Norepinephrine has <em>growth</em>-promoting effects in cardiac myocytes. The present study in cultured neonatal rat cardiac myocytes tested the hypothesis that norepinephrine also stimulates expression of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF), an important angiogenic <em>factor</em>. As assessed by polymerase chain reaction cardiac myocytes and non-myocytes expressed all three isoforms of rat VEGF, with the short isoform (VEGF <em>121</em> ) preferentially expressed in non-myocytes. When cardiac myocytes were stimulated with 1 micro M norepinephrine for 24 h in the presence or absence of the specific alpha - and beta -adrenoceptor antagonists prazosin and propranolol, respectively, VEGF mRNA levels and splice variant pattern did not change, whereas atrial natriuretic peptide mRNA levels increased 3 to 4-fold. CoCl(2) increased VEGF mRNA levels in cardiac myocytes five-fold. When cardiac myocytes were cultured with conditioned medium from non-myocytes that had been stimulated with norepinephrine for 24 h VEGF mRNA increased 2-fold. The increase was blocked by antibodies neutralizing TGF beta. These data suggest that norepinephrine stimulates myocardial angiogenesis by a paracrine mechanism that involves cardiac non-myocytes and TGF beta.

Ligand stimulation promotes downregulation of RTKs, a mechanism by which RTKs, through the ubiquitination pathway are removed from the cell surface, causing a temporary termination of RTK signaling. The molecular mechanisms governing RTK trafficking and maturation in the endoplasmic reticulum (ER)/Golgi compartments are poorly understood. <em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> receptor-2 (VEGFR-2) is a prototypic RTK that plays a critical role in physiologic and pathologic angiogenesis. Here we demonstrate that Ring Finger Protein <em>121</em> (RNF<em>121</em>), an ER ubiquitin E3 ligase, is expressed in <em>endothelial</em> cells and regulates maturation of VEGFR-2. RNF<em>121</em> recognizes newly synthesized VEGFR-2 in the ER and controls its trafficking and maturation. Over-expression of RNF<em>121</em> promoted ubiquitination of VEGFR-2, inhibited its maturation and resulted a significantly reduced VEGFR-2 presence at the cell surface. Conversely, the shRNA-mediated knockdown of RNF<em>121</em> in primary <em>endothelial</em> cells reduced VEGFR-2 ubiquitination and increased its cell surface level. The RING Finger domain of RNF<em>121</em> is required for its activity toward VEGFR-2, as its deletion significantly reduced the effect of RNF<em>121</em> on VEGFR-2. Additionally, RNF<em>121</em> inhibited VEGF-induced <em>endothelial</em> cell proliferation and angiogenesis. Taken together, these data identify RNF<em>121</em> as a key determinant of angiogenic signaling that restricts VEGFR-2 cell surface presence and its angiogenic signaling.

<AbstractText>Ramucirumab, a human IgG 1 antibody against <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> receptor 2, has been shown to improve progression-free survival and overall survival in patients with advanced gastric cancer in the second-line setting.</AbstractText><AbstractText>To compare progression-free survival for S-1 and oxaliplatin plus ramucirumab with that for S-1 and oxaliplatin plus placebo in patients with advanced gastric cancer.</AbstractText><AbstractText>This phase 2, double-blind randomized clinical trial (RAINSTORM [First-line S-1 Plus Oxaliplatin With or Without Ramucirumab Followed by Paclitaxel Plus Ramucirumab in Patients With Advanced Gastric Cancer]) was conducted from October 12, 2015, to April 11, 2018, at 36 sites in Japan, South Korea, and Taiwan. Participants were chemotherapy-naive patients (n = 189) with metastatic gastric or gastroesophageal adenocarcinoma. Analyses of the full analysis set and safety population were conducted between November 27, 2017, and June 4, 2018.</AbstractText><AbstractText>Patients randomized to the ramucirumab plus S-1 and oxaliplatin arm received S-1, 80 to 120 mg/d twice daily, on days 1 to 14 and oxaliplatin, 100 mg/m2, on day 1 with ramucirumab, 8 mg/kg, on days 1 and 8 in part A (21-day cycle). Patients randomized to the placebo plus S-1 and oxaliplatin arm received the same S-1 and oxaliplatin dosage as well as placebo on days 1 and 8 in part A. Eligible patients received second-line paclitaxel, 80 mg/m2, on days 1, 8, and 15 and ramucirumab, 8 mg/kg, on days 1 and 15 in part B (28-day cycle).</AbstractText><AbstractText>The primary end point was progression-free survival, analyzed using the stratified log-rank test; the hazard ratio (HR) was estimated using the stratified Cox proportional hazards regression model. Secondary end points included overall survival and adverse events.</AbstractText><AbstractText>In total, 189 patients were randomized and received treatment: 96 to the ramucirumab plus S-1 and oxaliplatin arm and 93 to the placebo plus S-1 and oxaliplatin arm. Among the 189 patients, <em>121</em> (64.0%) were male, and the median (range) age was 62.0 (26-84) years. Median progression-free survival was not prolonged in the ramucirumab plus S-1 and oxaliplatin arm compared with the placebo plus S-1 and oxaliplatin arm (6.34 [80% CI, 5.65-6.93] vs 6.74 [80% CI, 5.75-7.13] months; HR, 1.07; 80% CI, 0.86-1.33; P = .70). Median overall survival was 14.65 (80% CI, 12.39-15.67) months in the ramucirumab plus S-1 and oxaliplatin arm and 14.26 (80% CI, 13.83-17.31) months in the placebo plus S-1 and oxaliplatin arm (HR, 1.11; 80% CI, 0.89-1.40; P = .55). The most commonly reported grade 3 or higher treatment-emergent adverse events in the ramucirumab plus S-1 and oxaliplatin arm in part A were decreased neutrophil count (14 patients [14.6%]), hypertension (10 patients [10.4%]), and anemia (10 patients [10.4%]).</AbstractText><AbstractText>In this randomized clinical trial, the addition of ramucirumab to first-line S-1 and oxaliplatin treatment did not prolong progression-free survival or overall survival compared with S-1 and oxaliplatin alone among East Asian patients with advanced gastric cancer; no new safety signals for ramucirumab were identified.</AbstractText><AbstractText>ClinicalTrials.gov identifier: NCT02539225.</AbstractText>

