BACKGROUND

Interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) have important functions in injury and repair processes of glomerular intrinsic cells. A study was conducted to analyze the urinary VEGF/creatinine (CR) and IL-6/CR levels in simple hematuria patients after excluding the interference of creatinine. We aimed to investigate the function and relationships of the above indices in the glomerular pathological injury process, and to elaborate the values of urinary VEGF and IL-6 changes in the diagnosis of asymptomatic hematuria or hematuria with proteinuria.

METHODS

A total of 121 renal hematuria patients diagnosed by clinical and laboratory tests were included as research subjects. The midstream fresh morning urine was collected on the day renal biopsy was performed.

RESULTS

The IL-6/CR value of the group III was significantly greater than in group I (Z=-2.478, P<0.05), with a statistically significant difference between these 2 groups. The VEGF/CR value of group III was significantly greater than in group II (P<0.01). Compared with group I, the VEGF/CR of group III was significantly greater (Z=-4.65, P<0.01), with a statistically significant difference.

CONCLUSIONS

The VEGF/CR and IL-6/CR values in simple hematuria patients were positively correlated with glomerular pathological injury scores. VEGF/CR and IL-6/CR might be used as biological diagnostic indicators in determining the extent of simple hematuria glomerular injury.

To investigate the effect of 5'-(N-ethylcarboxamido) adenosine (NECA), an adenosine agonist, on triggering angiogenesis in transplanted human ovarian tissue, the expression of angiopoietin-1 (Ang1), Ang2, <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> <em>121</em> (VEGF-<em>121</em>) and VEGF-189 at both gene and protein levels as well as the density of vasculature were studied in tissue fragments grafted to NECA-treated and control groups of nude mice. The results showed that NECA treatment triggered down-regulation of Ang1, induced VEGF-189 expression, and stimulated neo<em>vascular</em>ization, highlighting the beneficial effect of NECA on the process of angiogenesis.

OBJECTIVE

To describe the microbiological profile and clinical outcome in the eyes with culture-proven exogenous endophthalmitis.

METHODS

A retrospective analysis of 495 eyes diagnosed as exogenous endophthalmitis was performed over a period of 10 years. In all, aseptically collected aqueous and vitreous aspirates were cultured for bacteria and fungus using standard microbiological techniques. Gram-stain and KOH preparation of the specimens were also performed. The antibiotic susceptibility testing for bacterial isolates was performed by Kirby-Bauer disk diffusion method. The treatment was modified according to the antibiotic sensitivity profile. The final clinical ocular condition was divided into improved, stable or deteriorated.

RESULTS

Of 148 culture-proven endophthalmitis eyes, 137 (92.57%) were referred from elsewhere, and 11 (7.43%) belonged to our institute. Aetiologically, 76 (51.35%) eyes were post-cataract surgery, 61 (41.22%) were post-traumatic, 5 (3.38%) eyes post-intravitreal anti-<em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> injection, 5 associated with corneal diseases and 1 bleb-related endophthalmitis. In 31 (20.95%) eyes, primary intravitreal antibiotics were given outside. The cultures revealed monomicrobial <em>growth</em> in 92.57% (n = 137) and polymicrobial <em>growth</em> in 7.43% (n = 11). Among the bacteria (n = <em>121</em>, 81.76%), Pseudomonas species dominated overall (n = 32, 27.11%) and post-operative (n = 26, 38.23%) endophthalmitis group. Staphylococcus epidermidis (n = 14, 28%) was prominent in post-traumatic endophthalmitis group. Ninety-two percent (n = 108 isolates) of bacteria were sensitive to vancomycin. In 78 (52.7%) eyes, the clinical ocular condition improved or remained stable while deteriorated in 51 (34.46%).

CONCLUSIONS

A bacterial predominance was observed among causative organisms of exogenous endophthalmitis with Pseudomonas species being the most common. The appropriate surgical intervention improved or stabilised the visual acuity in nearly 50% eyes.

BACKGROUND

Placental growth factor (PlGF), a member of the vascular endothelial growth factor (VEGF) family, has recently emerged as a predictor of survival and cardiovascular risk. Along with others, we have shown an independent association between PlGF and cardiovascular events in CKD patients, but not much is known about patients receiving dialysis.

METHODS

We studied 205 dialysis patients undergoing cardiac catheterization at the Nara Medical University between April 1, 2004, and December 31, 2012. Serum levels of PlGF and VEGF were measured with ELISA in all the patients.

