Vascular endothelial growth factor (<em>VEGF</em>) is a potent and specific mitogen for endothelial cells. <em>VEGF</em> is synthesized and secreted by many differentiated cells in response to a variety of stimuli including hypoxia. <em>VEGF</em> is expressed in a variety of tissues as multiple homodimeric forms (121, 165, 189, and 206 amino acids/monomer) resulting from alternative RNA splicing. <em>VEGF</em>121 is a soluble mitogen that does not bind heparin; the longer forms of <em>VEGF</em> bind heparin with progressively higher affinity. The higher molecular weight forms of <em>VEGF</em> can be cleaved by plasmin to release a diffusible form(s) of <em>VEGF</em>. We characterized the proteolysis of <em>VEGF</em> by plasmin and other proteases. Thrombin, elastase, and collagenase did not cleave <em>VEGF</em>, whereas trypsin generated a series of smaller fragments. The isolated plasmin fragments of <em>VEGF</em> were compared with respect to heparin binding, interaction with soluble <em>VEGF</em> receptors, and ability to promote endothelial cell mitogenesis. Plasmin yields two fragments of <em>VEGF</em> as indicated by reverse phase high performance liquid chromatography and SDS-polyacrylamide gel electrophoresis: an amino-terminal homodimeric protein containing receptor binding determinants and a carboxyl-terminal polypeptide which bound heparin. Amino-terminal sequencing of the carboxyl-terminal peptide identified the plasmin cleavage site as Arg110-Ala111. A heterodimeric form of <em>VEGF</em>165/110, was isolated from partial plasmin digests of <em>VEGF</em>165. The carboxyl-terminal polypeptide (111-165) displayed no affinity for soluble kinase domain region (KDR) or Fms-like tyrosine kinase (FLT-1) receptors. The various isoforms of <em>VEGF</em> (165, 165/110, and 121) bound soluble kinase domain region receptor with similar affinity (approximately 30 pM). In contrast, soluble FLT-1 receptor differentiated <em>VEGF</em> isoforms (165, 165/110, 110, and 121) with apparent affinities of 10, 30, 120, and 200 pM, respectively. Endothelial cell mitogenic potencies of <em>VEGF</em>110 and <em>VEGF</em>121 were decreased more than 100-fold compared to that of <em>VEGF</em>165. The present findings indicate that removal of the carboxyl-terminal domain, whether it is due to alternative splicing of mRNA or to proteolysis, is associated with a significant loss in bioactivity.

Fifteen patients with refractory AML were treated in a phase 1 study with SU11248, an oral kinase inhibitor of fms-like tyrosine kinase 3 (Flt3), Kit, vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF) receptors. Separate cohorts of patients received SU11248 for 4-week cycles followed by either a 2- or a 1-week rest period. At the starting dose level of 50 mg (n = 13), no dose-limiting toxicities were observed. The most frequent grade 2 toxicities were edema, fatigue, and oral ulcerations. Two fatal bleedings possibly related to the disease, one from a concomitant lung cancer and one cerebral bleeding, were observed. At the 75 mg dose level (n = 2), one case each of grade 4 fatigue, hypertension, and cardiac failure was observed, and this dose level was abandoned. All patients with FLT3 mutations (n = 4) had morphologic or partial responses compared with 2 of 10 evaluable patients with wild-type FLT3. Responses, although longer in patients with mutated FLT3, were of short duration. Reductions of cellularity and numbers of Ki-67(+), phospho-Kit(+), phospho-kinase domain-containing receptor-positive (phospho-KDR(+)), phospho-signal transducer and activator of transcription 5-positive (phospho-STAT5(+)), and phospho-Akt(+) cells were detected in bone marrow histology analysis. In summary, monotherapy with SU11248 induced partial remissions of short duration in acute myeloid leukemia (AML) patients. Further evaluation of this compound, for example in combination with chemotherapy, is warranted.

von Hippel-Lindau disease is a hereditary cancer syndrome characterized by the development of vascular tumors of the central nervous system and retina, clear cell renal carcinomas, pheochromocytomas, pancreatic islet cell tumors, endolymphatic sac tumors, and benign cysts affecting a variety of organs. VHL disease is caused by germline mutations of the von Hippel-Lindau tumor suppressor gene located on chromosome 3p25. Tumor development in this setting is due to inactivation or loss of the remaining wild-type allele in a susceptible cell. The highly vascular nature of VHL-associated neoplasms can be understood in light of the recent finding that the VHL gene product (pVHL) inhibits the accumulation of hypoxia-inducible mRNAs, such as the mRNA encoding vascular endothelial growth factor (VEGF), under normoxic conditions. This property of pVHL appears to be linked to its ability to bind to complexes containing elongin B, elongin C, and cullin 2 (Cul2). Elongin C and Cul2, based on their homology with Skp1 and Cdc53, respectively, are suspected of targeting certain proteins for covalent modification with ubiquitin and hence for degradation. One model, which remains to be tested, is that the binding of pVHL to elongins B/C and Cul2 affects the ubiquitination of RNA-binding proteins that regulate the stability of hypoxia-inducible mRNAs.