Conbercept is a novel anti-<em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> for the treatment of age-related macular degeneration (AMD). The most optimal injection strategy is unknown. To assess the effectiveness of intravitreal injection of conbercept using the 3 + pro re nata (PRN) and 3 + Q3 M strategies for the treatment of exudative AMD.From January 2015 to January 2018, patients confirmed with exudative AMD at Qilu Hospital of Shandong University were included in this retrospective study. Intravitreal injection of 0.5 mg of conbercept was conducted either with the 3 + PRN or 3 + Q3 M strategy. Best-corrected visual acuity (BCVA), intraocular pressure, and optical coherence tomography were conducted at 1 and 2 weeks, then every month. fundus fluorescein angiography examination was conducted every 3 months.There were 106 eyes from 106 patients. The number of follow-ups (3 + Q3 M: 12.4 ± 1.3 vs 3 + PRN: 12.9 ± 1.6, P = .079) and the follow-up time (3 + Q3 M: 12.7 ± 0.6 vs 3 + PRN: 12.5 ± 0.7 months, P = .<em>121</em>) were similar in the 2 groups. The number of injections was less in 3 + PRN than 3 + Q3 M (5.3 ± 1.0 vs 6.0 ± 0.0, P < .001) The BCVA at months 7 and 9 to 12 in the 3 + Q3 M (n = 51) group were lower than for 3 + PRN (n = 55) (all P < .05). The CRT at months 9 to 12 in the 3 + Q3 M group was lower than in the 3 + PRN group (all P < .05). There were no differences between the 2 groups regarding the exudation area during follow-up. No serious treatment-related ocular complications or serious systemic adverse events were found.The 3 + PRN and 3 + Q3 M strategies of intravitreal injection of conbercept are effective in treating exudative AMD. The 3 + Q3 M strategy needs more injection but is more effective in increasing visual acuity and reducing macular CRT than the 3 + PRN strategy.

Abstract Coronary artery disease (CAD), a leading cause of mortality, is a chronic disease in which blood flow to the myocardium is obstructed, leading to ischemia. Although coronary artery stenting and surgical bypass are successful with localized coronary lesions, patients with diffuse CAD have only pharmacologic options. In a mouse ischemic hind-limb model, AdVEGF-All6A+, an Ad5 vector expressing the cDNA/genomic hybrid of the <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) gene (expressing isoforms, <em>121</em>, 165, and 189) mediated recovery of blood flow at a dose of two logs less than that required with a single isoform. The objective of the current study was to ascertain the safety profile of good manufacturing practice (GMP)-grade AdVEGF-All6A+ in the adult rat ischemic heart model in support of a clinical study to treat humans with diffuse CAD. AdVEGF-All6A+ (10(5), 10(6), or 10(7) particle units), a control vector (AdNull, 10(7) particle units) with no translatable expression cassette and a vehicle sham control (phosphate buffered saline [PBS]) were administered separately to the left ventricle of rats immediately following acute coronary artery ligation to initiate myocardial infarction (MI), designed to evoke an extreme ischemic myocardium in cohorts (n=5 males; n=5 females), with sacrifice at 5, 14, or 30 days. Six cohorts received no ligation but were administered AdVEGF-All6A+ vector or PBS with sacrifice at 30 or 365 days. Although there were surgical-related abnormalities among the groups, blinded evaluation of gross and histopathology, complete blood count, and serum chemistry found no significant differences between control- and vector-treated groups and no adverse effects could be attributed to AdVEGF-All6A+. No changes in serum troponin-I levels persisted beyond those associated with the MI. Gross pathology and histopathology of all major organs showed no AdVEGF-All6A+-related changes. Overall, this safety profile suggests that AdVEGF-All6A+ or AdNull administration to the myocardium meets the criteria to proceed to clinical trial.

