OBJECTIVE

Restoration of coronary blood flow by angiogenesis may offer a new approach to intractable ischemic heart disease. In the present study, we investigated the angiogenic effects of gene transfer of vascular endothelial growth factor 165 on microvascular myocardial ischemia.

METHODS

A rabbit model of microvascular myocardial ischemia was created by plugging coronary microvessels with microspheres (15 microm in diameter, 2.8 x 10(5)/kg, n = 29). Gene transfer was performed by semi-selective intracoronary injection of recombinant adenovirus expressing vascular endothelial growth factor 165 forty minutes after microsphere injection (n = 9).

RESULTS

Microsphere injection reduced myocardial perfusion (78% +/- 9% of baseline tissue flow) and diminished myocardial contraction (61% +/- 12% of the baseline ejection fraction) and cardiac performance (elevated left ventricular end-diastolic pressure and decreased systemic flow) in the acute phase. At 17 +/- 3 days, gene transfer of vascular endothelial growth factor 165 had had the following effects: (1) promoted coronary angiogenesis as evidenced by myocardial flow above the baseline (121% +/- 24%), (2) increased vascular density revealed by synchrotron radiation microangiography and histologic analysis, (3) ameliorated the degree of myocardial ischemia as evidenced by myocardial lactate content and the extent of histologic necrosis, and (4) restored heart function as evidenced by increased ejection fraction (95% +/- 10%), reduced left ventricular end-diastolic pressure, and restored body weight.

CONCLUSIONS

In vivo vascular endothelial growth factor 165 gene transfer promoted angiogenesis and was an effective approach to treating microvascular myocardial ischemia.

OBJECTIVE

To determine the effect of 17beta-E(2), levonorgestrel, medroxyprogesterone acetate, and norethindrone on the expression of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) isoforms <em>121</em> and 165 in Ishikawa cells in vitro.

METHODS

Prospective basic research study.

METHODS

Basic research laboratory.

METHODS

None.

METHODS

Ishikawa cells were cultured in vitro. After 24 hours' incubation in serum-free media, 1.0, 0.1, and 0.01 microM concentrations of E(2), levonorgestrel, medroxyprogesterone acetate, and norethindrone were added for a further 24 hours of incubation.

METHODS

Isolation and identification of VEGF isoforms <em>121</em> and 165 using semiquantitative polymerase chain reaction, gel electrophoresis, with beta-actin as an internal control.

RESULTS

Estradiol stimulated VEGF isoforms <em>121</em> and 165. The progestins studied increased mRNA for VEGF isoforms <em>121</em> and 165 at all doses. Medroxyprogesterone acetate resulted in the greatest increase in both VEGF <em>121</em> and 165 compared with norethindrone and levonorgestrel.

CONCLUSIONS

Estradiol and progestins increased VEGF <em>121</em> and 165 isoform mRNA in Ishikawa cells in vitro. We hypothesize that differences in VEGF expression may be associated with the irregular bleeding during progestin use in clinical situations.

GenVec, in collaboration with Pfizer (formerly Parke-Davis), is developing AdGVVEGF<em>121</em>.10 (BioBypass), a gene therapy involving the <em>121</em>-residue isoform of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF), licensed from Scios, for the potential treatment of coronary artery disease (CAD) and peripheral <em>vascular</em> disease (PVD) [262000]. By March 2000, phase II trials in CAD had commenced [359531], [359532], [359538]. By August 2000, phase II trials were also underway for PVD [386293]. The initial phase II trial will include approximately 70 patients with severe CAD who are not candidates for bypass surgery and will assess exercise capacity and patient well-being, before and after treatment, as well as safety and drug tolerance [364137]. Scios licensed the gene transfer applications of VEGF<em>121</em> to GenVec in June 1996 [263381]. In September 1997, GenVec entered into an agreement with Parke-Davis, a subsidiary of Warner-Lambert (now Pfizer), to develop the therapy [262000]. In May 1999, Warner-Lambert signed an agreement with Bioscience for a device for the administration of AdGVVEGF<em>121</em>.10 1325443]. In May 2000, Merrill Lynch predicted a US filing in the first half of 2003 [375962]. In January 2001, AG Edwards predicted the product would generate $70 million in revenues to Pfizer and $12 million in royalties to GenVec in 2005. In February 1999, GenVec was awarded US-05846782, covering vectors for targeting the transfer of therapeutic genes to specific tissues in the human body [316038].

OBJECTIVE

Metabolic syndrome (MetS) increases the likelihood of developing coronary artery disease (CAD), and inflammation is involved in the pathogenesis of both these conditions. The present work was conducted to examine the relative expression of 18 key inflammatory genes associated with MetS and incident CAD in a representative group of patients.