RESULTS

During a median follow-up of 20 months, 121 participants died from any cause or experienced a cardiovascular event. In the fully adjusted analysis, having an above-median PlGF or VEGF level was associated with a hazards ratio for adverse outcomes of 2.55 (1.72-3.83) and 1.39 (0.95-2.04), respectively. Using a multimarker strategy in a model with age, serum albumin, history of coronary artery disease, brain natriuretic peptide and PlGF, patients with 2, 3 and 4 positive markers had a 3.82-, 5.77- and 6.59-fold higher risk of mortality or a cardiovascular event, respectively, compared to those with no positive markers. The model with PlGF had a significantly higher c-statistic, integrated discrimination improvement index and category-free net reclassification improvement index than the model without PlGF.

CONCLUSIONS

PlGF is independently associated with mortality and cardiovascular events, but the association between VEGF and adverse events was attenuated with covariate adjustment. The addition of PlGF to models with established clinical predictors provides additional useful prognostic information in patients receiving dialysis.

BACKGROUND

Breast angiosarcoma is rare and often associated with previous breast cancer treatment. The present study aimed to define long-term outcomes in relation to common prognostic factors. The expression of vascular endothelial growth factor receptor was also evaluated, as it may be a potential target for anti-angiogenic therapy.

METHODS

We retrospectively assessed outcomes in relation to age, association with previous breast-conserving treatment for breast cancer, tumor size, and grade in 19 patients without metastases at diagnosis. Vascular endothelial growth factor receptor was also assessed.

RESULTS

Median follow-up was 33 months (range, 1-121). There were 6 local recurrences and 6 deaths for disease progression. Five-year disease-free survival and overall survival were 53% (95% CI, 20-86%) and 49% (95% CI, 14-84%), respectively. No factor significantly affected survival. Vascular endothelial growth factor receptor was positive in 50% of cases and was more frequent in better differentiated cancer.

CONCLUSIONS

The association of vascular endothelial growth factor receptor with G1/G2 tumors requires further investigations. Our findings suggest that anti-angiogenic treatment in vascular endothelial growth factor receptor-positive cases be considered as a novel therapeutic modality in this rare and aggressive disease. Although information is still incomplete, we propose a multimodal therapeutic approach including surgery, radiotherapy and chemotherapy.

Pegaptanib sodium (Macugen) is a selective RNA aptamer that inhibits <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) 165 , the VEGF isoform primarily responsible for pathologic ocular neo<em>vascular</em>ization and <em>vascular</em> permeability, while sparing the physiological isoform VEGF <em>121</em> . After more than 10 years in development and preclinical study, pegaptanib was shown in clinical trials to be effective in treating choroidal neo<em>vascular</em>ization associated with age-related macular degeneration. Its excellent ocular and systemic safety profile has also been confirmed in patients receiving up to three years of therapy. Early, well-controlled studies further suggest that pegaptanib may provide therapeutic benefit for patients with diabetic macular edema, proliferative diabetic retinopathy and retinal vein occlusion. Notably, pegaptanib was the first available aptamer approved for therapeutic use in humans and the first VEGF inhibitor available for the treatment of ocular <em>vascular</em> diseases.

Endomucin (EMCN) is the type I transmembrane glycoprotein, mucin-like component of the <em>endothelial</em> cell glycocalyx. We have previously shown that EMCN is necessary for <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF)-induced VEGF receptor 2 (VEGFR2) internalization and downstream signaling. To explore the structural components of EMCN that are necessary for its function and the molecular mechanism of EMCN in VEGF-induced <em>endothelial</em> functions, we generated a series of mouse EMCN truncation mutants and examined their ability to rescue VEGF-induced <em>endothelial</em> functions in human primary <em>endothelial</em> cells (EC) in which endogenous EMCN had been knocked down using siRNA. Expression of the mouse full-length EMCN (FL EMCN) and the extracellular domain truncation mutants ∆21-81 EMCN and ∆21-<em>121</em> EMCN, but not the shortest mutant ∆21-161 EMCN, successfully rescued the VEGF-induced EC migration, tube formation, and proliferation. ∆21-161 EMCN failed to interact with VEGFR2 and did not facilitate VEGFR2 internalization. Deletion of COSMC (C1GalT1C1) revealed that the abundant mucin-type <i>O</i>-glycans were not required for its VEGFR2-related functions. Mutation of the two <i>N</i>-glycosylation sites on ∆21-<em>121</em> EMCN abolished its interaction with VEGFR2 and its function in VEGFR2 internalization. These results reveal ∆21-<em>121</em> EMCN as the minimal extracellular domain sufficient for VEGFR2-mediated <em>endothelial</em> function and demonstrate an important role for <i>N</i>-glycosylation in VEGFR2 interaction, internalization, and angiogenic activity.

