Human papillomavirus (HPV) 16 is involved in causing cervical cancer. The E6 and E7 proteins of HPV 16 immortalize human keratinocytes and this is due, at least in part, to inactivation of the tumor suppressor proteins p53 and pRB. These tumor suppressor proteins also regulate the expression of pro- and antiangiogenic <em>factors</em> by cells. For this reason, experiments were conducted to determine whether the expression of E6 and E7 in primary keratinocytes alters the phenotype of these cells such that they express diminished levels of antiangiogenic <em>factors</em> and/or increased levels of proangiogenic <em>factors</em>. To avoid variances in experimental observations, pools of human foreskin keratinocytes from multiple sources were infected with recombinant retrovirus expressing HPV 16 E6 and E7 or control retrovirus. Gene array analysis, RT-PCR, ELISAs and Western blotting showed that in cells expressing HPV 16 E6 and E7, expression levels of two potent angiogenesis inhibitors, thrombospondin-1 and maspin, were lower compared to controls. Additionally, major angiogenesis inducers, interleukin-8 and <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF), were increased relative to controls. VEGF can be produced as multiple splice variants, all of which are required for the formation of patent blood vessels. The expression of HPV 16 E6 and E7 in keratinocytes augmented expression of VEGF 121, <em>145</em>, 165 and 189. These observations show that HPV 16 E6 and E7 alter the phenotype of primary keratinocytes, diminishing expression of inhibitors and increasing expression of inducers of angiogenesis. This altered phenotype may permit keratinocytes infected by HPV 16 to play a role in the progression of cancer by permitting tumors to acquire a blood supply permissive of <em>growth</em> and spread.

The aim of the present study is to identify microRNAs (miRs) with high potential to be used as biomarkers in plasma and/or serum to clinically diagnose, or provide accurate prognosis for survival in, patients with atherosclerosis, coronary artery disease, and acute coronary syndrome (ACS). A systematic search of published original research yielded a total of 72 studies. After review of the risk of bias of the published studies, according to Cochrane Collaboration and the QUADUAS Group standards, 19 studies were selected. Overall 52 different miRs were reported. In particular, miR-133a/b (5 studies), miR-208a/b (6 studies), and miR-499 (7 studies) were well studied and found to be significant diagnostic and/or prognostic markers across different cardio<em>vascular</em> disease progression stages. miR-1 and miR-<em>145</em>b are potential biomarkers of ACS; miR-1 with higher sensitivity for all acute myocardial infarction (AMI), and miR-<em>145</em> for STEMI and worse outcome of AMI. But when miRs were studied across different ACS study populations, patients had varying degrees of coronary stenosis, which was identified as an important confounder that limited the ability to quantitatively pool the study results. The identified miRs were found to regulate <em>endothelial</em> function and angiogenesis (miR-1, miR-133), <em>vascular</em> smooth muscle cell differentiation (miR-133, miR-<em>145</em>), communication between <em>vascular</em> smooth muscle and <em>endothelial</em> cell to stabilize plaques (miR-<em>145</em>), apoptosis (miR-1, miR-133, miR-499), cardiac myocyte differentiation (miR-1, miR-133, miR-<em>145</em>, miR-208, miR-499), and to repress cardiac hypertrophy (miR-133). Their role in these processes may be explained by regulation of shared RNA targets such as cyclin-dependent kinase inhibitor 1A (or p21), ETS proto-oncogene 1, fascin actin-bundling protein 1, hyperpolarization-activated cyclic nucleotide-gated potassium channel 4, insulin-like <em>growth</em> <em>factor</em> 1 receptor LIM and SH3 protein 1, purine nucleoside phosphorylase, and transgelin 2. These mechanistic data further support the clinical relevance of the identified miRs. miR-1, miR-133a/b, miR-<em>145</em>, miR-208a/b, and miR-499(a) in plasma and/or serum show some potential for diagnosis of cardio<em>vascular</em> disease. However, biased selection of miRs in most studies and unexplained contrasting results are major limitations of current miR research. Inconsistencies need to be addressed in order to definitively identify clinically useful miRs. Therefore, this paper presents important aspects to improve future miR research, including unbiased selection of miRs, standardization/normalization of reference miRs, adjustment for patient comorbidities and medication, and robust protocols of data-sharing plans that could prevent selective publication and selective reporting of miR research outcomes.

OBJECTIVE

To evaluate the safety, maximum-tolerated dose, pharmacokinetics, and pharmacodynamics of vandetanib, an oral vascular endothelial growth factor receptor 2 (VEGFR2) and epidermal growth factor receptor inhibitor, administered once daily during and after radiotherapy in children with newly diagnosed diffuse intrinsic pontine glioma.

METHODS

Radiotherapy was administered as 1.8-Gy fractions (total cumulative dose of 54 Gy). Vandetanib was administered concurrently with radiotherapy for a maximum of 2 years. Dose-limiting toxicities (DLTs) were evaluated during the first 6 weeks of therapy. Pharmacokinetic studies were obtained for all patients. Plasma angiogenic factors and VEGFR2 phosphorylation in mononuclear cells were analyzed before and during therapy.