BACKGROUND

Profound hearing loss is a serious complication of neurofibromatosis type 2, a genetic condition associated with bilateral vestibular schwannomas, benign tumors that arise from the eighth cranial nerve. There is no medical treatment for such tumors.

METHODS

We determined the expression pattern of vascular endothelial growth factor (VEGF) and three of its receptors, VEGFR-2, neuropilin-1, and neuropilin-2, in paraffin-embedded samples from 21 vestibular schwannomas associated with neurofibromatosis type 2 and from 22 sporadic schwannomas. Ten consecutive patients with neurofibromatosis type 2 and progressive vestibular schwannomas who were not candidates for standard treatment were treated with bevacizumab, an anti-VEGF monoclonal antibody. An imaging response was defined as a decrease of at least 20% in tumor volume, as compared with baseline. A hearing response was defined as a significant increase in the word-recognition score, as compared with baseline.

RESULTS

VEGF was expressed in 100% of vestibular schwannomas and VEGFR-2 in 32% of tumor vessels on immunohistochemical analysis. Before treatment, the median annual volumetric growth rate for 10 index tumors was 62%. After bevacizumab treatment in the 10 patients, tumors shrank in 9 patients, and 6 patients had an imaging response, which was maintained in 4 patients during 11 to 16 months of follow-up. The median best response to treatment was a volumetric reduction of 26%. Three patients were not eligible for a hearing response; of the remaining seven patients, four had a hearing response, two had stable hearing, and one had progressive hearing loss. There were 21 adverse events of grade 1 or 2.

CONCLUSIONS

VEGF blockade with bevacizumab improved hearing in some, but not all, patients with neurofibromatosis type 2 and was associated with a reduction in the volume of most growing vestibular schwannomas.

The clinical benefit conferred by vascular endothelial growth factors (VEGF)-targeted therapies is variable, and tumors from treated patients eventually reinitiate growth. Here, we identify a glycosylation-dependent pathway that compensates for the absence of cognate ligand and preserves angiogenesis in response to VEGF blockade. Remodeling of the endothelial cell (EC) surface glycome selectively regulated binding of galectin-1 (Gal1), which upon recognition of complex N-glycans on VEGFR2, activated VEGF-like signaling. Vessels within anti-VEGF-sensitive tumors exhibited high levels of α2-6-linked sialic acid, which prevented Gal1 binding. In contrast, anti-VEGF refractory tumors secreted increased Gal1 and their associated vasculature displayed glycosylation patterns that facilitated Gal1-EC interactions. Interruption of β1-6GlcNAc branching in ECs or silencing of tumor-derived Gal1 converted refractory into anti-VEGF-sensitive tumors, whereas elimination of α2-6-linked sialic acid conferred resistance to anti-VEGF. Disruption of the Gal1-N-glycan axis promoted vascular remodeling, immune cell influx and tumor growth inhibition. Thus, targeting glycosylation-dependent lectin-receptor interactions may increase the efficacy of anti-VEGF treatment.

OBJECTIVE

Angiogenesis is a critical component of neoplastic and chronic inflammatory disorders, but whether angiogenesis also occurs in inflammatory bowel disease (IBD) has yet to be established. We assessed mucosal vascularization, expression of endothelial alphaVbeta3 integrin, angiogenic factors, and their bioactivity in Crohn's disease (CD) and ulcerative colitis (UC) mucosa.

METHODS

Mucosal endothelium was immunostained for CD31 and factor VIII and quantified by digital morphometry. alphaVbeta3 expression was studied in vivo by confocal microscopy and in vitro by flow cytometric analysis of human intestinal microvascular endothelial cells (HIMECs). Vascular endothelial growth factor (VEGF), interleukin (IL)-8, and bFGF levels were measured in mucosal extracts and cells and angiogenic bioactivity shown by induction of HIMEC migration and the corneal and chorioallantoic membrane angiogenesis assays.

RESULTS

Microvessel density was increased in IBD mucosa. Endothelial alphaVbeta3 was strongly expressed in IBD but only sporadically in normal mucosa and was up-regulated in HIMECs by VEGF, tumor necrosis factor alpha, and bFGF. IBD mucosal extracts induced a significantly higher degree of HIMEC migration than control mucosa, and this response was mostly dependent on IL-8 and less on basic fibroblast growth factor or vascular endothelial growth factor. Compared with normal mucosa, IBD mucosal extracts induced a potent angiogenic response in both the corneal and chorioallantoic membrane assays.