Chronic wounds and in particular diabetic foot ulcers (DFUs) are a <em>growing</em> clinical challenge, but the underlying molecular pathophysiological mechanisms are unclear. Recently, we reported reduced levels of the immunomodulating and antimicrobial S100A8/A9 in non-healing venous leg ulcers (VLUs), while another study found increased S100A8/A9 in DFUs. To clarify these apparently contradictory findings, we compared S100A8/A9 as well as an inducer, lipopolysaccharide (LPS) and selected innate immune response mediators in wound fluids from non-healing DFUs and VLUs with healing wounds. Wound fluids were collected from neuropathic DFUs (n = 6) and VLUs (n = 9) of median 2-year duration, and split-thickness skin graft donor site wounds (n = 10) by standardized method. None of the patients had ischaemic extremities or clinically infected wounds. LPS was determined by limulus amoebocyte lysate test, and S100A8/A9, granulocyte colony-stimulating <em>factor</em> (G-CSF), interleukin (IL)-10 and <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) by immunospecific quantitative assays. LPS levels were median 8.7 (interquartile range 5.4-21.2) ng/ml in DFUs compared with <em>121</em> (22-2000) ng/ml in VLUs. S100A8/A9 was higher (p = 0.020) in DFUs [718 (634-811) µg/ml] than in VLUs [303 (252-533) µg/ml]. Neither G-CSF nor IL-10 wound fluid levels differed significantly between the chronic wound groups. VEGF levels correlated with LPS (r = 0.758, p = 0.011, n = 10) and were higher (p = 0.024) in VLU wound fluids. LPS (p < 0.0001), S100A8/A9 (p = 0.005), G-CSF (p = 0.003), IL-10 (p = 0.003) and VEGF (p = 0.005) were increased in chronic wound fluids combined compared with the sterile donor site wound fluids. The protein alterations in the wounds were not reflected in the patients' sera. Low S100A8/A9 levels may contribute to poor wound healing in colonized chronic wounds with striking difference between DFUs and VLUs.

The contribution of VEGF (<em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em>) to angiogenesis and tumor aggressiveness has been shown in several tumor types, but no comparative data regarding the impact of the VEGF-subtypes on <em>endothelial</em> and tumor cell protection are available. Therefore, we analysed the cytoprotective effects of the two major soluble VEGF-subtypes, VEGF-<em>121</em> and -165, on HUVEC cell cultures (human umbilical vein <em>endothelial</em> cells) and squamous cell carcinoma (SCC) cell lines after ionizing radiation. We performed clonogenic analyses and proliferation assays for evaluation of cell survival and proliferative activity. Experiments were performed employing different intensities up to 8 Gy with or without added recombinant VEGF-<em>121</em> or -165 and compared to non-irradiated cultures. To evaluate a possible contribution of the VEGF-receptors, we performed immunohistochemical stainings of VEGF-R1 (flt), -R2 (KDR,flk), and Neuropilin-1. In the SCC cell lines and HUVEC cultures, cell survival and proliferative activity were reduced in a dosage-dependent manner. The addition of VEGF-<em>121</em> yielded a significant increase of resistance from 1.2 to 2.7 Gy (+125%) for killing 50% of subjected cells, while VEGF-165 was less effective in this regard (+83%). Conversely, in HUVEC cultures, 50%-survival was increased more strongly by VEGF-165 compared to VEGF-<em>121</em> (+100% vs. +43%). Accordingly, proliferation was more intensely stimulated in HUVECs by VEGF-165 than by VEGF-<em>121</em>. VEGF-receptors were expressed in all cell cultures analysed at comparable levels. We conclude that the two VEGF-subtypes differentially increase the survival of tumor and <em>endothelial</em> cells after irradiation in vitro. We hypothesise, that the release of VEGF by the tumor protects tumor cells and endothelium resulting in increased radiation resistance.

The healing of ischemic wounds is a particularly difficult clinical challenge. In this study, rabbit dermal fibroblasts transduced retrovirally with human platelet-derived <em>growth</em> <em>factor</em> B (PDGF-B) and human <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> <em>121</em> (VEGF<em>121</em>) genes were used to treat wounds in a rabbit ischemic ear model. The PDGF-B and VEGF<em>121</em> genes were obtained from human umbilical vein <em>endothelial</em> cells (HUVECs) by reverse transcription-polymerase chain reaction, cloned into retroviral vectors under control of the beta-actin promoter, and introduced into primary rabbit dermal fibroblast cells. In vitro results demonstrated that rabbit dermal fibroblasts are transduced and selected readily using retroviral vectors, and are engineered to secrete PDGF-B and VEGF<em>121</em> at steady-state levels of 150 ng per 10(6) cells per 24 hours and 230 ng per 10(6) cells per 24 hours respectively. These cells were then seeded onto polyglycolic acid (PGA) scaffold matrices and used to treat ischemic rabbit ear wounds. Immunohistochemistry showed intense staining for PDGF-B and VEGF<em>121</em> in the wounds treated with these transduced cells compared with the control treatment groups. For the relatively more ischemic distal ear wounds, granulation tissue deposition was increased significantly in the wounds treated with PDGF-B- and VEGF<em>121</em>-transduced cells compared with wounds treated with PGA alone. These results demonstrate that gene augmentation of rabbit dermal fibroblasts with the PDGF-B and VEGF<em>121</em> genes introduced into this ischemic wound model via PGA matrices modulates wound healing, and may have clinical potential in the treatment of ischemic wounds.