RESULTS

Twenty-one patients were administered 50 (n = 3), 65 (n = 3), 85 (n = 3), 110 (n = 6), and 145 mg/m(2) (n = 6) of vandetanib. Only one patient developed DLT (grade 3 diarrhea) at dosage level 5. An expanded cohort of patients were treated at dosage levels 4 (n = 10) and 5 (n = 4); two patients developed grade 4 hypertension and posterior reversible encephalopathy syndrome while also receiving high-dose dexamethasone. Despite significant interpatient variability, exposure to vandetanib increased with higher dosage levels. The bivariable analysis of vascular endothelial growth factor (VEGF) before and during therapy showed that patients with higher levels of VEGF before therapy had a longer progression-free survival (PFS; P = .022), whereas patients with increases in VEGF during treatment had a shorter PFS (P = .0015). VEGFR2 phosphorylation was inhibited on day 8 or 29 of therapy compared with baseline (P = .039).

CONCLUSIONS

The recommended phase II dose of vandetanib in children is 145 mg/m(2) per day. Close monitoring and management of hypertension is required, particularly for patients receiving corticosteroids.

Recent evidence shows that hypoxia-inducible <em>factor</em> 2 alpha (HIF-2α) may have critical roles in the <em>growth</em> and progression of neuroblastoma (NB) under non-hypoxic conditions. However, the underlying mechanisms and clinical potentials of normoxic HIF-2α expression in NB still remain largely unknown. In this study, HIF-2α immunostaining was identified in 26/42 NB tissues, which was correlated with clinicopathological features. In subtotal 20 NB cases, microRNA-<em>145</em> (miR-<em>145</em>) was downregulated and inversely correlated with HIF-2α expression. Bioinformatics analysis revealed a putative miR-<em>145</em> binding site in the 3'-untranslated region (3'-UTR) of HIF-2α messenger RNA (mRNA). Overexpression or knockdown of miR-<em>145</em> responsively altered both the mRNA and protein levels of HIF-2α and its downstream genes, cyclin D1, matrix metalloproteinase 14 and <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em>, in normoxically cultured NB cell lines SH-SY5Y and SK-N-SH. In a luciferase reporter system, miR-<em>145</em> downregulated the luciferase activity of HIF-2α 3'-UTR, and these effects were abolished by a mutation in the putative miR-<em>145</em>-binding site. Overexpression of miR-<em>145</em> suppressed the <em>growth</em>, invasion, metastasis and angiogenesis of SH-SY5Y and SK-N-SH cells in vitro and in vivo, while restoration of HIF-2α expression rescued the tumor cells from miR-<em>145</em>-mediated defects in these biological features. Furthermore, anti-miR-<em>145</em> inhibitor rescued the HIF-2α knockdown-mediated repression on the <em>growth</em>, migration, invasion and angiogenesis of NB cells. These data indicate that miR-<em>145</em> suppresses HIF-2α expression via the binding site in the 3'-UTR under normoxic conditions, thus inhibiting the aggressiveness and angiogenesis of NB.

BACKGROUND

Neovascularization mediated by growth factors produced by tumors is critical for the growth of tumors. Vascular endothelial growth factor (VEGF) is one such growth factor. A neutralizing anti-VEGF antibody (A4.6.1) was recently shown in vivo to inhibit tumor angiogenesis and growth of the human rhabdomyosarcoma cell line A673. The antibody profoundly changed the growth characteristics of the tumor line from a rapidly growing malignancy to a dormant microcolony.

METHODS

In the present study, we evaluated the effects of A4.6.1 (100 microg twice weekly, i.p.) on growth and angiogenic activity of spheroids of the human prostatic cell line DU 145 (diameter 700 microm at implantation) implanted in dorsal skinfold chambers in nude mice (n = 11). An antibody of the same isotype (n = 5) or saline (n = 5) was used as control. Tumor cells were prelabeled with a fluorescent vital dye (CMTMR), which allowed measurement of size of the implanted tumor spheroids throughout a two week observation period. FITC-dextran was used for plasma enhancement to visualize angiogenic activity.

RESULTS

Tumors of control animals induced a neo-vasculature with high vascular density (350+/-12 cm[-1]). In animals treated with the anti-VEGF antibody, there was complete inhibition of neovascularization of the micro tumors and complete inhibition of tumor growth after the initial prevascular angiogenesis independent growth phase.

CONCLUSIONS

These results demonstrate that inhibition of the key regulatory paracrine growth factor for endothelial cells, VEGF, results in complete suppression of prostate cancer induced angiogenesis and prevents tumor growth beyond the initial prevascular growth phase.

BACKGROUND

A function for the microRNA (miRNA) pathway in vascular development and angiogenesis has been firmly established. miRNAs with selective expression in the vasculature are attractive as possible targets in miRNA-based therapies. However, little is known about the expression of miRNAs in microvessels in vivo. Here, we identified candidate microvascular-selective miRNAs by screening public miRNA expression datasets.

METHODS

Bioinformatics predictions of microvascular-selective expression were validated with real-time quantitative reverse transcription PCR on purified microvascular fragments from mouse. Pericyte expression was shown with in situ hybridization on tissue sections. Target sites were identified with 3' UTR luciferase assays, and migration was tested in a microfluid chemotaxis chamber.