METHODS

A total of 178 male patients, including 57 with CAD and <em>121</em> without CAD, were enrolled in the study. The participants without CAD were characterized for the presence of MetS using modified criteria specific for Asian Indians, which included a lower cut-off for waist circumference (≥90 cm for men). The expression of 18 inflammatory genes was evaluated in peripheral whole blood by quantitative polymerase chain reaction method.

RESULTS

Of the <em>121</em> participants without CAD, 53 (43.8%) had three or more risk factors (MetS group), 50 (41.3%) had one or two risk factors (non-MetS group), while 18 (14.8%) did not have any risk factors (control group). High nuclear factor-kappa B (NF-κB) expression levels and low interleukin-10 (IL-10) levels were observed in MetS patients. Linear association was seen between NF-κB and vascular endothelial growth factor A (VEGFA) expression and with increase in MetS components. Comparison of gene expression pattern between CAD and MetS revealed significantly higher expression of leukotriene genes - arachidonate 5-lipoxygenase (ALOX5), arachidonate 5-lipoxygenase activating protein (ALOX5 AP), leukotriene A4 hydrolase (LTA4H) and leukotriene C4 synthase (LTC4S), and lower expression of NF-κB, interleukin 1 beta (IL-1β), monocyte chemoattractant protein-1 (MCP-1/CCL2) and signal transducer and activator of transcription 3 (STAT3) genes in CAD. There was linear increase in expression of LTA4H, LTC4S, IL-8 and VEGFA genes across the four groups, namely from controls, non-MetS, MetS and CAD.

CONCLUSIONS

A distinct gene expression pattern was seen in MetS and CAD implying a well-orchestrated inflammatory and immune activity. Specifically, NF-κB might be playing an active role in MetS, allowing further expansion of the inflammatory process with resolution of inflammation in full-blown CAD, wherein other gene players such as leukotrienes may dominate.

Introduction of specific mutations into a synthetic internal ribosome entry site (IRES(GTX)) derived from the GTX homeodomain protein revealed additional transcriptional activity. This novel synthetic P(GTX) promoter exhibited consensus core promoter modules such as the initiator (Inr) and the partial downstream promoter elements (DPE) and mediated high-level expression of a variety of transgenes including the human <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> <em>121</em> (VEGF(<em>121</em>)), the human placental secreted alkaline phosphatase (SEAP), and the Bacillus stearothermophilus-derived secreted alpha-amylase (SAMY) in Chinese hamster ovary cells (CHO-K1) and a variety of other mammalian and human cell lines. The spacing between Inr and DPE modules was found to be critical for promoter performance since introduction of a single nucleotide (resulting in P(GTX2)) doubled the SEAP expression levels in CHO-K1. P(GTX2) reached near 70% of P(SV40)-driven expression levels and outperformed constitutive phosphoglycerate kinase (P(PGK)) and human ubiquitin C (P(hUBC)) promoters in CHO-K1. Also, P(GTX2) was successfully engineered for macrolide-inducible transgene expression. Owing to its size of only 182 bp, P(GTX2) is one of the smallest eukaryotic promoters. Although P(GTX2) was found to be a potent promoter, it retained its IRES(GTX)-specific translation-initiation capacity. Synthetic DNAs, which combine multiple activities in a most compact sequence format may foster advances in therapeutic engineering of mammalian cells.

OBJECTIVE

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> has been used in preclinical studies and phase 1 and 2 clinical trials as a potent mediator of therapeutic angiogenesis; however, its ability to enhance the <em>vascular</em> permeability may be a source of potential complications. The objective of this work was to evaluate the effects of the intramuscular injection of an adenovirus vector coding for the <em>121</em>-amino acid form of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (Ad.VEGF(<em>121</em> )) on <em>vascular</em> permeability and edema development in rabbits and rats.

METHODS

Different concentrations of Ad.VEGF(<em>121</em> ) ranging from 10(5) to 10(10) plaque-forming units/mL (3 x 10(6)-3 x 10(11) particles/mL) were injected into hind limb or forelimb muscles of Wistar rats or rabbits. The size of the scrotum, the circumferences of limbs, and the concentration of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> in the serum were measured daily after injection.

RESULTS

The injection of different concentrations of Ad.VEGF(<em>121</em> ) into the hind limb muscles of rabbits led to a dose-dependent scrotal edema in rabbits at concentrations higher than 10(7) plaque-forming units/mL (P =.002). The edema developed slowly, reached its maximum level 6 days after the injection, and spontaneously resolved thereafter. At concentrations higher than 10(9) plaque-forming units/mL the scrotal edema was accompanied by skin necrosis (P =.0001). No scrotal edema was observed in rats.