CONCLUSIONS

These results provide morphological, phenotypic and functional evidence of potent angiogenic activity in both CD and UC mucosa, indicating that the local microvasculature undergoes an intense process of inflammation-dependent angiogenesis. Thus, angiogenesis appears to be an integral component of IBD pathogenesis, providing the practical and conceptual framework for anti-angiogenic therapies in IBD.

Hypervascularity, focal necrosis, persistent cerebral edema, and rapid cellular proliferation are key histopathologic features of glioblastoma multiforme (GBM), the most common and malignant of human brain tumors. By immunoperoxidase and immunofluorescence, we definitively have demonstrated the presence of vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFr) in five out of five human glioma cell lines (U-251MG, U-105MG, D-65MG, D-54MG, and CH-235MG) and in eight human GBM tumor surgical specimens. In vitro experiments with glioma cell lines revealed a consistent and reliable relation between EGFr activation and VEGF production; namely, EGF (1-20 ng/ml) stimulation of glioma cells resulted in a 25-125% increase in secretion of bioactive VEGF. Conditioned media (CM) prepared from EGF-stimulated glioma cell lines produced significant increases in cytosolic free intracellular concentrations of Ca2+ ([Ca2+]i) in human umbilical vein endothelial cells (HUVECs). Neither EGF alone or CM from glioma cultures prepared in the absence of EGF induced [Ca2+]i increases in HUVECs. Preincubation of glioma CM with A4.6.1, a monoclonal antibody to VEGF, completely abolished VEGF-mediated [Ca2+]i transients in HUVECs. Likewise, induction by glioma-derived CM of von Willebrand factor release from HUVECs was completely blocked by A4.6.1 pretreatment. These observations provide a key link in understanding the basic cellular pathophysiology of GBM tumor angiogenesis, increased vascular permeability, and cellular proliferation. Specifically, EGF activation of EGFr expressed on glioma cells leads to enhanced secretion of VEGF by glioma cells. VEGF released by glioma cells in situ most likely accounts for pathognomonic histopathologic and clinical features of GBM tumors in patients, including striking tumor angiogenesis, increased cerebral edema and hypercoagulability manifesting as focal tumor necrosis, deep vein thrombosis, or pulmonary embolism.

The epidermal growth factor receptor (EGFR) family comprehends four different tyrosine kinases (EGFR, ErbB-2, ErbB-3, and ErbB-4) that are activated following binding to epidermal growth factor (EGF)-like growth factors. It has been long established that the EGFR system is involved in tumorigenesis. These proteins are frequently expressed in human carcinomas and support proliferation and survival of cancer cells. However, activation of the EGFR in non-malignant cell populations of the neoplastic microenvironment might also play an important role in cancer progression. EGFR signaling regulates in tumor cells the synthesis and secretion of several different angiogenic growth factors, including vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), and basic fibroblast growth factor (bFGF). Overexpression of ErbB-2 also leads to increased expression of angiogenic growth factors, whereas treatment with anti-EGFR or anti-ErbB-2 agents produces a significant reduction of the synthesis of these proteins by cancer cells. EGFR expression and function in tumor-associated endothelial cells has also been described. Therefore, EGFR signaling might regulate angiogenesis both directly and indirectly. In addition, activation of EGFR is involved in the pathogenesis of bone metastases. Within the bone marrow microenvironment, cancer cells stimulate the synthesis of osteoclastogenic factors by residing stromal cells, a phenomenon that leads to bone destruction. It has been shown that EGFR signaling regulates the ability of bone marrow stromal cells to produce osteoclastogenic factors and to sustain osteoclast activation. Taken together, these findings suggest that the EGFR system is an important mediator, within the tumor microenvironment, of autocrine and paracrine circuits that result in enhanced tumor growth.

NKX2.1 is a homeodomain transcriptional factor expressed in thyroid, lung, and parts of the brain. We demonstrate that septation of the anterior foregut along the dorsoventral axis, into distinct tracheal and esophageal structures, is blocked in mouse embryos carrying a homozygous targeted disruption of the Nkx2.1 locus. This is consistent with the loss of Nkx2.1 expression, which defines the dorsoventral boundary within the anterior foregut in wild-type E9 embryos. Failure in septation between the trachea and the esophagus in Nkx2.1(-/-) mice leads to the formation of a common lumen that connects the pharynx to the stomach, serving both as trachea and as esophagus, similar in phenotype to a human pathologic condition termed tracheoesophageal fistula. The main-stem bronchi bifurcate from this common structure and connect to profoundly hypoplastic lungs. The mutant lungs fail to undergo normal branching embryogenesis, consist of highly dilated sacs that are not capable of sustaining normal gas exchange functions, and lead to immediate postnatal death. In situ hybridization suggests reduced Bmp-4 expression in the mutant lung epithelium, providing a possible mechanistic clue for impaired branching. Functional deletion of Nkx2. 1 blocks pulmonary-specific epithelial cell differentiation marked by the absence of pulmonary surfactant protein gene expression. Altered expression of temporally regulated genes such as Vegf demonstrates that the lung in Nkx2.1(-/-) mutant embryos is arrested at early pseudoglandular (E11-E15) stage. These results demonstrate a critical role for Nkx2.1 in morphogenesis of the anterior foregut and the lung as well as in differentiation of pulmonary epithelial cells.