RESULTS

miR-145, miR-126, miR-24, and miR-23a were selectively expressed in microvascular fragments isolated from a range of tissues. In situ hybridization and analysis of Pdgfb retention motif mutant mice demonstrated predominant expression of miR-145 in pericytes. We identified the Ets transcription factor Friend leukemia virus integration 1 (Fli1) as a miR-145 target, and showed that elevated levels of miR-145 reduced migration of microvascular cells in response to growth factor gradients in vitro.

CONCLUSIONS

miR-126, miR-24 and miR-23a are selectively expressed in microvascular endothelial cells in vivo, whereas miR-145 is expressed in pericytes. miR-145 targets the hematopoietic transcription factor Fli1 and blocks migration in response to growth factor gradients. Our findings have implications for vascular disease and provide necessary information for future drug design against miRNAs with selective expression in the microvasculature.

The majority of deaths from prostate cancer occur in patients with androgen-insensitive metastatic disease. An important early event in the development of the metastatic phenotype is the induction of genes that promote angiogenesis, such as <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) and interleukin-8 (IL-8), which are released from tumor cells into their microenvironment. Coincident with progression from prostatic carcinoma in situ to metastatic disease is an increase in the number of tumor cells exhibiting neuroendocrine (NE) differentiation. NE cells express a variety of peptide hormones, including the bombesin (BBS)-like peptide, gastrin-releasing peptide (GRP), and its cognate receptor, GRP-R. Although there is a strong positive correlation between the degree of NE differentiation and the metastatic potential of prostate cancers, a mechanistic link between increased expression of peptide hormone receptors, such as GRP-R, and proangiogenic gene expression has not been established. Here we report that BBS stimulates nuclear <em>factor</em> kappa B (NF kappa B) activation and proangiogenic gene expression in the androgen-insensitive prostate cancer cells lines, PC-3 and DU-<em>145</em>. In PC-3 cells, BBS stimulation of GRP-R resulted in the up-regulation of IL-8 and VEGF expression through a NF kappa B-dependent pathway. We show that BBS treatment induced inhibitor of NF kappa B degradation, NF kappa B translocation to the cell nucleus, increased NF kappa B binding to its DNA consensus sequence, and increased IL-8 and VEGF mRNA expression and protein secretion. Treatment with the proteasome inhibitor, MG-132, blocked BBS-stimulated NF kappa B DNA binding, and IL-8 and VEGF expression and secretion. Finally, media collected from PC-3 cell cultures, after BBS treatment, stimulated an NF kappa B-dependent migration of human umbilical <em>vascular</em> <em>endothelial</em> cells in vitro. Together, our data demonstrate a role for BBS and GRP-R in the NF kappa B-dependent up-regulation of proangiogenic gene expression, and suggest a possible molecular mechanism linking NE differentiation and the increased metastatic potential of androgen-insensitive prostate cancers.

Low-oxygen tension is an important component of the stem cell microenvironment. In rodents, renal resident stem cells have been described in the papilla, a relatively hypoxic region of the kidney. In the present study, we found that CD133(+) cells, previously described as renal progenitors in the human cortex, were enriched in the renal inner medulla and localized within the Henle's loop and thin limb segments. Once isolated, the CD133(+) cell population expressed renal embryonic and stem-related transcription <em>factors</em> and was able to differentiate into mature renal epithelial cells. When injected subcutaneously in immunodeficient mice within Matrigel, CD133(+) cells generated canalized structures positive for renal specific markers of different nephron segments. Oct4A levels and differentiation potential of papillary CD133(+) cells were higher than those of CD133(+) cells from cortical tubuli. Hypoxia was able to promote the undifferentiated phenotype of CD133(+) progenitors from papilla. Hypoxia stimulated clonogenicity, proliferation, <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> synthesis, and expression of CD133 that were in turn reduced by epithelial differentiation with parallel HIF-1α downregulation. In addition, hypoxia downregulated microRNA-<em>145</em> and promoted the synthesis of Oct4A. Epithelial differentiation increased microRNA-<em>145</em> and reduced Oct4 level, suggesting a balance between Oct4 and microRNA-<em>145</em>. MicroRNA-<em>145</em> overexpression in CD133(+) cells induced downrelation of Oct4A at the protein level, inhibited cell proliferation, and stimulated terminal differentiation. This study underlines the role of the hypoxic microenvironment in controlling the proliferation and maintaining a progenitor phenotype and stem/progenitor properties of CD133(+) cells of the nephron. This mechanism may be at the basis of the maintenance of a CD133(+) population in the papillary region and may be involved in renal regeneration after injury.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) has five isoforms (VEGF206, 189, 165, <em>145</em> and 121). Increased VEGF expression in renal cell carcinoma (RCC) is associated with angiogenesis, but, it is not apparent which isoform is involved in this effect. We examined the isoform patterns of VEGF by reverse transcription-polymerase chain reaction (RT-PCR) in 47 RCCs. All showed increased VEGF expression as compared with extraneoplastic renal tissue. Four of the 47 RCCs showed VEGF121 alone, 10 showed VEGF121 + 165, and 33 showed the VEGF121 + 165 + 189 pattern. Patients with pathological stage pT3-4 RCC showed the VEGF121 + 165 + 189 isoform pattern at a significantly higher incidence (10/10, 100%) than those with pT0-2 (23/37, 62%) (P < 0.022). The VEGF121 + 165 + 189 isoform pattern was also significantly associated with high vessel counts and density (P = 0.0002, Mann-Whitney U test). These observations suggested that the VEGF189 mRNA isoform is closely associated with angiogenesis and results in the <em>growth</em> of RCC.