CONCLUSIONS

The massive species-specific scrotal edema accompanied by skin ulceration and necrosis was observed only in rabbits treated with Ad.VEGF(<em>121</em> ) in concentrations exceeding therapeutic doses. The therapeutic doses of Ad.VEGF(<em>121</em> ) resulted in only moderate transient scrotal edema in rabbits, suggesting that the potential for side effects of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> therapy as a result of increased <em>vascular</em> permeability should not be very alarming for generally healthy patients and may not cause a significant clinical problem in the treatment of peripheral <em>vascular</em> diseases.

OBJECTIVE

To investigate the feasibility of endothelialization of bioprosthesis by transfer of vascular endothelial growth factor (VEGF) gene.

METHODS

Bovine pericardium treated with glutaraldehyde and L-glutamic acid was positioned into the pig right atrium. pcD(2)/hVEGF(121) gene (1 mg) was transferred into the right ventricular myocardium using surgical sutures Reverse transcri ption polymerase chain reaction (RT PCR) was employed to evaluate the expression of myocardial VEGF mRNA. The determination of concentrations of VEGF protein in blood from both the right atrium and peripheral vein, and histological and ultrastructural analysis of implanted bovine pericardium were completed simultaneously.

RESULTS

The concentration of VEGF derived from the right atrium in pcD(2)/hVEGF(121) group was significantly higher than that in the pcD(2) group 10 days after VEGF gene transfer (P < 0.01). The expression of myocardial VEGF mRNA in pcD(2)/hVEGF(121) group was much higher in comparison with that in the pcD(2) group. The morphological analysis demonstrated that the coverage rate of host endothelium in the pcD(2)/hVEGF(121) group was 2.6 times as fast as that in the pcD(2) group at 16 days after VEGF(121) gene transfer (P < 0.01). Entire endothelialization occurred at 30 days after VEGF gene transfer. In addition, higher expression of myocardial VEGF mRNA was still available.

CONCLUSIONS

VEGF gene transfer by surgical suture can remarkably accelerate endothelialization of bioprosthesis, which may provide a new approach for inhibiting biological valve calcification and improve biocompatibility and long-term durability of the bioprosthesis.

Angiogenesis is very important not only for embryogenesis and wound healing but also for tumor <em>growth</em> in vivo because vessels supply oxygen and nutrition to the tumor mass. In this study, we focused on <em>Vascular</em> <em>Endothelial</em> <em>Growth</em> <em>Factor</em> (VEGF), a newly characterized endothel-specific <em>growth</em> <em>factor</em> and investigated the expression of VEGF in 13 ovarian tumors and 3 normal ovaries by using polymerase chain reaction (PCR) analysis and Northern blot analysis. Further, we examined the expression pattern of 4 alternatively spliced forms of VEGF in these tissues. The level of VEGF mRNA was higher in 77% of ovarian tumors when compared with that in normal ovaries. Among subtypes of VEGF, <em>121</em>-, 165- and 189-amino acid types were detected but 206-amino acid type was not observed in ovarian tumors. The most abundant form of VEGF was <em>121</em>-amino acid type and the relative amounts of the various forms of VEGF were <em>121</em>-amino acid type>> 165-amino acid type>>) 189-amino acid type. Expression of flt-1, a receptor for VEGF was detectable by PCR but not by Northern blot analysis. These results suggest that like other epithelial cell-derived carcinomas, ovarian tumors use the VEGF/flt-1 system for tumor angiogenesis.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) is a multifunctional cytokine which plays a major role in angiogenesis. Alternative splicing causes the production of several different isoforms (VEGF-A <em>121</em>, 145, 165, 189, 206). VEGF is essential for tumor angiogenesis and several studies have correlated elevated VEGF levels with tumor stage, metastases and progression. Antibody phage display was employed to isolate two scFv antibody fragments, D8 and F10, with specificity for the VEGF165 isoform. It was shown by ELISA and competitive immunohistochemistry that each clone bound to VEGF165 but not VEGF<em>121</em>. Immunohistochemistry with D8 and F10 on colorectal carcinoma and adenoma sections revealed positive staining similar to that shown by a polyclonal VEGF antibody. The scFv antibody fragments, D8 and F10, will be useful in specifically detecting circulating VEGF165 in cancer patients as most studies to date have quantified the total level of circulating VEGF (<em>121</em> and 165). These reagents will allow further elucidation of the role of VEGF in tumor angiogenesis.

