJECTIVE

To study whether vascular endothelial growth factor messenger RNA (VEGF mRNA) in the hepatocellular carcinoma (HCC) tissues obtained after curative resection has a prognostic value.

METHODS

Using a reverse-transcription polymerase chain reaction (RT-PCR)-based assay, VEGF mRNA was determined prospectively in liver tissues of 50 controls and in HCC tissues of 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 HCC tissue correlated with a significant risk of HCC recurrence (P=0.038) and significantly with recurrence-related mortality (P=0.045); 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 variables of recurrence related mortality consisted of vascular permeation (P=0.045), and cellular dedifferentiation (P=0.053). The level of VEGF mRNA in HCC tissues, however, did not significantly correlate with tumor size, cellular differentiation, capsule, daughter nodules, vascular permeation, necrosis and hemorrhage of tumors.

CONCLUSIONS

The expression of VEGF mRNA, especially isoform VEGF(165), in HCC tissues, may play a significant and independent role in the prediction of postoperative recurrence of HCC.

Primary effusion lymphomas (PEL), rare lymphomas associated with Kaposi's sarcoma-associated herpesvirus (KSHV or HHV-8) infection, present as malignant lymphomatous effusions in body cavities. We have recently found that PEL effusions contain high levels of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em>/<em>vascular</em> permeability <em>factor</em> (VEGF/VPF). VEGF/VPF, an important regulator of tumor-angiogenesis in vivo, exerts its effects acting through the receptors KDR/Flk-1 and Flt-1 on the <em>endothelial</em> cell membrane. In vitro, the PEL cell lines BC-1, BCP-1 and BCBL-1 produce high levels of VEGF. RT-PCR analysis of RNA from the PEL cell lines amplified the three VEGF/VPF secreted isoforms, VEGF/VPF(<em>121</em>), VEGF/VPF(145) and VEGF/VPF(165). Two of the PEL cell lines express the VEGF/VPF receptor Flt-1, but VEGF did not stimulate proliferation in these cells. SCID/beige mice inoculated intraperitoneally with BCBL-1 cells developed effusion lymphoma of human cell origin with prominent bloody ascites. In contrast, none of the mice treated with a neutralizing anti-human VEGF/VPF antibody developed ascites and effusion lymphoma. Although the precise mechanisms by which VEGF/VPF can promote <em>vascular</em> permeability are not fully understood, VEGF/VPF stimulation of <em>vascular</em> leakage may be critical to the pathogenesis of PEL.

Preeclampsia (PE) is a life-threatening hypertensive disorder during pregnancy associated with decreased circulating aldosterone levels. However, the molecular mechanisms underlying aldosterone reduction in PE remain unidentified. Here we demonstrate that reduced circulating aldosterone levels in preeclamptic women are associated with the presence of angiotensin II type 1 receptor agonistic autoantibody and elevated soluble Fms-like tyrosine kinase-1, 2 prominent pathogenic <em>factors</em> in PE. Using an adoptive transfer animal model of PE, we provide in vivo evidence that the injection of IgG from women with PE, but not IgG from normotensive individuals, resulted in hypertension, proteinuria, and a reduction in aldosterone production from 1377 ± 272 pg/mL to 544 ± 92 pg/mL (P<0.05) in pregnant mice. These features were prevented by coinjection with an epitope peptide that blocks antibody-mediated angiotensin type 1 receptor activation. In contrast, injection of IgG from preeclamptic women into nonpregnant mice induced aldosterone levels from 213 ± 24 pg/mL to 615 ± 48 pg/mL (P<0.05). These results indicate that maternal circulating autoantibody in preeclamptic women is a detrimental <em>factor</em> causing decreased aldosterone production via angiotensin type 1 receptor activation in a pregnancy-dependent manner. Next, we found that circulating soluble Fms-like tyrosine kinase-1 was only induced in autoantibody-injected pregnant mice but not nonpregnant mice. As such, we further observed <em>vascular</em> impairment in adrenal glands of pregnant mice. Finally, we demonstrated that infusion of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em>(<em>121</em>) attenuated autoantibody-induced adrenal gland <em>vascular</em> impairment resulting in a recovery in circulating aldosterone (from 544 ± 92 to 1110 ± 269 pg/mL; P<0.05). Overall, we revealed that angiotensin II type 1 receptor agonistic autoantibody-induced soluble Fms-like tyrosine kinase-1 elevation is a novel pathogenic mechanism underlying decreased aldosterone production in PE.