Primary effusion lymphomas (PELs), which are rare lymphomas associated with Kaposi's sarcoma-associated herpesvirus (or human herpesvirus-8) infection, present as malignant lymphomatous effusions in body cavities. Because PELs prefer liquid <em>growth</em>, we hypothesized that increased <em>vascular</em> permeability would be required for effusions to form. We found that the PEL cell lines BC-1, BCP-1, and BCBL-1 produce high levels of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em>/<em>vascular</em> permeability <em>factor</em> (VEGF/VPF). Reverse transcriptase-polymerase chain reaction analysis of RNA from the PEL cell lines amplified the 3 VEGF-secreted isoforms: VEGF/VPF(121), VEGF/VPF(<em>145</em>), and VEGF/VPF(165). Two of the PEL cell lines expressed the VEGF/VPF receptor Flt-1, but VEGF/VPF did not stimulate proliferation in these cells. Most (13/14) control SCID/beige mice inoculated intraperitoneally with BCBL-1 cells and subsequently observed or treated with control antibodies developed effusion lymphoma of human cell origin with prominent bloody ascites. In contrast, none (0/9) of the mice treated with a neutralizing antihuman VEGF/VPF antibody developed ascites and effusion lymphoma. These results demonstrate that VEGF/VPF is critical to BCBL-1 <em>growth</em> as effusion lymphoma in mice and suggest that VEGF/VPF stimulation of <em>vascular</em> permeability may be critical to the pathogenesis of PELs.

BACKGROUND

Hypoxia is a critical aspect of the glioma microenvironment and has been associated with poor prognosis and resistance to various therapies. However, the mechanisms responsible for hypoxic survival of glioma cells remain unclear. Recent studies strongly suggest that microRNAs act as critical mediators of the hypoxic response. We thus hypothesized their prominent role in hypoxia resistance in glioblastoma (GBM) and aimed to identify those.

RESULTS

With this study, we present the first detailed analysis of small RNA transcriptome of cell line U87MG, a grade IV glioma cell line, and its alteration under hypoxic condition. Based on deep sequencing and microarray data, we identify a set of hypoxia regulated microRNAs, with the miR-210-3p and its isomiRs showing highest induction in GBM cell lines U87MG and U251MG. We show miR-210-3p, miR-1275, miR-376c-3p, miR-23b-3p, miR-193a-3p and miR-<em>145</em>-5p to be up-regulated, while miR-92b-3p, miR-20a-5p, miR-10b-5p, miR-181a-2-3p and miR-185-5p are down-regulated by hypoxia. Interestingly, certain hypoxia-induced miRNAs are also known to be over-expressed in GBM tumors, suggesting that hypoxia may be one of the <em>factors</em> involved in establishing the miRNA signature of GBM. Transcription <em>factor</em> binding sites for Hypoxia inducible <em>factor</em> 1 A (HIF1A) were identified in the promoter region (5 kb upstream) of 30 hypoxia-induced miRNAs. HIF-1A over-expression and silencing studies show regulation of specific miRNAs, including miR-210-3p, to be HIF1A dependent. On the other hand, miR-210-3p leads to an increase in transcriptional activity of HIF and its target genes <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) and carbonic anhydrase 9 (CA9). MiR-210-3p levels were found to be high in GBM patient samples and showed good correlation with the known hypoxia markers CA9 and VEGF. We show that miR-210-3p promotes hypoxic survival and chemoresistance in GBM cells and targets a negative regulator of hypoxic response, HIF3A. Additionally, a total of 139 novel miRNAs were discovered by the analysis of deep sequencing data and three of these were found to be differentially expressed under hypoxia.

CONCLUSIONS

Overall, our study reveals a novel miRNA signature of hypoxia in GBM and suggests miR-210-3p to be an oncogenic player and a novel potential intrinsic marker of hypoxia in glioblastoma.

MicroRNAs are important gene regulators that play a profound role in tumorigenesis. MicroRNA-<em>145</em> (miR-<em>145</em>), an important member in the family of microRNAs, is under-expressed in several types of tumors and acts as a tumor suppressor. The role and probable pathways of miR-<em>145</em> in osteosarcoma carcinogenesis are still unknown. In this study, we found that miR-<em>145</em> was significantly under-expressed in osteosarcoma tissues, and the over-expression of miR-<em>145</em> could inhibit invasion and angiopoiesis of osteosarcoma cells. Furthermore, the results showed that <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) expression was down-regulated in osteosarcoma cells after miR-<em>145</em> transfection. On the basis of these results, we performed the luciferase assay and verified that miR-<em>145</em> could down-regulate VEGF at the translational level by partially binding to VEGF 3' untranslated region (3'UTR). Therefore, it can be concluded that miR-<em>145</em> can inhibit invasion and metastasis of osteosarcoma cells. One of the mechanisms is the down-regulation of VEGF expression by miR-<em>145</em> by binding to the 3'UTR of VEGF mRNA specifically. These novel findings may have extensive implications for an effective gene therapy of osteosarcoma.