Keywords: EMCN; VEGF; VEGFR2; angiogenesis; glycosylation; mucin.

OBJECTIVE

To evaluate changes in expression levels of vascular endothelial growth factor (VEGF) mRNA in human endometrial explants in a chicken chorioallantoic membrane model of endometriosis.

METHODS

Experimental prospective study.

METHODS

University hospital.

METHODS

Endometrial biopsy samples were obtained from healthy, ovulating women undergoing elective surgery.

METHODS

Endometrial fragments were placed on the chicken chorioallantoic membrane and removed for analysis after 0, 24, 48, and 72 hours.

METHODS

Expression of different VEGF mRNA splice variants was tested. Expression of VEGF(165) mRNA was assessed by using competitive polymerase chain reaction and normalized to expression of the housekeeping gene human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA.

RESULTS

After 0, 24, 48, and 72 hours of incubation, all grafts expressed VEGF(121), VEGF(145), VEGF(165), and VEGF(189) mRNA. Expression of VEGF(165) mRNA increased up to 10-fold at 24 to 72 hours compared with precultivation values.

CONCLUSIONS

Levels of VEGF(165) mRNA in endometrial grafts increase after implantation on chicken chorioallantoic membrane. Hypoxic induction of VEGF mRNA expression in endometrial cell cultures has been reported previously. Induction of VEGF expression might indicate relative hypoxia of the specimen due to insufficient vascularization. Expression of VEGF may assist in vascularization of endometrial explants after retrograde menstruation.

Circulating natural killer (NK) cells have been shown to adopt a type 1 innate lymphoid cell (ILC1)-like phenotype in response to TGF-β and secrete VEGF-A when exposed to hypoxia. Although these changes are often considered to be linked attributes of tissue residency, it has yet to be determined if TGF-β and hypoxia work in concert to coordinate NK cellular phenotype and angiogenic potential. Examination of human circulating NK cells treated with TGF-β demonstrated heterogeneity in their potential to adopt an ILC1-like phenotype, as indicated by the upregulation of CD9 and CD103 on only a subset of cells in culture. Culturing NK cells in chronic hypoxia did not induce a similar ILC1-like conversion and did not enhance the degree of conversion for cells exposed to TGF-β. Similarly, hypoxic culture of circulating NK cells induced VEGF-A secretion, but this production was not enhanced by TGF-β. Fluorescent <i>in-situ</i> hybridization flow cytometry demonstrated that hypoxia-induced VEGF-A production was uniform across all NK cells in culture and was not a selective feature of the cellular subset that adopted an ILC1-like phenotype in response to TGF-β. Examination of VEGF-A isoforms demonstrated that hypoxia induces the production of pro-angiogenic VEGF-A isoforms, including VEGF-A₁₆₅ and VEGF-A_<em>121</em>, and does not stimulate any meaningful production of anti-angiogenic isoforms, such as VEGF-A_b transcriptional variants or VEGF-Ax. In summary, TGF-β-mediated ILC1-like conversion and hypoxia-induced VEGF-A production are discrete processes in NK cells and are not part of a linked cellular program associated with tissue residency.

Keywords: angiogenesis; natural killer cells; tissue residency; transforming growth factor-β; vascular endothelial growth factor-A.