Despite a clinically recognized association between the lymphatics and metastasis, the biology of tumor-lymphatic interaction is not clearly understood. We report here that functional lymphatic capillaries are absent from the interior of a solid tumor, despite the presence within the tumor of the lymphangiogenic molecule vascular endothelial growth factor (VEGF)-C and endothelial cells bearing its receptor, VEGF receptor 3. Functional lymphatics, enlarged and VEGF receptor 3 positive, were detected in some tumors only at the tumor periphery (within 100 microm of the interface with normal tissue). We conclude that although lymphangiogenic factors are present, formation of functional lymphatic vessels is prevented, possibly due to collapse by the solid stress exerted by growing cancer cells.

OBJECTIVE

To investigate the role of vascular endothelial growth factor (VEGF) in the pathogenesis of the first phase of retinopathy of prematurity (ROP) and to examine the mechanism by which supplemental oxygen therapy might inhibit neovascularization in the second phase of ROP.

METHODS

A novel combination of fluorescein-dextran perfusion and colorimetric whole-retina in situ hybridization was used to evaluate the expression of VEGF messenger RNA in relationship to the location of blood vessels in retinas from neonatal mice that were exposed to hyperoxia. Northern blot and immunoblot analyses were used to quantify the changes in VEGF messenger RNA and protein expression caused by hyperoxia. The ability of VEGF to prevent hyperoxia-induced vaso-obliteration was evaluated by injecting exogenous VEGF into the vitreous cavity prior to oxygen exposure.

RESULTS

Vascular endothelial growth factor messenger RNA was produced in a reticular pattern just anterior to the developing blood vessels in normal retina on postnatal day 7. The expression of VEGF in the peripheral retina was down-regulated by hyperoxia in conjunction with the arrest of growth and the loss of some of the developing vasculature. Total VEGF messenger RNA and protein levels in retinas from animals on postnatal day 7 were decreased 55% and 85%, respectively, after 6 hours in 75% oxygen. Vaso-obliteration was inhibited 57% by pretreatment of animals with exogenous VEGF. In animals with retinal ischemia secondary to loss of vasculature, treatment with supplemental oxygen therapy decreased stimulated retinal VEGF levels by approximately 70%.

CONCLUSIONS

Down-regulation of VEGF expression by hyperoxia may be partly responsible for the vaso-obliteration and cessation of normal retinal blood vessel growth observed in premature infants in whom ROP develops. Hyperoxia also has the potential to be used therapeutically to down-regulate VEGF expression in hypoxic retina in the hope of limiting the neovascular complications of ROP. Based on these findings about the regulation of VEGF expression in the retina, an explanation of the pathogenesis of ROP is proposed.

Vascular endothelial growth factor (VEGF), a multifunctional cytokine, potently stimulates angiogenesis including tumor neovascularization. Although well established in solid tumors, the role of VEGF in bone marrow neoangiogenesis and paracrine tumor-stromal cell interactions in lymphohematopoietic malignancies has not been fully elucidated. In multiple myeloma (MM), marrow neovascularization parallels disease progression. This parallel prompted us to investigate the expression and secretion of VEGF by myeloma cells and its potential effects in myeloma-marrow stroma interactions. The biologically active splice variants VEGFVEGFVEGF receptors FLT-1 and FLK-1/KDR, indicating that autocrine stimulation is unlikely. In contrast, FLK-1/KDR was abundantly expressed by marrow stromal cells. Therefore, we studied the effects of VEGF on marrow stroma, focusing on the secretion of interleukin-6 (IL-6), a potent growth factor for myeloma cells and an inhibitor of plasma cell apoptosis. Exposure of stromal and microvascular endothelial cells to recombinant human (rh) VEGFVEGFVEGF expression and secretion in myeloma cell lines (40%-60%; P <.05) and to a variable degree (up to 5.3-fold; P <.005) in plasma cells purified from the marrow of patients with MM. This mutual stimulation suggests paracrine interactions between myeloma and marrow stromal cells triggered by VEGF and IL-6. (Blood. 2000;95:2630-2636)