Notch signaling pathway plays critical roles in human cancers, including osteosarcoma, suggesting that the discovery of specific agents targeting Notch would be extremely valuable for osteosarcoma. Our previous studies have shown that diallyl trisulfide (DATS) inhibits proliferation of osteosarcoma cells by triggering cell cycle arrest and apoptosis in vitro. However, the underlying mechanism is still unclear. In this study, we found that DATS suppressed cell survival, wound-healing capacity, invasion and angiogenesis in osteosarcoma cells. These effects were associated with decreased expression of Notch-1 and its downstream genes, such as <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> and matrix metalloproteinases, as well as increased expression of a panel of tumor-suppressive microRNAs (miRNAs), including miR-34a, miR-143, miR-<em>145</em> and miR-200b/c that are typically lost in osteosarcoma. We also found that reexpression of miR-34a and miR-200b by transfection led to reduced expression of Notch-1, resulting in the inhibition of osteosarcoma cell proliferation, invasion and angiogenesis. These results clearly suggest that DATS inhibited osteosarcoma <em>growth</em> and aggressiveness via a novel mechanism targeting a Notch-miRNA regulatory circuit. Our data provide the first evidence that the downregulation of Notch-1 and reexpression of miRNAs by DATS may be an effective approach for the treatment of osteosarcoma.

Increased bone marrow angiogenesis and <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) levels are adverse prognostic features in patients with acute myeloid leukemia (AML) or myelodysplastic syndromes (MDSs). VEGF is a soluble circulating angiogenic molecule that stimulates signaling via receptor tyrosine kinases (RTKs), including VEGF receptor 2 (VEGFR-2). AML blasts may express VEGFR-2, c-kit, and FLT3. SU5416 is a small molecule RTK inhibitor (RTKI) of VEGFR-2, c-kit, and both wild-type and mutant FLT3. A multicenter phase 2 study of SU5416 was conducted in patients with refractory AML or MDS. For a median of 9 weeks (range, 1-55 weeks), 55 patients (33 AML: 10 [30%] primary refractory, 23 [70%] relapsed; 22 MDS: 15 [68%] relapsed) received <em>145</em> mg/m2 SU5416 twice weekly intravenously. Grade 3 or 4 drug-related toxicities included headaches (14%), infusion-related reactions (11%), dyspnea (14%), fatigue (7%), thrombotic episodes (7%), bone pain (5%), and gastrointestinal disturbance (4%). There were 11 patients (20%) who did not complete 4 weeks of therapy (10 progressive disease, 1 adverse event); 3 patients (5%) who achieved partial responses; and 1 (2%) who achieved hematologic improvement. Single agent SU5416 had biologic and modest clinical activity in refractory AML/MDS. Overall median survival was 12 weeks in AML patients (range, 4-41 weeks) and not reached in MDS patients. Most observed toxicities were attributable to drug formulation (polyoxyl 35 castor oil or hyperosmolarity of the SU5416 preparation). Studies of other RTKI and/or other antiangiogenic approaches, with correlative studies to examine biologic effects, may be warranted in patients with AML/MDS.

OBJECTIVE

Chronic atrial fibrillation (AF) is characterized by a remodeling process which involves the development of fibrosis. Since angiotensin II has been suspected to be involved in this process, the aim of our study was to investigate a possible influence of an ACE-I therapy in patients with chronic AF regarding the occurrence of left atrial structural remodeling.

METHODS

Atrial tissue samples were obtained from patients with lone chronic AF or sinus rhythm (SR). Collagen I, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) protein expression were measured by quantitative Western Blotting techniques and calculated as mean +/- SEM. Histological tissue samples were used for calculating microvessel density (microvessel/mm(2) +/- SEM).

RESULTS

In AF, the collagen amount was higher (1.78 +/- 0.21; p = 0.01) vs. SR (0.37 +/- 0.07) accompanied by declining microcapillary density (AF: 145 +/- 13 vs. SR: 202 +/- 9; p = 0.01). Additionally, a negative correlation (p = 0.01) between collagen content and microcapillary density was observed. To investigate the influence of an ACE-I therapy on this remodeling process, patient groups were divided into AF and SR both with or without ACE-I. Interestingly, there was a significantly lower expression of collagen I in AF with ACE-I (1.04 +/- 0.26) vs. AF without ACE-I treatment (2.07 +/- 0.24, p = 0.02). The microcapillaries were not diminished in AF with ACE-I (180 +/- 15) vs. SR with ACE-I (196 +/- 9), but there was a significant rarification in AF without ACE-I (123 +/- 18; p = 0.03). The expression of VEGF and bFGF did not reveal any significant differences.

CONCLUSIONS

In patients undergoing ACE-I treatment: atrial structural remodeling was attenuated and the loss of atrial microcapillaries was prevented.

OBJECTIVE

We have previously demonstrated that celecoxib, a selective COX-2 inhibitor, reaches the retina following repeated oral administrations and inhibits diabetes-induced vascular endothelial growth factor (VEGF) mRNA expression and vascular leakage in a rat model. The aim of this study was to quantify the relative retinal bioavailability of celecoxib from the subconjunctival route compared to a systemic route.