During angiogenesis, <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> A (VEGFA) regulates <em>endothelial</em> cell (EC) survival, tip cell formation, and stalk cell proliferation via VEGF receptor 2 (VEGFR2). VEGFR2 can interact with VEGFR2 co-receptors such as heparan sulfate proteoglycans (HSPGs) and neuropilin 2 (NRP2), but the exact roles of these co-receptors, or of sulfatase 2 (SULF2), an enzyme that removes sulfate groups from HSPGs and inhibits HSPG-mediated uptake of very low density lipoprotein (VLDL), in angiogenesis and tip cell biology are unknown. In the present study, we investigated whether the modulation of binding of VEGFA to VEGFR2 by knockdown of <i>SULF2</i> or <i>NRP2</i> affects sprouting angiogenesis, tip cell formation, proliferation of non-tip cells, and EC survival, or uptake of VLDL. To this end, we employed VEGFA splice variant <em>121</em>, which lacks an HSPG binding domain, and VEGFA splice variant 165, which does have this domain, in in vitro models of angiogenic tip cells and <em>vascular</em> sprouting. We conclude that VEGFA₁₆₅ and VEGFA_<em>121</em> have similar inducing effects on tip cells and sprouting in vitro, and that the binding of VEGFA₁₆₅ to HSPGs in the extracellular matrix does not seem to play a role, as knockdown of <i>SULF2</i> did not alter these effects. Co-binding of NRP2 appears to regulate VEGFA-VEGFR2-induced sprout initiation, but not tip cell formation. Finally, as the addition of VLDL increased sprout formation but not tip cell formation, and as VLDL uptake was limited to non-tip cells, our findings suggest that VLDL plays a role in sprout formation by providing biomass for stalk cell proliferation.

Keywords: HSPG; NRP2; SULF2; VEGFA; angiogenesis; endothelial cells; tip cells.

OBJECTIVE

To compare the mRNA expression of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) <em>121</em> and 165 isoforms and thrombospondin-1 (TSP-1) after incubation with tibolone and tibolone metabolites 3alpha-hydroxytibolone, 3beta-hydroxytibolone, Delta4-tibolone, and 17beta-estradiol (E2) in cultured Ishikawa cells.

METHODS

Ishikawa cells (immortalized from a well-differentiated human adenocarcinoma cell line) were cultured in vitro to confluence. Tibolone, 3alpha-hydroxytibolone, 3beta-hydroxytibolone, Delta4-tibolone and E2 at concentrations of 1.0, 0.1 and 0.01 micromol/L were added to confluent cells and further cultured for an additional 24 h. Control cells were treated with medium in absence of hormone. Total RNA was extracted from control and treated Ishikawa cells. After reverse transcription, VEGF, TSP-1 and the housekeeping gene, beta-actin cDNAs, were amplified in a polymerase chain reaction spiked with 33p-dCTP. Relative abundance of VEGF <em>121</em> and 165 isoforms and TSP-1 mRNA was measured by scintillation spectroscopy.

RESULTS

E2, tibolone, 3alpha-hydroxytibolone, and 3beta-hydroxytibolone increased both VEGF <em>121</em> and 165 mRNA compared with the control. However, Delta4-tibolone had no effect on either VEGF <em>121</em> or 165 mRNA compared with the control. Delta4-Tibolone increased TSP-1 mRNA expression compared with control levels. E2, tibolone, 3alpha-hydroxytibolone, and 3beta-hydroxytibolone did not increase TSP-1 mRNA expression at any concentration.

CONCLUSIONS

Tibolone and the 3alpha- and 3beta-tibolone metabolites with E2 increased VEGF <em>121</em> and 165 isoforms. Conversely, Delta4-tibolone, which is reported to have progestational-like activity, did not stimulate VEGF <em>121</em> and VEGF 165 but increased TSP-1 mRNA synthesis in cultured Ishikawa cells. We hypothesize, based on these data, that the clinical finding of no endometrial <em>growth</em> in women using tibolone may be partly related to alterations in these angiogenic <em>factor</em>s.

Identifying genes of major effect for wool <em>growth</em> would offer strategies for improving the quality and increasing the yield of fine wool. In this study, we employed the Agilent Sheep Gene Expression Microarray and proteomic technology to investigate the gene expression patterns of body side skin (more wool <em>growing</em>) in Aohan fine wool sheep (a Chinese indigenous breed) in comparison with groin skin (no wool <em>growing</em>) at the anagen stage of the wool follicle. A microarray study revealed that 4772 probes were differentially expressed, including 2071 upregulated and 2701 downregulated probes, in the comparisons of body side skin vs. groin skin (S/G). The microarray results were verified by means of quantitative PCR. A total of 1099 probes were assigned to unique genes/transcripts. The number of distinct genes/transcripts (annotated) was 926, of which 352 were upregulated and 574 were downregulated. In S/G, 13 genes were upregulated by more than 10 fold, whereas 60 genes were downregulated by more than 10 fold. Further analysis revealed that the majority of the genes possibly related to the wool <em>growth</em> could be assigned to categories including regulation of cell division, intermediate filament, cytoskeletal part and <em>growth</em> <em>factor</em> activity. Several potential gene families may participate in hair <em>growth</em> regulation, including fibroblast <em>growth</em> <em>factors</em>, transforming <em>growth</em> <em>factor</em>-β, WNTs, insulin-like <em>growth</em> <em>factor</em>, <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factors</em> and so on. Proteomic analysis also revealed 196 differentially expressed protein points, of which <em>121</em> were identified as single protein points.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> <em>121</em> (VEGF(<em>121</em>)) was expressed as inclusion bodies by recombinant Escherichia coli. High concentrations of both biomass (46 g dry cell/L) and VEGF(<em>121</em>) inclusion bodies (4.5 g/L) were obtained by applying a high-cell-density culture. After the inclusion bodies were washed and dissolved, VEGF(<em>121</em>) was refolded at 0.2 mg/ml by ultrafiltration in refolding buffer with a yield of 81%. Renatured VEGF(<em>121</em>) was purified by anion chromatography and Sephacry S-100 chromatography with purity higher than 95% and final purification yield of 31%. The purified VEGF(<em>121</em>) could stimulate the proliferation of human umbilical vein <em>endothelial</em> cells as demonstrated by a biological activity assay.