BACKGROUND

Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis and is implicated in the development of diabetic microvascular and macrovascular disease.

METHODS

The expression of total VEGF, VEGF splice variants (VEGF(121), VEGF(145), VEGF(148), VEGF(165), VEGF(183) and VEGF(189)), VEGFR-1 and VEGFR-2, was investigated in biopsies from the right atrium and left internal mammary artery (LIMA) of 32 non-diabetic and 20 diabetic patients undergoing coronary artery bypass grafting.

RESULTS

Diabetes was independently negatively correlated to total VEGF mRNA expression in atrium. Total VEGF, VEGF(121) and VEGF(165) mRNA levels were upregulated in the LIMA of diabetics vs. non-diabetics. The expression of VEGF receptors in atrium and LIMA was similar between these groups. VEGF(121) and VEGF(165) were the major variants expressed, followed by VEGF(189) and VEGF(183), while VEGF(148) and VEGF(145) were detected in small amounts. The expression profile of VEGF splice variants displayed significant heterogeneity between the examined tissues.

CONCLUSIONS

This is the first study to quantify VEGF splice variants expression in cardiac and vascular tissue. Our results could help elucidate the role of VEGF splice variants in diabetic complications.

BACKGROUND

Our objective was to observe the role of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) <em>121</em> gene transfer in promoting <em>vascular</em> reconstruction and bone repair in femur head necrosis of rabbits.

METHODS

The femoral head necrosis model was induced by injection with ethanol. The necrotic femoral head was transfected with a human adenoviral vector expressing VEGF (Ad-hVEGF<em>121</em>). Bone formation in the subchondral necrotic region was analyzed using histology, by measuring the bone mineral density value, and by observing bone trabecular morphology using image analysis.

RESULTS

Revascularization level, bone formation rate, bone quality and quantity, and mineralization level in the subchondral necrotic region of the gene transfection group were significantly higher than the control groups. The control groups had more subchondral bone resorption compared with the gene transfection group.

CONCLUSIONS

VEGF might promote bone formation and revascularization in the subchondral necrotic region of the femoral head, indirectly protecting the necrotic bone trabecula from absorption and avoiding a reduction in the mechanical function of the subchondral region.

Plant expression systems offer a valuable alternative to traditional systems for the production of recombinant biopharmaceuticals. A highly efficient polyethyleneglycol (PEG)-mediated transient expression system for secreted recombinant proteins in plants has been developed. The human <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> <em>121</em> (rhVEGF) has been successfully expressed and efficiently secreted into the culture medium by transiently transformed moss protoplasts. In order to obtain secretion efficiency data, different expressed signal peptides were analysed and time course studies were performed with expression constructs containing different promoters. The transformation procedure was optimised for high level expression (up to 10 microg/ml) and successfully performed even with a transgenic glyco-engineered strain lacking plant-specific immunogenic sugar residues in N-glycans. The amount of rhVEGF was produced in such quantity that it allowed for the analysis of biological activity, silver-staining and Western blotting, revealing the correct formation and processing of the homodimer. This fast and flexible transient expression system enables feasibility studies and construct optimisation to be concluded within a few days, thus avoiding the time consuming step of having to generate stably transformed lines.

BACKGROUND

The fusion protein VEGF(<em>121</em>)/rGel composed of the <em>growth</em> <em>factor</em> VEGF(<em>121</em>) and the plant toxin gelonin targets the tumor neovasculature and exerts impressive anti-<em>vascular</em> effects. We have previously shown that VEGF(<em>121</em>)/rGel is cytotoxic to <em>endothelial</em> cells overexpressing VEGFR-2 but not to <em>endothelial</em> cells overexpressing VEGFR-1. In this study, we examined the basis for the specific toxicity of this construct and assessed its intracellular effects in vitro and in vivo.

METHODS

We investigated the binding, cytotoxicity and internalization profile of VEGF(<em>121</em>)/rGel on <em>endothelial</em> cells expressing VEGFR-1 or VEGFR-2, identified its effects on angiogenesis models in vitro and ex vivo, and explored its intracellular effects on a number of molecular pathways using microarray analysis.