METHODS

The plasma and ocular tissue distribution of celecoxib was determined in male Sprague-Dawley rats following subconjunctival and intraperitoneal administrations of drug suspension at a dose of 3 mg/rat. The animals were sacrificed at 0.5, 1, 2, 3, 4, 8, and 12 h post-dosing, the blood was collected, and the eyes were enucleated and frozen. The plasma, sclera, retina, vitreous, lens, and the cornea were isolated and celecoxib levels were determined using an HPLC method. The tissue exposure of the drug was measured as the area under the curve (AUC(0-infinity)) of the concentration vs. time profiles. The relative bioavailability was estimated as the AUC(0-infinity) ratio between subconjunctival and intraperitoneal groups.

RESULTS

For the subconjunctivally dosed (ipsilateral) eye, the AUC(0-infinity) ratios between subconjunctival and intraperitoneal groups were 0.8 +/- 0.1, 53 +/- 4, 54 +/- 8, 145 +/- 21, 61 +/- 16, and 52 +/- 6 for plasma, sclera, retina, vitreous, lens, and cornea, respectively. For the contralateral ocular tissues, the AUC0-infinity ratios were 1.2 +/- 03, 11 +/- 0.3, 1.1 +/- 0.4, 1.0 +/- 0.3, and 1.2 +/- 0.3 in the sclera, retina, vitreous, lens, and the cornea, respectively, between the subconjunctival and the intraperitoneal groups. Assuming that the drug AUCs in contralateral eye were equal to the systemic pathway contribution to AUCs in the ipsilateral eye, the percent contribution of local pathways as opposed to systemic circulation for celecoxib delivery to the ipsilateral eye tissues was estimated to be 98% or greater.

CONCLUSIONS

The retinal delivery of celecoxib was substantially higher following subconjunctival administration compared to the intraperitoneal route. The transscleral pathway almost completely accounts for the retinal celecoxib delivery following subconjunctival administration.

BACKGROUND

Vascular endothelial growth factor (VEGF) is a key regulator in angiogenesis. Preclinical and clinical data support the role of VEGF and angiogenesis in renal cell carcinoma (RCC), melanoma (M), and soft tissue sarcoma (STS). The tyrosine kinase inhibitor SU5416 is a potent inhibitor of the VEGF receptors 1 and 2.

METHODS

We investigated 145 mg/m(2) SU5416 twice weekly in patients with advanced or metastatic RCC, M, and STS. The primary objectives were efficacy and safety. Disease assessments were performed after 4 and 8 weeks of treatment and every 2 months thereafter. Documented stable disease (SD) lasting for>> or =3 months was considered an antitumor response.

RESULTS

A group of 29 patients was entered in the RCC trial, 20 patients in the M trial, and 31 patients in the STS trial. Response was observed in 6 (1 minor response and 5 SDs) of 24 evaluable patients (25%) in the RCC group, 6 (1 minor response and 5 SDs) of 26 patients (23%) in the STS group, and none of the patients in the M group. Progression-free survival ranged from 7 to 252 days (median 59 days) in the RCC group, from 7 to 260 days (median 60 days) in the STS group, and from 14 to 139 days (median 41 days) in the M group. Toxicities observed were those reported previously for SU5416.

CONCLUSIONS

SU5416 single agent is well tolerated. The antitumor response was low in patients with RCC and STS, whereas no responses were seen in patients with M.

In an effort to develop a new therapy for prostate cancer (PCa) bone metastases, we have created Ad.dcn, a recombinant oncolytic adenovirus carrying the human decorin gene. Infection of PC-3 and DU-<em>145</em>, the human prostate tumor cells, with Ad.dcn or a non-replicating adenovirus Ad(E1-).dcn resulted in decorin expression; Ad.dcn produced high viral titers and cytotoxicity in human prostate tumor cells. Adenoviral-mediated decorin expression inhibited Met, the Wnt/β-catenin signaling axis, <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> A, reduced mitochondrial DNA levels and inhibited tumor cell migration. To examine the antitumor response of Ad.dcn, PC-3-luc cells were inoculated in the left heart ventricle to establish bone metastases in nude mice. Ad.dcn, in conjunction with control replicating and non-replicating vectors were injected via tail vein. The real-time monitoring of mice, once a week, by bioluminescence imaging and X-ray radiography showed that Ad.dcn produced significant inhibition of skeletal metastases. Analyses of the mice at the terminal time point indicated a significant reduction in the tumor burden, osteoclast number, serum tartrate-resistant acid phosphatase 5b levels, osteocalcin levels, hypercalcemia, inhibition of cancer cachexia and an increase in the animal survival. Based on these studies, we believe that Ad.dcn can be developed as a potential new therapy for PCa bone metastasis.

OBJECTIVE

SU5416 (semaxanib) is a small molecule inhibitor of the vascular endothelial growth factor (VEGF) receptor-2 and KIT receptor tyrosine kinases. This Phase II study was conducted to investigate the safety and efficacy of SU5416 for patients with soft tissue sarcomas.