BACKGROUND

Endothelial cell hyperpermeability is a proposed mechanism of increased lipid insudation into the vessel walls of allografts. Vascular endothelial growth factor (VEGF) is a potent inducer of vascular permeability and its expression is upregulated in human heart allografts. The goal of these experiments was to investigate the effects of VEGF on low-density lipoprotein (LDL) permeability through confluent monolayers of human cardiac microvascular endothelial cells (HCMEC) in vitro.

METHODS

VEGF mRNA and protein expression was characterized in coronary arteries from cardiac allograft vasculopathy patients as compared with healthy controls using in situ hybridization and immunohistochemical staining of sub-adjacent sections. HCMEC were grown to confluence and treated with VEGF-A(121) or VEGF-A(165). Permeability of LDL in confluent endothelial monolayers was measured using fluorometry. Transendothelial electrical resistance (TER) measurements were used to indirectly measure the tight junctional status. Immunocytochemical staining was performed to visualize changes in CD31 and zonula occludens-1.

RESULTS

We observed significant increases in VEGF expression within the superficial and deep intima and media of coronaries from allografts, as compared with controls. In vitro treatment with VEGF-A(121) and VEGF-A(165) significantly increased LDL passage through endothelial monolayers. We further showed that VEGF-A(121) and VEGF-A(165) caused significant decreases in TER at 2 to 4 hours post-treatment. Also, VEGF induced disruption of tight junctions, resulting in an increase in the intercellular gap formation.

CONCLUSIONS

These results demonstrate that VEGF increases low-density lipoprotein permeability through endothelial monolayers, and this effect is correlated with VEGF-induced disruption of endothelial tight junctions resulting in the formation of intercellular gaps.

OBJECTIVE

Improvements in ventricular function after cellular cardiomyoplasty appear to be limited by the poor survival of the cellular implants. Angiogenic pretreatment of infarcted myocardium may improve implanted cell survival and consequently myocardial function.

METHODS

Fischer 344 rats underwent coronary artery ligation and injection of an adenovirus encoding <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> <em>121</em> or of saline solution at increasing intervals after ligation. Myocardial perfusion and mass preservation were assessed. On the basis of these data, four groups of animals underwent coronary ligation and adenovirus with or without syngeneic skeletal myoblast administration: (1) adenovirus at ligation and myoblasts 3 weeks later (n = 7), (2) saline solution at ligation and myoblasts 3 weeks later (n = 8), (3) saline solution at ligation and 3 weeks later (n = 8), and (4) saline solution at ligation and adenovirus with myoblasts 3 weeks later (n = 5). Left ventricular ejection fraction was analyzed by echocardiography before coronary ligation and 3 and 5 weeks later, after which cell survival was assessed in harvested tissues.

RESULTS

Myocardial infarct perfusion was at least 50% greater in animals treated with adenoviral vector than with saline solution immediately after ligation (P < .02). In comparison, delayed adenovirus administration did not significantly diminish infarct perfusion but resulted in decreased myocardial preservation (P < .05). Accordingly, adenovirus administration nearly tripled implanted myoblast survival relative to saline solution-treated animals (P = .004). Left ventricular ejection fraction was improved, however, only after cell implantation with adenovirus pretreatment (P = .027).