RESULTS

Incubation of PAE/VEGFR-2 and PAE/VEGFR-1 cells with (125)I-VEGF(<em>121</em>)/rGel demonstrated binding specificity that was competed with unlabeled VEGF(<em>121</em>)/rGel but not with unlabeled gelonin. Assessment of the effect of VEGF(<em>121</em>)/rGel on blocking tube formation in vitro revealed a 100-fold difference in IC(50) levels between PAE/VEGFR-2 (1 nM) and PAE/VEGFR-1 (100 nM) cells. VEGF(<em>121</em>)/rGel entered PAE/VEGFR-2 cells within one hour of treatment but was not detected in PAE/VEGFR-1 cells up to 24 hours after treatment. In <em>vascular</em>ization studies using chicken chorioallantoic membranes, 1 nM VEGF(<em>121</em>)/rGel completely inhibited bFGF-stimulated neo<em>vascular</em> <em>growth</em>. The cytotoxic effects of VEGF(<em>121</em>)/rGel were not apoptotic since treated cells were TUNEL-negative with no evidence of PARP cleavage or alteration in the protein levels of select apoptotic markers. Microarray analysis of VEGF(<em>121</em>)/rGel-treated HUVECs revealed the upregulation of a unique "fingerprint" profile of 22 genes that control cell adhesion, apoptosis, transcription regulation, chemotaxis, and inflammatory response.

CONCLUSIONS

Taken together, these data confirm the selectivity of VEGF(<em>121</em>)/rGel for VEGFR-2-overexpressing <em>endothelial</em> cells and represent the first analysis of genes governing intoxication of mammalian <em>endothelial</em> cells by a gelonin-based targeted therapeutic agent.

BACKGROUND

We hypothesized that adenovirus-mediated transfer of the vascular endothelial growth factor (VEGF121) complementary DNA (cDNA) to murine laparotomy fascial wounds would enhance vascularity and bursting strength.

METHODS

Microfibrillar collagen sponges saturated with adenovirus (Ad) vectors encoding for the human VEGF121 cDNA (Ad(CU)VEGF121.1), a control marker gene (Ad beta gal, AdLuc) or no transgene (AdNull) were sutured to fascial edges during laparotomy closure in normal mice and mice treated with dexamethasone. Endpoints addressed included transgene expression in the fascia and surrounding tissue, the number of blood vessels in the healing wound determined using immunostaining, and wound bursting strength using a calibrated tensinometer.

RESULTS

Transgene expression was detected readily in the fascial edges, but only marginally detectable in neighboring tissues. In normal mice and mice treated with dexamethasone, no differences were observed at 7 days. Strikingly, however, 21 days after wound closure/therapy, significantly more blood vessels were present in the wounds that received the VEGF121 vector compared with controls (normal: AdNull: 4.2 +/- 1.8; Ad(CU)VEGF121.1: 11.2 +/- 1.2; P <.05; dexamethasone: AdNull: 1.4 +/- 0.8; Ad(CU)VEGF121.1: 5.4 +/- 1.2; P <.05), and bursting strength was significantly higher in VEGF121-treated wounds (normal: AdNull: 665 +/- 68 mN/mm; Ad(CU)VEGF121.1: 956 +/- 82 mN/mm; P <.0005; dexamethasone: AdNull: 234 +/- 111 mN/mm; Ad(CU)VEGF121.1: 592 +/- 121 mN/mm; P <.03).

CONCLUSIONS

Adenovirus-mediated gene transfer to healing fascial wounds is achieved readily using a microfibrillar collagen sponge, with transfer of the human VEGF121 cDNA significantly enhancing wound vascularity and bursting strength in normal mice, as well as in mice treated with dexamethasone.

Enforced expression of c-mpl in embryonic stem (ES) cells inactivated for this gene results in protein expression in all the ES cell progeny, producing cells that do not belong to the megakaryocytic lineage and are responsive to PEG-rhuMGDF, a truncated form of human thrombopoietin (TPO) conjugated to polyethylene glycol. These include a primitive cell called BL-CFC, thought to represent the equivalent of the hemangioblast, and all myeloid progenitor cells. In this model, PEG-rhuMGDF was able to potentiate the stimulating effects of other <em>growth</em> <em>factors</em>, including <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em>, on BL-CFC and a combination of cytokines on the <em>growth</em> of granulocyte macrophage-colony-forming units. The importance of the C-terminal domain of Mpl and of mitogen-activated protein kinase (MAPK) activation in TPO-dependent megakaryocytic differentiation has been well studied in vitro. Here, the role of this domain and the involvement of MAPK in upstream and nonmegakaryocytic cells are examined by using 2 truncated mutants of Mpl (Delta34, deletion of residues 71 to <em>121</em> in the C-terminal domain; and Delta3, deletion of residues 71-94) and specific inhibitors of the MAPK pathway. The 2 deleted regions support different functions, mediated by different signals. Residues 71 to <em>121</em> were required for PEG-rhuMGDF-dependent <em>growth</em> of BL-CFC, for megakaryocytic and other myeloid progenitors, and for megakaryocyte polyploidization. These responses were mediated by the ERK1-ERK2 MAPK pathway. In contrast, the only function of the sequence comprising residues 71 to 94 was to mediate the synergistic effects of PEG-rhuMGDF with other hematopoietic <em>growth</em> <em>factors</em>. This function is not mediated by MAPK activation.