METHODS

Thirteen patients with locally advanced or metastatic soft tissue sarcomas were treated with SU5416 via intravenous infusion at a dose of 145 mg/m(2) twice weekly. In selected cases tumor biopsies were taken before and after 2 months of treatment.

RESULTS

The median progression-free survival was 1.8 months. Median overall survival was 22.8 months. No objective tumor responses were observed. There was evidence of shorter survival among patients with high baseline urine VEGF levels (P = 0.04). No grade 4 toxicities were observed. The most common grade 3 toxicities were headache and thrombosis. Other less serious toxicities included fatigue, nausea, and abdominal pain. The median systolic blood pressure increased from 118 mmHg at baseline to 133 after 1 month of treatment (P = 0.01). Post-treatment tumor biopsies showed no significant decreases in VEGF receptor phosphorylation compared with baseline in 3 evaluable patients. One patient with gastrointestinal stromal tumor who had rapid progression during SU5416 treatment was subsequently treated with another KIT inhibitor, imatinib mesylate, and had a partial response lasting >36 months.

CONCLUSIONS

SU5416 was relatively well tolerated but did not demonstrate significant antitumor activity against advanced soft tissue sarcoma. Correlative studies suggest that VEGF receptor or KIT inhibition was incomplete in at least some cases, providing a possible explanation for the observed lack of activity.

OBJECTIVE

Vascular endothelial growth factor (VEGF) is expressed in up to 70% of renal cell carcinomas (RCCs) and is a rational therapeutic target. SU5416 is a small molecule inhibitor of VEGF-mediated signaling through Flk-1, a transmembrane tyrosine kinase. IFN-alpha also possesses dose- and schedule-dependent antiangiogenic effects at doses lower than those used for RCC therapy. We hypothesized that SU5416 plus low dose IFN-alpha 2B (Intron-A) would result in a 1-year event-free survival (EFS), exceeding 20% in patients with metastatic RCC using the results of a randomized immunotherapy trial as historical control. Efficacy was correlated with serial plasma VEGF and plasminogen activator inhibitor-1 levels and with positron emission tomography scans.

METHODS

Thirty patients were treated with SU5416 145 mg/m(2) i.v. twice weekly plus Intron-A 1 million units s.c. twice daily, cycled every 6 weeks.

RESULTS

Fifteen patients (50%) had stable disease (SD) at 12 weeks, including 1 minor response and 8 with progressive disease (27%). Median survival time was 10 months, and 1-year EFS was 6% (95% confidence interval, 1-35). The most common grade 3 or 4 toxicities included fatigue and lymphopenia, among others. There were 3 on-study deaths, 2 of which were infection-related. Significant declines in median plasma levels of VEGF pre- and posttherapy were observed. In 5 patients with paired FDG and O-15 positron emission tomography scans, tumor metabolism and perfusion were unchanged in 3 patients with SD, increased in 1 patient with progression, and decreased in 1 patient with SD.

CONCLUSIONS

Although SU5146 plus low-dose IFN exhibits biological activity in RCC as evidenced by significant declines in serial VEGF and plasminogen activator inhibitor-1 plasma levels, the 1-year EFS of 6% and adverse toxicity profile diminishes enthusiasm for additional studies with this combination in advanced RCC.

OBJECTIVE

To assess the activity of the antiangiogenic agent and VEGFR2 inhibitor SU5416 in hormone-refractory prostate cancer.

METHODS

Thirty-six chemotherapy naïve patients were randomized to treatment with SU5416 (<em>145</em> mg/m(2)) and dexamethasone premedication or dexamethasone alone. Patients in the control arm could cross over to experimental therapy after progression. Prostate-specific antigen (PSA) was measured every 2 weeks, and radiological evaluation was performed every 8 weeks. In vitro assessment of SU5416 on PSA secretion was assessed in the LNCaP cell line. Baseline serum basic fibroblast <em>growth</em> <em>factor</em> and plasma <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) were explored as prognostic <em>factors</em>.

RESULTS

VEGF receptor-2 expression is detectable in prostate cancer cell lines, and SU5416 inhibited in vitro PSA secretion. No effect of SU5416 on PSA secretion or time to progression is detectable in patients. VEGF and basic fibroblast growth factor were not prognostic. Headache and fatigue were the most common SU5416 toxicities, but hyperglycemia, hyponatremia, lymphopenia, infection, and adrenal suppression, all attributable to steroids and the required central line, were common.

CONCLUSIONS

No disease modifying effects of SU5416 were detectable in this small study. Modest toxicity, an inconvenient administration schedule, and availability of other VEGFR-targeted agents support the decision to halt further evaluation of SU5416 in prostate cancer.

OBJECTIVE

Tumor angiogenesis, expressed by the microvessel count (MVC), and its mediators (i.e. vascular endothelial growth factor) significantly correlate with metastases in surgically treated non-small cell lung carcinoma/cancer (NSCLC). SCLC is rarely treated by surgery, as a consequence, few specimens are available to perform a biological characterization. We reviewed our experience in the surgical treatment of SCLC with particular reference to the angiogenetic expression and its correlation to the stage of disease and prognosis.