CONCLUSIONS

Angiogenic strategies can help to preserve myocardium jeopardized by acute coronary occlusions. Angiogenic pretreatment enhances the efficacy of cellular cardiomyoplasty.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> A (VEGF-A) is essential for maintaining the glomerular filtration barrier. Absolute renal levels of VEGF-A change in patients with diabetic nephropathy and inflammatory kidney diseases, but whether changes in the renal splicing patterns of VEGF-A play a role remains unclear. In this study, we investigated mRNA splicing patterns of pro-angiogenic isoforms of VEGF-A in glomeruli and whole kidney samples from human patients with kidney disease and from mouse models of kidney disease. Kidney biopsies were obtained from patients with acute rejection following kidney transplantation, patients with diabetic nephropathy, and control subjects. In addition, kidney samples were obtained from mice with lupus nephritis, mice with diabetes mellitus, and control mice. The relative expression of each VEGF-A splice variant was measured using RT-PCR followed by quantitative fragment analysis. The pattern of renal VEGF-A splice variants was unchanged in diabetic nephropathy and lupus nephritis and was stable throughout disease progression in acute transplant rejection and diabetic nephropathy; these results suggest renal VEGF-A splicing stability during kidney disease. The splicing patterns were species-specific; in the control human kidney samples, VEGF-A <em>121</em> was the dominant isoform, whereas VEGF-A 164 was the dominant isoform measured in the mouse kidney samples.

OBJECTIVE

We hypothesized that paracrine factors from human umbilical cord blood mononuclear cells (hUCBC) activate in injured cardiomyocytes the survival protein kinase Akt and limit activation of death protein kinases JNK and p38.

METHODS

We treated hUCBC with H2O2 and measured growth factors and cytokines secreted by hUCBC. We then treated cardiomyocytes with H2O2 for 24 h and measured Akt, JNK and p38 activation by means of Western blots. We also measured myocyte viability and apoptosis with the use of fluorescence-activated cell-sorting cytometry. We then investigated myocytes treated for 24 h with H2O2 plus hUCBC and myocytes without hUCBC or H2O2. Four million hUCBC were placed in transwells permeable only to hUCBC paracrine factors, and the transwells were placed in flasks with H2O2 + Dulbecco's modified Eagle's medium or in flasks with myocytes plus H2O2+Dulbecco's modified Eagle's medium.

RESULTS

hUCBC increased secretion during H2O2 of hepatocyte growth factor by 338%, insulin-like growth factor by 200%, interleukin-4 by 200%, vascular endothelial cell growth factor by 192%, placental growth factor by 150%, interleukin-10 by 150% and angiogenin by 121%. H2O2 increased myocyte JNK activation by 237% and p38 activation by 60%, decreased myocyte viability by 38% and increased necrosis by 34% (all P < 0.01). hUCBC paracrine factors increased in myocytes with H2O2 Akt activation by ≥ 25%, decreased JNK and p38 activation by>> 35%, increased viability by>> 22% and decreased apoptosis by>> 33% (all P < 0.05). Akt inhibitor API-1 prevented the effects of hUCBC and enhanced H2O2 decrease of myocyte viability. Addition of JNK inhibitor SP600125 or p38 inhibitor SB203580 to myocytes plus H2O2 prevented H2O2 decrease in viability and increased hUCBC beneficial effects.

CONCLUSIONS

During free radical stress, hUCBC paracrine factors activate myocyte Akt, which increases myocyte viability by decreasing activation of death-promoting protein kinases JNK and p38.

OBJECTIVE

To characterize a new alternative splicing isoform of human vascular endothelial growth factor (VEGF) gene.

METHODS

The total RNA was extracted from the lung tissue of a legally aborted 4-month-old fetus and amplified by RT-PCR. The amplified product was cloned into the plasmid pMD18-T and plasmid pcDNA3.1- for sequence analysis.

RESULTS

Electrophoresis of the RT-PCR products displayed one short band for VEGF(121) (487 bp) and a long band. The latter was characterized to contain two fragments: one was normal VEGF(165) (619 bp), and the other (639 bp) had an identical nucleotide sequence to VEGF(165) with a 20 bp fragment inserted between exons 3 and 4. Sequence analysis showed that this 20-bp nucleotide was inserted from the 3' end of the third intron containing a splicing signal, thus causing shift mutation in the reading frame of VEGF gene and early appearance of the stop codon UAG in the middle of exon 4.

CONCLUSIONS

A new alternative splicing isoform of VEGF probably exists in the lung tissue of a legally aborted human fetus, and its biological significance remains to be further investigated.