The rate of transcription of several genes encoding proteins involved in O(2) and energy homeostasis is controlled by hypoxia-inducible factor-1 (HIF-1), a heterodimeric DNA binding complex composed of alpha and beta subunits. HIF-1 is considered the primary trans-acting factor for the erythropoietin (EPO) and vascular endothelial growth factor (VEGF) genes. Since EPO gene expression is inhibited by the proinflammatory cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), while no such effect has been reported with respect to the VEGF gene, we investigated the effects of IL-1beta and TNF-alpha on the activation of the HIF-1 DNA-binding complex and the amount of HIF-1alpha protein in human hepatoma cells in culture. Under normoxic conditions, both cytokines caused a moderate activation of HIF-1 DNA binding. In hypoxia, cytokines strongly increased HIF-1 activity compared with the effect of hypoxia alone. Only IL-1beta increased HIF-1alpha protein levels. In transient transfection experiments, HIF-1-driven reporter gene expression was augmented by cytokines only under hypoxic conditions. In contrast to their effect on EPO synthesis, neither IL-1beta nor TNF-alpha decreased VEGF production. The mRNA levels of HIF-1alpha and VEGF were unaffected. Thus, cytokine-induced inhibition of EPO production is not mediated by impairment of HIF-1 function. We propose that HIF-1 may be involved in modulating gene expression during inflammation.

Differentiation of functional dendritic cells (DCs) critically depends on the microenvironment. DCs differentiate in hypoxic tumor sites and inflamed or damaged tissue. Because local concentrations of adenosine reach high physiologically relevant levels in these conditions, we assessed the expression of adenosine receptors and the effect of their activation on differentiation of human monocytes and mouse peritoneal macrophages and hematopoietic progenitor cells (HPCs) into myeloid DCs. Stimulation of adenosine receptors skews DC differentiation toward a distinct cell population characterized by expression of both DC and monocyte/macrophage cell surface markers. Pharmacologic analysis and experiments with cells from A(2B) adenosine receptor knockout mice identified A(2B) receptor as the mediator of adenosine effects on DCs. Unlike normal myeloid DCs, adenosine-differentiated DCs have impaired allostimulatory activity and express high levels of angiogenic, pro-inflammatory, immune suppressor, and tolerogenic factors, including VEGF, IL-8, IL-6, IL-10, COX-2, TGF-beta, and IDO. They promoted tumor growth if injected into tumors implanted in mice. Using adenosine desaminase knockout animals, we showed that DCs with proangiogenic phenotype are highly abundant under conditions associated with elevated levels of extracellular adenosine in vivo. Adenosine signaling through A(2B) receptor is an important factor of aberrant DC differentiation and generation of tolerogenic, angiogenic, and proinflammatory cells.

OBJECTIVE

The late stages of age-related maculopathy (ARM), especially neovascular macular degeneration (ARMD), can severely affect central vision and are the main cause of blindness in the elderly in the Western world. It has been shown that angiogenic growth factors are present in neovascular membranes in ARMD. However, it is not known if angiogenic growth factors play a role in the onset of neovascularisation.

METHODS

In order to elucidate the involvement of angiogenic growth factors in the initiation of neovascularisation in early stages of ARM, the expression patterns of VEGF, TGF-beta, b-FGF, and PDGF-AA on 18 human maculae with ARM, and on 11 control specimens were investigated immunohistochemically.

RESULTS

A significantly increased expression of VEGF (p = 0.00001) and TGF-beta (p = 0.019) was found in the retinal pigment epithelium (RPE) of maculae with ARM compared with control maculae. Furthermore, an increased expression of VEGF and PDGF was found in the outer nuclear layer of maculae with ARM.

CONCLUSIONS

These results demonstrate an increased expression of VEGF in the RPE, and in the outer nuclear layer in maculae with ARM, that could be involved in the pathogenesis of neovascular macular degeneration. Furthermore, enhanced TGF-beta expression in the RPE cells of maculae with early stages of ARM was shown.

Vascular endothelial growth factor (VEGF), a factor that is critical for development of the vascular system in mouse embryos, exists as at least three isoforms, VEGFVEGFVEGFVEGF receptors, VEGFR-1 (Flt-1), VEGFR-2 (Flk-1), and neuropilin-1, suggesting that different VEGF isoforms may play distinct roles in vascular development. To determine whether there are differences in the organ-specific expression patterns that would support this concept, we used a quantitative RNase protection assay (RPA) to determine the distribution of different VEGF isoform mRNA in developing and adult mouse organs. Results revealed that the ratios of the three VEGF isoforms changed during organ development and that adult organs expressed different levels of the three VEGF isoforms. Because the lung expressed the highest levels of VEGFVEGF isoform-specific in situ hybridization in the developing lung and determined that type II alveolar epithelial cells were expressing high levels of VEGFVEGF gene revealed that mice that developed in the absence of the heparan sulfate binding isoforms VEGFVEGFVEGF isoforms have distinct functions in vascular development.