METHODS

We retrospectively investigated a homogenous cohort of 87 patients with SCLC, who were primarily operated on and then underwent adjuvant chemotherapy between 1980 and 1998. Their median age was 62 years (range 34-73). All the patients were completely staged. The surgical procedures included: 32 pneumonectomies and 55 lobectomies. There were 46 N0, 17 N1 and 24 N2-disease. The adjuvant chemotherapy consisted of four to six courses of cyclophosphamide, epidoxorubicine and etoposide. The MVC was determined highlighting the microvessels with anti-CD34 monoclonal antibodies. Immunostaining for VEGF was performed using the ABC method with anti-VEGF monoclonal antibodies. The p53 protein expression was assessed by NCL-DO7 anti-p53 monoclonal antibody.

RESULTS

With a median follow-up of 109.6 months (range 25-238), 37 patients are alive and well, two are alive with systemic metastases. Forty-four patients died of local (n=5) or systemic (n=39) relapse, while four patients died from other causes. The median MVC was 59 (range 18-145). Among the clinico-pathological parameters, metastatic nodal-involvement (P=0.002) and advanced stage of disease (P=0.005) were associated with a worse overall survival (OS). MVC and VEGF protein expression significantly affected the survival (P<0.001 and P=0.0008, respectively). No statistical association was found between p53 alterations and OS as well as no association was found among p53 alterations, MVC and VEGF expression. On multivariate analysis only the VEGF expression (P=0.003) was an independent prognostic factor.

CONCLUSIONS

Angiogenesis plays a role in the metastatic process of the SCLC as well as NSCLC. SCLC has a higher vascularization than NSCLC as results from the higher number of microvessels; however, tumor angiogenesis tested by the MVC and the VEGF protein expression correlates with the prognosis also in SCLC. SCLC may be an ideal field to test new antiangiogenic drugs associated to chemotherapy.

OBJECTIVE

Increased bone marrow angiogenesis and vascular endothelial growth factor (VEGF) levels are of adverse prognostic significance in patients with multiple myeloma (MM). VEGF, a soluble circulating angiogenic molecule, acts via receptor tyrosine kinases, including VEGF receptor 2. SU5416 is a small molecule VEGF receptor 2 inhibitor.

METHODS

Adult patients with advanced MM were entered on a multicenter phase II study.

RESULTS

Twenty-seven patients (median age 69, range 39-79), median 4 (0-10) lines of prior therapy, 14 with prior thalidomide therapy, received SU5416 at 145 mg/m(2) twice weekly i.v. for a median of two 4-week cycles (range 0.2-9). Grade 3/4 toxicities were rarely observed; the most frequent was thrombocytopenia (12%). Mild-to-moderate toxicities included nausea (63%), headache (56%), diarrhea, vomiting (both 37%), and fatigue (33%). There were three thromboembolic episodes and five cases of new onset hypertension. Two (7%) patients did not complete the first 4-week cycle of therapy because of adverse events (pneumonia and headache). There were no objective responses. Four patients had disease stabilization for>>/==" BORDER="0">4 months. A decrease in median VEGF plasma levels was observed in patients with stable disease (n = 7) compared with patients with progressive disease (n = 5). Overall median survival was 42 weeks (range 3-92+).

CONCLUSIONS

Although SU5416 had minimal clinical activity, signs of biological activity (decrease in plasma VEGF levels) suggest that angiogenic modulation may be of value in patients with MM.

Pregnant women who develop preeclampsia exhibit higher circulating levels of the soluble VEGF receptor-1 (sFlt-1). Recent findings suggest that soluble Flt-1 may contribute to the pathogenesis of preeclampsia by binding and neutralizing <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factors</em> (VEGF) and placental <em>growth</em> <em>factor</em> (PlGF). Existing literature identifies sFlt-1 as a 100 kDa glycoprotein, a product of an mRNA splice variant. We hypothesized that sFlt-1 expression may be more complex with multiple variants of sFlt-1 as well as multiple sources during normal pregnancy and preeclampsia. Using a combination of affinity purification of sFlt-1 by heparin-agarose and epitope specific antibodies, we performed Western blot analysis with epitope specific antibodies for sFlt-1. Plasma of preeclamptic women exhibits significantly higher amounts of a novel <em>145</em> kDa variant of sFlt-1, along with the 100 kDa isoform. We identified sFlt-1 variants in the conditioned medium from placental explant cultures that are hypoxia responsive with varying sizes, including 185, <em>145</em>,100 and 60 kDa forms, as well as antigenicity. The <em>145</em> kDa was similar in antigenicity to the 100 kDa found in plasma whereas the 185 and 60 kDa sFlt-1 demonstrated different epitopes. Deglycosylation studies also confirm that there are multiple sFlt-1 polypeptides. Co-immunoprecipitation with VEGF suggests that these different sFlt isoforms can bind VEGF and therefore, may be of functional importance. Finally, comparison of sFlt-1 in the conditioned medium obtained from cultured cytotrophoblasts, peripheral blood mononuclear cells (PBMCs) and human uterine micro<em>vascular</em> cells (HUtMVECs) exhibit mainly the100 kDa sFlt-1. Collectively these data suggest the presence of multiple isoforms of sFlt-1 in the circulation of women with preeclampsia as well as in uncomplicated pregnancies and the possibility of multiple sources. Placental hypoxia may contribute to sFlt-1 over expression but other regulatory mechanisms cannot be ruled out.