Arteriogenic erectile dysfunction is associated with impairment of <em>vascular</em> perfusion to the erectile components of the penis. Animal studies have identified insulin-like <em>growth</em> <em>factor</em> (IGF-I) and <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) as penile angiogenic <em>growth</em> <em>factors</em>, but the role of these <em>factors</em> in humans is not well understood. We evaluated the ex vivo expression of IGF-I, VEGF, and their receptors (IGF-IR, Flt-1, and KDR) in human penile cavernosal smooth muscle cells (HCSMCs) to identify cellular and molecular pathways involved in the regulation of penile tissue <em>vascular</em>ity. Primary culture was initiated with explants of human corpora cavernosa, and early passage (3-5) cells were used for these evaluations. Cultures were examined to verify the presence of smooth muscle cells and the absence of <em>endothelial</em> cell contamination. Specific monoclonal antibodies were used to localize <em>growth</em> <em>factors</em> and their receptors. To evaluate gene expression of VEGF, Flt-1, and KDR, total RNA was extracted from cavernosal cells and subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) using custom synthesized primers. To study the effect on cell proliferation, 10000 cells/well were exposed to varying concentrations of VEGF (0-50 ng/mL). At specified time periods the cells were trypsinized and counted. IGF-I and VEGF and their receptors were localized in the cultures, which were positive for the presence of smooth muscle cells and negative for <em>endothelial</em> cell contamination. RT-PCR evaluation revealed the expression of four splice variants of VEGF messenger RNA (VEGFs <em>121</em>, 145, 165, and 189) and two of its receptors (Flt-1 and KDR). VEGF165 and VEGF<em>121</em> were the most abundant forms of messenger RNA and Flt-1 appeared to be the most prominent receptor type in these cells. Exposure to VEGF elicited a twofold to threefold increase in the proliferation of HCSMCs. HCSMCs express both IGF-I and VEGF and their receptors, which may be important in the control of <em>vascular</em>ity in human penile architecture.

The present study was conducted to examine changes of mRNAs encoding <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) and its receptors (KDR/Flk-1 and Flt-1), and CD34, which is known to be a specific marker for <em>endothelial</em> cells, during the development and maintenance of the caprine corpora lutea (CL). Effects of a potent GnRH antagonist (GA), which was previously shown to suppress release of luteinizing hormone (LH), on expressions of those mRNAs during the CL development were also investigated. Goats were divided into control (n = 12) and GA-treated groups (n = 6). The goats were treated with saline or GA (50 microg/kg, sc) on days 0 (day of ovulation), 4, and 8 (control only), and CL collected on a subset of goats (n = 3 for each day) on days 0 (no saline), 4, 8, or 14 (control only). Ribonuclease protection assay was performed to quantitate the mRNAs in the CL using specific cRNA probes generated by RT-PCR and in vitro transcription. Level of CD34 mRNA significantly increased from day 0 to 8 (CL development) in the control group (P < 0.05). Long and short forms were detected in the caprine CL by RT-PCR for VEGF mRNA and analyses of their sequences showed that they correspond to mRNAs encoding VEGF(165) and VEGF(<em>121</em>), respectively. Level of VEGF(165) mRNA significantly increased from day 4 to 8 and day 8 to 14 (CL maintenance) in the control group (P < 0.05) while VEGF(<em>121</em>) mRNA did not change during the whole period. Level of KDR/Flk-1 mRNA significantly increased from day 0 to 8 (P < 0.05) while Flt-1 mRNA significantly increased from day 8 to 14 (P < 0.005) in the control group. In the GA-treated group, levels of all of the mRNAs did not alter remarkably as compared with those in the control group. These results suggest that rise of KDR/Flk-1 and VEGF(165) mRNAs during the caprine CL development may be associated with enhanced angiogenesis and that increment of VEGF(165) and Flt-1 mRNAs during the CL maintenance may play nonangiogenic roles. The present study also indicates that the changes of VEGF(165) and KDR/Flk-1 mRNAs during the CL development are probably not regulated by LH.