OBJECTIVE

Colorectal cancers (CRC) with high-level micro-satellite instability (MSI-H) show reduced metastatic potential and better prognosis compared to stage-matched stable (MSS) cancers. Angiogenesis/lymphangiogenesis, central to tumour growth and spread, is mediated by vascular endothelial growth factor (VEGF) cytokines, but little is known of their relationship to MSI.

METHODS

In this study, 67 sporadic CRC with identified MSI status, and 8 samples of normal colon were analysed for VEGF-A soluble isoforms (VEGF-121/VEGF-165) and VEGF-C gene transcription (by RT-PCR and scanning densitometry), and blood vessel density (BVD; measuring angiogenesis) and VEGF-C protein expression (measuring lymphangiogenesis).

RESULTS

Compared to normal colon, VEGF-165 transcription was reduced (P < 0.05), but VEGF-121 transcription was higher in MSS (P < 0.06) and MSI-L (P < 0.01) cancers (but similar in MSI-H). VEGF-165 transcription was unrelated to MSI, but VEGF-121 transcription was elevated in MSI-L (P < 0.06). There was a weak inverse correlation with VEGF-121 transcription and Dukes stage (P < 0.09), and with BVD and MSI (P < 0.09). With a singular di-nucleotide loci mutation (MSI-L), VEGF-121 (P < 0.03) and VEGF-C (P < 0.04) transcription was elevated.

CONCLUSIONS

MSI-H cancers have reduced angiogenic/lymphangiogenic potential, and transcription of VEGF-121 may be important in early growth and spread of CRC. Elevated VEGF-121 and VEGF-C transcription with singular di-nucleotide mutations may aid in the identification of distinct MSI-L cancers.

Mifepristone has been used for both medical termination of pregnancy and emergency contraception. Mifepristone may have both an antiovulatory activity and an antiproliferative effect on the endometrium. We have evaluated the effect of mifepristone on <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) and thrombospondin-1 (TSP-1) using Ishikawa cells in vitro. Mifepristone, progesterone and 17beta-estradiol at concentrations of 1.0, 0.1 and 0.01 microM, were added to confluent cells and further cultured for additional 24 h. Total RNA was extracted from control and treated cells. After reverse transcription, VEGF, TSP-1 and beta-actin cDNAs were amplified with polymerase chain reaction spiked with 33p-dCTP. The relative abundance of VEGF <em>121</em> and 165 isoforms and TSP-1 mRNA were measured by scintillation spectroscopy. Mifepristone and progesterone did not stimulate VEGF mRNA <em>121</em> and 165 isoforms, while 17beta-estradiol increased both VEGF isoforms. Mifepristone did not stimulate TSP-1 mRNA at any concentration, but progesterone increased TSP-1 mRNA, and this effect was inhibited with mifepristone. 17beta-Estradiol did not increase TSP-1 expression. We hypothesized, based on these data, that the clinical finding of endometrial antiproliferative effect and low vaginal bleeding rate observed in women using mifepristone may be related to lack of stimulation of these angiogenic <em>factors</em>.

OBJECTIVE

To investigate the effects of two histone deacetylase (HDAC) inhibitors, valproic acid (VPA) and TSA, on the expression of vascular endothelial growth factor (VEGF) and its receptor KDR of the leukemia cell line Kasumi-1 cells, and to explore their potential mechanism in leukemia angiogenesis.

METHODS

Kasumi-1 cells were treated with VPA and TSA at different concentrations for 3 days. The mRNA and protein expression levels of VEGF and KDR were determined by semi-quantitative RT-PCR and Western blot, and the bFGF mRNA by semi-quantitative RT-PCR.

RESULTS

As compared with that of control groups, VPA at 3 mmol/L downregulated the VEGF mRNA expression level for VEGF(121) from 0.632 ± 0.014 to 0.034 ± 0.004 and for VEGF(165) from 0.526 ± 0.021 to 0.015 ± 0.001, for KDR mRNA from 0.258 ± 0.034 to 0.038 ± 0.000, and for bFGF mRNA from 0.228 ± 0.017 to 0.086 ± 0.015. TSA downregulated the VEGF mRNA and KDR mRNA at concentration of 100 nmol/L, but its effect on bFGF mRNA only at higher concentration.

CONCLUSIONS

HDAC inhibitors might inhibit the leukemia angiogenesis by regulating the expression of VEGF and its recptor.