BACKGROUND

Preeclampsia is characterized clinically by hypertension and proteinuria. Soluble Flt-1 (sFlt-1; also known as soluble vascular endothelial growth factor receptor-1 [VEGFR-1]) and soluble endoglin (sEng) are elevated in preeclampsia, and their administration to pregnant rats elicits preeclampsia-like symptoms. Heme oxygenase-1 (HO-1) and its metabolite carbon monoxide (CO) exert protective effects against oxidative stimuli. Thus, we hypothesized that HO-1 upregulation may offer protection against preeclampsia by inhibiting sFlt-1 and sEng release.

RESULTS

Preeclamptic villous explants secreted high levels of sFlt-1 and sEng. Adenoviral overexpression of HO-1 in endothelial cells inhibited VEGF-mediated sFlt-1 release and interferon-gamma- and tumor necrosis factor-alpha-induced sEng release, whereas HO-1 inhibition potentiated sFlt-1 and sEng production from endothelial cells and placental villous explants. Consistent with these findings, mice lacking HO-1 produced higher levels of sFlt-1 and sEng compared with wild-type mice. Using selective ligands (VEGF-E and placental growth factor) and a receptor-specific inhibitor (SU-1498), we demonstrated that VEGF-induced sFlt-1 release was VEGFR-2 dependent. Furthermore, CO-releasing molecule-2 (CORM-2) or CO decreased sFlt-1 release and inhibited VEGFR-2 phosphorylation. Treatment of endothelial cells with statins upregulated HO-1 and inhibited the release of sFlt-1, whereas vitamins C and E had no effect.

CONCLUSIONS

The present study demonstrates that the HO-1/CO pathway inhibits sFlt-1 and sEng release, providing compelling evidence for a protective role of HO-1 in pregnancy, and identifies HO-1 as a novel target for the treatment of preeclampsia.

Monocytes attracted by tumor-induced chronic inflammation differentiate to APCs, the type of which depends on cues in the local tumor milieu. In this work, we studied the influence of human cervical cancer cells on monocyte differentiation and showed that the majority of cancer cells either hampered monocyte to dendritic cell differentiation or skewed their differentiation toward M2-like macrophages. Blocking studies revealed that M2 differentiation was caused by tumor-produced PGE(2) and IL-6. TGF-β, IL-10, VEGF, and macrophage colony-stimulating factor did not play a role. Notably, these CD14(+)CD163(+) M2 macrophages were also detected in situ. Activation of cancer cell-induced M2-like macrophages by several TLR-agonists revealed that compared with dendritic cells, these M2 macrophages displayed a tolerogenic phenotype reflected by a lower expression of costimulatory molecules, an altered balance in IL-12p70 and IL-10 production, and a poor capacity to stimulate T cell proliferation and IFN-γ production. Notably, upon cognate interaction with Th1 cells, these tumor-induced M2 macrophages could be switched to activated M1-like macrophages that expressed high levels of costimulatory molecules, produced high amounts of IL-12 and low amounts of IL-10, and acquired the lymphoid homing marker CCR7. The effects of the interaction between M2 macrophages and Th1 cells could partially be mimicked by activation of these APCs via CD40 in the presence of IFN-γ. Our data on the presence, induction, and plasticity of tumor-induced tolerogenic APCs in cervical cancer suggest that tumor-infiltrated Th1 cells can stimulate a tumor-rejecting environment by switching M2 macrophages to classical proinflammatory M1 macrophages.