The transplantation of skeletal myoblasts (SkM) might improve cardiac function after myocardial infarction via paracrine action. We used scaffold-based cell transfer by using <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF)-overexpressing myoblasts. Skeletal myoblasts were isolated and expanded from newborn Lewis rats. Cells were transfected with pCINeo-VEGF(<em>121</em>) and seeded on polyurethane (PU) scaffolds. The seeded scaffolds were epicardially implanted in rats 2 weeks after myocardial infarction (group: PU-VEGF-SkM). Before this intervention and 6 weeks later, pressure/volume loops were analyzed followed by histology. Additional study groups (n = 10 per group) were injected with VEGF-overexpressing myoblasts (Inj-VEGF-SkM) or unmodified myoblasts (Inj-SkM) or underwent a sham operation. Overexpression of VEGF was verified in vitro. The transplantation of <em>growth</em> <em>factor</em> producing myoblast-seeded scaffolds resulted in enhanced angiogenesis of ischemically damaged myocardium in vivo. However, the infarction size was not reduced. In group Inj-SkM, hemodynamics remained unchanged. Systolic function as measured by dP/dt(max) was not significantly altered in PU-VEGF-SkM (preinterventionally 2,156 ± 1,222 mmHg vs. postinterventionally 2,134 ± 850 mmHg). Other systolic function and diastolic function parameters as measured by dP/dt(min), tau, and pressure half-time were not restored in groups PU-VEGF-SkM and Inj-VEGF-SkM either. Transplantation of VEGF-overexpressing skeletal myoblasts leads to neo<em>vascular</em>ization in infarcted hearts. No functional myocardial recovery was observed. Scaffold-based transfer of genetically-modified cells remains a useful tool to study paracrine stem cell action.

Increased <em>vascular</em> permeability and <em>endothelial</em> cell <em>growth</em> are important in the pathogenesis of otitis media with effusion (OME) and the <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) is known to play an important role in the increased <em>vascular</em> permeability and angiogenesis. To date, at least five isoforms of the VEGF family have been identified as VEGF transcripts, encoding polypeptides of 206, 189, 165, 145 and <em>121</em>, but their physiological roles are unclear. The purpose of this study was to investigate the expression of VEGF, in both endotoxin-induced OME of the rat and human otitis media. We instilled endotoxin and saline as a control into the middle ear cavity of the rat. Middle ear mucosa were taken at 0 h, 1 h, 3 h, 6 h, 12 h, 1 day, 3 days, 7 days and 14 days and the expression of VEGF mRNA and VEGF protein was evaluated using semi-quantitative RT-PCR and immunohistochemistry. Expression of VEGF164 mRNA and VEGF120 mRNA was first identified 1 h after endotoxin instillation and was dramatically increased over the period 6 h-1 day and then progressively decreased by day 7. The level of expression of VEGF120 mRNA was slightly higher than that of VEGF164 mRNA and that of VEGF164 mRNA was much higher than that of VEGF188 mRNA. Immunostaining revealed expression of VEGF during 6 h to day 3 and its expression was localized to ciliated cells and some inflammatory cells. We also performed RT-PCRs of cDNA from middle ear fluids of 8 human OME patients and middle ear mucosa of 4 chronic otitis media patients for the identification of VEGF mRNA expression. VEGF<em>121</em> mRNA was highly expressed in all samples compared with VEGF165 mRNA. These results suggest that VEGF may be primarily responsible for increased <em>vascular</em> permeability and <em>endothelial</em> cell <em>growth</em> in OME and that VEGF seems to play a significant role in the pathogenesis of OME.

Expression of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) in oral tissues was assessed using different antibodies. Quantitative and topographical differences were observed between paraffin and cryostat sections. Two polyclonal antibodies (PC36, PC37) differing in their cross-reactivity with VEGF<em>121</em> (not recognized by PC36), were used to stain serial cryostat sections of normal oral mucosa (n = 8) and squamous cell carcinoma (n = 7). The expression of VEGF in the epithelium was overall higher with PC37 than with PC36, the difference being significant in normal oral mucosa (p = 0.001) but not in squamous cell carcinoma samples (p = 0.094). With PC36, VEGF expression was significantly higher in squamous cell carcinoma than in normal oral mucosa specimens, whereas the opposite was true with PC37. Our results suggest that the relative levels of isoform <em>121</em> to that of 165 (and possibly others) may be different in the tissues examined, with VEGF<em>121</em> preferentially expressed in normal oral mucosa. Previously published conflicting results may, therefore, be due to the presence of variable ratios of VEGF isoforms in the tissues examined, combined with differences in the cross-reactivity of the antibodies used. VEGF isoforms <em>121</em>, 165 and (for the first time) 189 were detected in two frozen oral tissues by polymerase chain reaction amplification. Quantification of specific VEGF isoforms will be required in future studies concerned with the clinical value of VEGF expression.