The malignancy of endometrial carcinoma (EC) largely results from its high invasive feature. The regulation of the mRNA splicing of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> A (VEGF-A) is critical for EC-associated cancer <em>vascular</em>ization and invasion. Recently, we have reported that poorly prognostic EC had high levels of YT521, a newly defined RNA splicing protein. However, whether YT521 may similarly regulate the splicing of VEGF-A in EC is unknown. Here, we showed that EC specimens contained significantly higher levels of YT521, compared to the adjacent non-tumor endometrial tissue. Higher levels of YT521 were detected in EC specimens with metastases. High-YT521 EC is associated with poor patient survival. In order to examine whether YT521 may regulate VEGF-A mRNA splicing in EC, we transfected an EC cell line HEC-1A with different doses of YT521 mimics. We found that YT521 dose-dependently increased the ratio of VEGF-165 vs VEGF-<em>121</em> at both mRNA and protein level, suggesting that YT521 may promote VEGF-A mRNA splicing to favor a VEGF-165 isoform. Moreover, the increases in the ratio of VEGF-165 vs VEGF-<em>121</em> by YT521 overexpression resulted in increases in EC cell invasion, while decreases in the ratio of VEGF-165 vs VEGF-<em>121</em> by YT521 depletion resulted in decreases in EC cell invasion in a transwell cell migration assay. Further, overexpression of VEGF-165, but not overexpression of VEGF-<em>121</em>, increased EC cell invasiveness. Finally, a strong correlation was detected between the ratio of VEGF-165 vs VEGF-<em>121</em> and the levels of YT521 in EC specimens. Together, these data suggest that YT521 may promote EC metastases by regulating mRNA splicing of VEGF-A.

OBJECTIVE

To construct a retroviral vector carrying human <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (hVEGF (<em>121</em>)) cDNA for evaluation of the possibility of VEGF gene therapy in ischemic bone disease.

METHODS

hVEGF(<em>121</em>) cDNA was obtained from the plasmid pCDI/VEGF(<em>121</em>) and cloned into retroviral plasmid pLXSN. Recombinant plasmid was transferred to the retro virus packaging cell, PT-67, by lipofectamine mediated gene transfer. Mouse bone marrow stromal cells (MSCs) were transfected by the retrovirus. The integration of the hVEGF(<em>121</em>) cDNA into MSC genomic DNA and expression of the VEGF gene was detected. Proliferation assays of human umbilical vein <em>endothelial</em> cells (HUVECs) by VEGF(<em>121</em>) in culture medium were performed.

RESULTS

Recombinant pLXSN/VEGF(<em>121</em>) was correctly constructed and confirmed by restriction endonuclease analysis and DNA sequencing analysis. hVEGF(<em>121</em>) gene was integrated into MSC genomic DNA after transfection, and the VEGF(<em>121</em>) protein was expressed. Proliferation assays showed VEGF(<em>121</em>) in culture medium was a biologically active protein and had a mitogenic effect on HUVEC.

CONCLUSIONS

Recombinant retroviral vector carrying hVEGF(<em>121</em>) cDNA was successfully constructed. VEGF (<em>121</em>) protein expressed by MSCs had mitogenic effect biologically. This provides a further foundation for VEGF gene therapy for bone ischemic disease and bone tissue engineering.

OBJECTIVE

To investigate whether vascular endothelial growth factor (VEGF) gene plasmid carried by polytetrafluoroethylene (PTFE) vascular graft materials could transfect endothelial cells (ECs) and promote their growth.

METHODS

PTFE vascular graft materials carried with pCDI-hVEGF(121), pCDI or pEGFP were incubated in Tris-buffer solution and the values of optical density of 260 nm at different time were plotted, then the DNA controlled release curve was made. ECs derived from human umbilical vein were seeded on the pCDI-hVEGF(121)/pCDI/pEGFP-PTFE materials or tissue culture plates, ECs numbers were counted and VEGF protein concentrations at different time were measured by enzyme-linked immunoadsorbent assay method. Green fluorescent protein (GFP) expression in ECs on pEGFP-PTFE materials was examined with fluorescence microscopy.

RESULTS

The controlled release curve showed that the gene released from PTFE materials was rapid within 8 h, then slowed down and that the gene released continuously even after 72 h. At 24, 72 and 120 h, ECs number and proliferation rate of pCDI-hVEGF(121)-PTFE materials were higher than those of pCDI or pEGFP-PTFE materials (P<0.05). VEGF protein concentration of pCDI-hVEGF(121)-PTFE materials was higher than that of pCDI or pEGFP-PTFE materials at 6, 24, 72 and 120 h (P<0.01). GFP expression in ECs on the pEGFP-PTFE materials could be detected by fluorescence microscopy.

CONCLUSIONS

PTFE graft can be used as a carrier of VEGF gene plasmid, VEGF gene carried by PTFE can transfect ECs and promote ECs growth.