The transforming growth factor-alpha/epidermal growth factor receptor (TGF-alpha-EGFR) autocrine pathway, which is involved in the development and the progression of human epithelial cancers, controls, in part, the production of angiogenic factors. These angiogenic factors, including vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), are secreted by cancer cells to stimulate normal endothelial cell growth through paracrine mechanisms. ZD1839 (Iressa) is a p.o.-active, selective EGFR-tyrosine kinase inhibitor (TKI) in clinical trials in cancer patients. In this study, we evaluated the antiangiogenic and antitumor activity of ZD1839 in human colon (GEO, SW480, and CaCo2), breast (ZR-75-1 and MCF-7 ADR), ovarian (OVCAR-3), and gastric (KATO III and N87) cancer cells that coexpress TGF-alpha and EGFR. ZD1839 treatment determined a dose- and time-dependent growth inhibition accompanied by the decrease of VEGF, bFGF and TGF-alpha production in vitro. Treatment of immunodeficient mice bearing well-established, palpable GEO xenografts with ZD1839 determined a cytostatic dose-dependent tumor growth inhibition. Immunohistochemical analysis of GEO tumor xenografts after ZD1839 treatment revealed a significant dose-dependent reduction of TGF-alpha, bFGF, and VEGF expression in cancer cells and of neoangiogenesis, as determined by microvessel count. Furthermore, the antitumor activity of ZD1839 was potentiated in combination with the cytotoxic drug paclitaxel in GEO tumor xenografts. Tumor regression was observed in all mice after treatment with ZD1839 plus paclitaxel, and it was accompanied by a significant potentiation in inhibition of TGF-alpha, VEGF, and bFGF expression with a few or no microvessels. Furthermore, 6 of 16 mice bearing well-established, palpable GEO xenografts had no histological evidence of GEO tumors at the end of treatment with ZD1839 plus paclitaxel. These results demonstrate that the antitumor effect of ZD1839 is accompanied by inhibition in the production of autocrine and paracrine growth factors that sustain autonomous local growth and facilitate angiogenesis, and that this effect can be potentiated by the combined treatment with certain cytotoxic drugs, such as paclitaxel.

Neuropilin 1 (Nrp1) is a coreceptor for vascular endothelial growth factor A165 (VEGF-A165, VEGF-A164 in mice) and semaphorin 3A (SEMA3A). Nevertheless, Nrp1 null embryos display vascular defects that differ from those of mice lacking either VEGF-A164 or Sema3A proteins. Furthermore, it has been recently reported that Nrp1 is required for endothelial cell (EC) response to both VEGF-A165 and VEGF-A121 isoforms, the latter being incapable of binding Nrp1 on the EC surface. Taken together, these data suggest that the vascular phenotype caused by the loss of Nrp1 could be due to a VEGF-A164/SEMA3A-independent function of Nrp1 in ECs, such as adhesion to the extracellular matrix. By using RNA interference and rescue with wild-type and mutant constructs, we show here that Nrp1 through its cytoplasmic SEA motif and independently of VEGF-A165 and SEMA3A specifically promotes alpha5beta1-integrin-mediated EC adhesion to fibronectin that is crucial for vascular development. We provide evidence that Nrp1, while not directly mediating cell spreading on fibronectin, interacts with alpha5beta1 at adhesion sites. Binding of the homomultimeric endocytic adaptor GAIP interacting protein C terminus, member 1 (GIPC1), to the SEA motif of Nrp1 selectively stimulates the internalization of active alpha5beta1 in Rab5-positive early endosomes. Accordingly, GIPC1, which also interacts with alpha5beta1, and the associated motor myosin VI (Myo6) support active alpha5beta1 endocytosis and EC adhesion to fibronectin. In conclusion, we propose that Nrp1, in addition to and independently of its role as coreceptor for VEGF-A165 and SEMA3A, stimulates through its cytoplasmic domain the spreading of ECs on fibronectin by increasing the Rab5/GIPC1/Myo6-dependent internalization of active alpha5beta1. Nrp1 modulation of alpha5beta1 integrin function can play a causal role in the generation of angiogenesis defects observed in Nrp1 null mice.

The aim of this study was to explore further the hypothesis that early stages of normal human hematopoiesis might be coregulated by autocrine/paracrine regulatory loops and by cross-talk among early hematopoietic cells. Highly purified normal human CD34(+) cells and ex vivo expanded early colony-forming unit-granulocyte-macrophage (CFU-GM)-derived, burst forming unit-erythroid (BFU-E)-derived, and CFU-megakaryocyte (CFU-Meg)-derived cells were phenotyped for messenger RNA expression and protein secretion of various growth factors, cytokines, and chemokines to determine the biological significance of this secretion. Transcripts were found for numerous growth factors (kit ligand [KL], FLT3 ligand, fibroblast growth factor-2 [FGF-2], vascular endothelial growth factor [VEGF], hepatocyte growth factor [HGF], insulinlike growth factor-1 [IGF-1], and thrombopoietin [TPO]); cytokines (tumor necrosis factor-alpha, Fas ligand, interferon alpha, interleukin 1 [IL-1], and IL-16); and chemokines (macrophage inflammatory protein-1alpha [MIP-1alpha], MIP-1beta, regulated upon activation, normal T cell expressed and secreted [RANTES], monocyte chemotactic protein-3 [MCP-3], MCP-4, IL-8, interferon-inducible protein-10, macrophage-derived chemokine [MDC], and platelet factor-4 [PF-4]) to be expressed by CD34(+) cells. More importantly, the regulatory proteins VEGF, HGF, FGF-2, KL, FLT3 ligand, TPO, IL-16, IGF-1, transforming growth factor-beta1 (TGF-beta1), TGF-beta2, RANTES, MIP-1alpha, MIP-1beta, IL-8, and PF-4 were identified in media conditioned by these cells. Moreover, media conditioned by CD34(+) cells were found to inhibit apoptosis and slightly stimulate the proliferation of other freshly isolated CD34(+) cells; chemo-attract CFU-GM- and CFU-Meg-derived cells as well as other CD34(+) cells; and, finally, stimulate the proliferation of human endothelial cells. It was also demonstrated that these various hematopoietic growth factors, cytokines, and chemokines are expressed and secreted by CFU-GM-, CFU-Meg-, and BFU-E-derived cells. It is concluded that normal human CD34(+) cells and hematopoietic precursors secrete numerous regulatory molecules that form the basis of intercellular cross-talk networks and regulate in an autocrine and/or a paracrine manner the various stages of normal human hematopoiesis.