VEGF(<em>121</em>), the <em>121</em>-amino acid form of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> is a homodimer with nine cysteine residues per monomer. While three intramolecular and two intermolecular disulfide bonds have been mapped, the state of the ninth cysteine, Cys116, is not known. In this study, we determined that human VEGF(<em>121</em>) contains a third interchain disulfide bond between Cys116 of each monomer. We also isolated a VEGF(<em>121</em>) variant with two extra cysteines bound to each Cys116. No evidence was found for the exsistence of Cys116 in the reduced state. In fact, selective reduction of the Cys116 interchain disulfide bond yielded an unstable VEGF(<em>121</em>) molecule, which reoxidized quickly. Biological activities of VEGF(<em>121</em>) Cys116 variants were assessed. The oxidative state of Cys116 has no effect on binding or proliferation activities but may be important for overall stability of the molecule.

Recent evidence demonstrates that masked hypertension (MH) is a significant predictor of cardio<em>vascular</em> disease, while apelin and relaxin are two novel <em>factors</em> with a significant role in <em>vascular</em> regulation. Apelin is an adipokine that elicits endothelium-dependent vasorelaxation and reduces arterial blood pressure, while relaxin is a protein hormone that induces the production of nitric oxide and <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> and inhibits endothelin and angiotensin II. This study aimed to investigate whether apelin and relaxin plasma levels are affected in patients with MH and compare the findings with those of healthy normotensives. One hundred-thirty (60 men, 70 women) healthy patients with a mean age of 45±12 years who had clinic blood pressure <140/90 mmHg were studied. The whole study population underwent 24-hour ambulatory blood pressure monitoring (ABPM). According to the ABPM recordings, 24 individuals (8 men, 16 women) had MH and the remaining 106 patients (52 men, 54 women) had normal ABPM recordings. Apelin and relaxin plasma levels were determined in both groups (enzyme-linked immunosorbent assay method). The apelin (220±<em>121</em> vs 315±147 pg/mL, P=.001) and relaxin (35.2±6.7 vs 56.8±13.6 pg/mL, P<.001) plasma levels were significantly lower in the masked hypertensive group compared with normotensive controls. Our findings suggest that patients with masked hypertension have significantly lower apelin and relaxin levels. This observation may have prognostic significance for future cardio<em>vascular</em> events in patients with MH and needs further investigation.

BACKGROUND

Intra-articular glucocorticoid treatment (IAGC) is widely used for symptom relief in arthritis. However, knowledge of factors predicting treatment outcome is limited. The aim of the present study was to identify response predictors of IAGC for knee synovitis in patients with rheumatoid arthritis (RA).

METHODS

In this study 121 RA patients with synovitis of the knee were treated with intra-articular injections of 20 mg triamcinolone hexacetonide. They were followed for six months and the rate of clinical relapse was studied. Non-responders (relapse within 6 months) and responders were compared regarding patient characteristics and knee joint damage as determined by the Larsen-Dale index. In addition, matched samples of serum and synovial fluid were analysed for factors reflecting the inflammatory process (C-reactive protein, interleukin 6, tumour necrosis factor alpha, vascular endothelial growth factor), joint tissue turnover (cartilage oligomeric matrix protein, metalloproteinase 3), and autoimmunity (antinuclear antibodies, antibodies against citrullinated peptides, rheumatoid factor).

RESULTS

During the observation period, 48 knees relapsed (40%). Non-responders had more radiographic joint damage than responders (P = 0.002) and the pre-treatment vascular endothelial growth factor (VEGF) level in synovial fluid was significantly higher in non-responders (P = 0.002).

CONCLUSIONS

Joint destruction is associated with poor outcome of IAGC for knee synovitis in RA. In addition, higher levels of VEGF in synovial fluid are found in non-responders, suggesting that locally produced VEGF is a biomarker for recurrence of synovial hyperplasia and the risk for arthritis relapse.