Pre-eclampsia is a common and potentially dangerous disorder of human pregnancy. The maternal syndrome of hypertension, proteinuria and oedema is part of a severe systemic inflammatory response that includes leukocyte and endothelial cell activation. Although the origins of pre-eclampsia remain unclear, a major cause is the failure to develop an adequate blood supply to the placenta, leading to placental oxidative stress. This results in the excess release of placental factors, such as syncytiotrophoblast debris or soluble fms-like tyrosine kinase-1 (sFlt-1), the soluble receptor for vascular endothelial growth factor (VEGF), into the maternal circulation, where they trigger an inflammatory response and endothelial dysfunction. Alternatively, pre-eclampsia can develop in the presence of a normal placenta in women that are susceptible to systemic inflammation, such as with chronic cardiovascular disease or diabetes. While clinical management of pre-eclampsia does not currently include anti-inflammatory agents, current research is focusing on ways to reduce inflammation and oxidative stress.

OBJECTIVE

The purpose of this study was to assess trends in endothelial coverage and recovery among leading polymer-based drug-eluting stents (DES).

BACKGROUND

Autopsy studies of human U.S. Food and Drug Administration (FDA)-approved DES implanted coronary arteries suggest that complications of late stent thrombosis are associated with incomplete endothelial coverage of struts.

METHODS

Rabbits received sirolimus-eluting stents (SES), paclitaxel-eluting stents (PES), zotarolimus-eluting stents (ZES), and everolimus-eluting stents (EES) for 14 or 28 days along with MULTI-LINK (ML) Vision control stents. Endothelial coverage above and between struts was measured by morphometric analysis of images acquired through en face scanning electron microscopy. Dual fluorescent immunolabeling was performed for platelet-endothelial cell adhesion molecule (PECAM)-1 and thrombomodulin (TM), factors involved in cell-to-cell contact and thrombogenicity, respectively. In a separate analysis, the endothelial mitogen, vascular endothelial growth factor (VEGF), was also assessed.

RESULTS

Varying rates of endothelialization among comparator DES were most notable at 14 days, where coverage above struts remained poor in SES, PES, and ZES (<or=30%) relative to EES and ML Vision controls >>or=70%), whereas no significant differences were observed at 28 days. Select DES with poor endothelialization showed a further reduced expression of PECAM-1. All DES showed an absence or weak expression of the antithrombotic cofactor TM. Incomplete endothelialization in select DES was further associated with increased <em>VEGF</em> secretion and messenger ribonucleic acid levels at 14 days, providing evidence of a transitional healing surface.

CONCLUSIONS

The present study marks the first comparator analysis of endothelial coverage in leading polymeric DES, supporting disparities in arterial healing based on endothelial regrowth and recovery, favoring newer designs over the current generation of FDA-approved stents.

Angiogenesis is an important process in cell development, especially in cancer. Vascular endothelial growth factor (VEGF) signaling is an important regulator of angiogenesis. Several therapies that act against VEGF signal transduction have been developed, including YN968D1, which is a potent inhibitor of the VEGF signaling pathway. This study investigated the antitumor activity of YN968D1 (apatinib mesylate) in vitro and in vivo. YN968D1 potently suppressed the kinase activities of VEGFR-2, c-kit and c-src, and inhibited cellular phosphorylation of VEGFR-2, c-kit and PDGFRβ. YN968D1 effectively inhibited proliferation, migration and tube formation of human umbilical vein endothelial cells induced by FBS, and blocked the budding of rat aortic ring. In vivo, YN968D1 alone and in combination with chemotherapeutic agents effectively inhibited the growth of several established human tumor xenograft models with little toxicity. A phase I study of YN968D1 has shown encouraging antitumor activity and a manageable toxicity profile. These findings suggest that YN968D1 has promise as an antitumor drug and might have clinical benefits.