Angiogenesis or formation of new blood vessels is required for regeneration of the endometrium after its breakdown during each menstruation. <em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF), a family of recently discovered angiogenic <em>factors</em>, may be involved in the repair and <em>growth</em> of the endometrium. In this study reverse transcription-polymerase chain reaction (PCR) was used to confirm the presence of VEGF mRNA and restriction enzyme digestion to confirm the identity of PCR products generated from different VEGF isoforms in cultured human endometrial stromal cells. The shortest isoform, VEGF <em>121</em>, was the most abundant in quiescent stromal cells. It was about one-and-a-half times that of VEGF 165. The longest isoform, VEGF 206, was not detected; only a relatively weak signal for VEGF 189 was detectable. The mRNA for VEGF increased 2-fold after stimulation by 17beta-oestradiol (10 nM) for 30 min. A further increase to 3-fold above baseline occurred after 2 h incubation and remained steady at 6 h incubation, but decreased to 2-fold of baseline after 15 h. There was no differential stimulation of mRNA for VEGF isoforms: the ratio of VEGF <em>121</em> to 165 remained constant at 3:2 during the course of the incubation, with the exception at 15 h incubation when the ratio was 2:1. The VEGF protein, determined by specific enzyme immunoassay, increased from undetectable at baseline to 79.8 +/- 18.9 pg/ml (n = 4, mean +/- SD, 9.6 cm2/well/ml) after 2 h, with a further significant increase to 249.5 +/- 27.3 pg/ml after 15 h and 695.0 +/- 41.4 pg/well after 39 h. At 15 h incubation, the specific oestradiol antagonist ICI 182,780 (1 microM) significantly reduced VEGF secretion by 25% from 249.5 +/- 27.3 to 189.0 +/- 26.6 pg/ml. Thus, VEGF showed specific patterns of isoform expression in the human endometrial stromal cells; oestradiol (10 microM) stimulated, but not differentially, the mRNA for VEGF isoforms.

To further elucidate the role of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) in the pathogenesis of chronic myeloid leukemia (CML), we transfected K562 cells with a VEGF(<em>121</em>)cDNA sense vector (S), an antisense (AS) vector or vector (V) alone. The <em>growth</em> of transfected cells was investigated by MTT and colony-formation assays, and apoptosis was measured by flow cytometry (FCM) of Annexin-V-FITC/PI dual labeled cells. Transfected cells were subcutaneously transplanted into nude mice and the microvessel density (MVD) of tumor masses was determined by vWF immunohistochemistry staining. We tested the supernatant of different transfected K562 cells against human bone marrow <em>endothelial</em> cells (BMECs), and examined the synergic effects of antisense VEGF(<em>121</em>)cDNA and IFNalpha or STI571 on the proliferation and apoptosis of K562 cells. We found that K562/AS transfectants exhibited a 49% reduction in VEGF secretion, whereas K562/S transfectants exhibited a 3-fold increase in VEGF secretion, all in comparison to the vector controls. K562 cells transfected with antisense VEGF(<em>121</em>)cDNA showed <em>growth</em> retardation in vitro. In transplanted nude mice in vivo, transfection of implanted cells with antisense VEGF(<em>121</em>)cDNA resulted in decreased tumor MVD, and increased apoptosis in the presence of IFNalpha. Taken together, these results suggest that VEGF may be involved in the pathogenesis of CML through autocrine and paracrine mechanisms, and that anti-VEGF therapy alone or in combination with conventional treatment may be beneficial for CML patients.

OBJECTIVE

To investigate the relationship between vascular endothelial growth factor (VEGF) microvascular density (MVD), lymph node metastasis and prognosis of breast carcinoma (BC).

METHODS

VEGF protein expression and MVD in 92 cases of BC and VEGF mRNA expression in part of the cases were studied by immunohistochemistry methods and reverse-transcription polymerase chain reaction (RT-PCR) technique.

RESULTS

VEGF mRNA expression in BC tumor tissues were higher than those in adjacent normal tissues. Increment of both VEGF(121) and VEGF(165) showed significant difference (P < 0.05 approximately 0.01). In addition, VEGF(145) expression was also observed in BC. There was a close positive correlation between VEGF and MVD (r = 0.702, P < 0.01). VEGF protein expression and MVD correlated significantly with lymph node metastasis and tumor relapse (P < 0.05 approximately 0.01). The time period of relapse-free-survival (RFS) in the patient group with high VEGF expression and MVD was significantly lower than RFS in the group with low VEGF expression and MVD (P < 0.01).

CONCLUSIONS

VEGF is highly related to angiogenesis of BC. The increase of VEGF and MVD may promote lymph node metastasis and relapse of BC. VEGF and MVD may have prognostic value in RFS of BC patients.

We hypothesized that the regulation of micro<em>vascular</em> functions and angiogenesis in breast tissue, a well known target of ovarian steroid action, is dependent on the hormonal exposure of the breast. Relative expression levels of VEGF-A (<em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> A), a putative key regulator of angiogenesis in breast cancer, were analysed in the tumour and the adjacent non-neoplastic breast tissue of 19 breast cancer patients by quantitative reverse transcriptase polymerase chain reaction. In non-neoplastic breast specimens the expression levels of all detected VEGF-A-isoforms (189, 165, <em>121</em>) were significantly higher in premenopausal compared to post-menopausal women (P = 0.02) and were inversely correlated with the patient's age (P = 0.006). In contrast, in cancerous tissues menopausal status had no influence on VEGF-A-expression levels. Benign and malignant tissues exhibited a similar expression pattern of VEGF-A-isoforms relative to each other. Thus, the regulation of the vasculature in normal breast tissue, as opposed to breast cancer tissue, appears to be hormonally dependent. Endogenous and therapeutically used hormonal steroids might, therefore, cause clinically relevant changes of the angiogenic phenotype of the human breast.