<strong class="sub-title"> Background: </strong> In this study, <em>Vascular</em> <em>Endothelial</em> <em>Growth</em> <em>Factor</em> <em>121</em> expressed abundantly in endometrial stromal cells is encapsulated with poly-l-lactide and characterized the properties for endometrial angiogenesis. We studied the migration, proliferation and the protein levels of human immortalized endometrium stromal cells after treating the cells with recombinant <em>Vascular</em> <em>Endothelial</em> <em>Growth</em> <em>Factor</em> (200 and 500 nanogram), and poly-l-lactide loaded <em>Vascular</em> <em>Endothelial</em> <em>Growth</em> <em>Factor</em> <em>121</em> (day 1, 20 and 30). The present study explains endometrium angiogenesis because endometrium plays an important role in pregnancy.

<strong class="sub-title"> Results: </strong> Migration and proliferation studies in endometrium cells proved the efficiency of <em>Vascular</em> <em>Endothelial</em> <em>Growth</em> <em>Factor</em> and poly-l-lactide loaded <em>Vascular</em> <em>Endothelial</em> <em>Growth</em> <em>Factor</em> <em>121</em>. This proliferated and increased the migration of the cells in vitro and also activated the Protein kinase B, Phosphatidylinositol-4, 5-Bisphosphate 3-Kinase Catalytic Subunit Beta, α-Smooth muscle actin and vascular endothelial growth factor receptor 2 pathways. Western blot analysis showed the increased expression levels of kinases, smooth muscle actin and vascular endothelial growth factor receptor 2 after the treatment with <em>Vascular</em> <em>Endothelial</em> <em>Growth</em> <em>Factor</em> and poly-l-lactide loaded <em>Vascular</em> <em>Endothelial</em> <em>Growth</em> <em>Factor</em> <em>121</em> particles in comparison to the control group. The elevated levels of α-Smooth muscle actin in endometrium cells with <em>Vascular</em> <em>Endothelial</em> <em>Growth</em> <em>Factor</em> prove the regulation of angiogenesis in vitro.

Conclusion: Endometrium thickness is one of the important factors during implantation of embryo and pregnancy. Slow release of VEGF from PLA encapsulated microparticle further controls the endothelial cell proliferation and migration and helps in the promotion of angiogenesis. The combined effect studied in vitro could be used as a pro-angiogenic drug on further in vivo confirmation.

Keywords: Angiogenesis; Endometrium; Human endometrium stromal cells; Migration; Proliferation; VEGF.

Alternative splicing leads to the secretion of multiple forms of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em>-A (VEGF-A) that differ in their activity profiles with respect to neo<em>vascular</em>ization. FSAP (<em>factor</em> VII activating protease) is the zymogen form of a plasma protease that is activated (FSAPa) upon tissue injury via the release of histones. The purpose of the study was to determine if FSAPa regulates VEGF-A activity in vitro and in vivo. FSAP bound to VEGF₁₆₅, but not VEGF_<em>121</em>, and VEGF₁₆₅ was cleaved in its neuropilin/proteoglycan binding domain. VEGF₁₆₅ cleavage did not alter its binding to VEGF receptors but diminished its binding to neuropilin. The stimulatory effects of VEGF₁₆₅ on <em>endothelial</em> cell proliferation, migration, and signal transduction were not altered by FSAP. Similarly, proliferation of VEGF receptor-expressing BAF3 cells, in response to VEGF₁₆₅, was not modulated by FSAP. In the mouse matrigel model of angiogenesis, FSAP decreased the ability of VEGF₁₆₅, basic fibroblast <em>growth</em> <em>factor</em> (bFGF), and their combination, to induce neo<em>vascular</em>ization. Lack of endogenous FSAP in mice did not influence neo<em>vascular</em>ization. Thus, FSAP inhibited VEGF₁₆₅-mediated angiogenesis in the matrigel model in vivo, where VEGF's interaction with the matrix and its diffusion are important.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> A (VEGF-A) and its binding to VEGFRs is an important angiogenesis regulator, especially the earliest-known isoform, VEGF-A_165a. Yet several additional splice variants play prominent roles in regulating angiogenesis in health and in <em>vascular</em> disease, including VEGF-A_<em>121</em> and an anti-angiogenic variant, VEGF-A_165b. Few studies have attempted to distinguish these forms from their angiogenic counterparts, experimentally. Previous studies of VEGF-A:VEGFR binding have measured binding kinetics for VEGFA₁₆₅ and VEGF-A_<em>121</em>, but binding kinetics of the other two pro- and all anti-angiogenic splice variants are not known. We measured the binding kinetics for VEGF-A₁₆₅, -A_165b, and -A_<em>121</em> with VEGFR1 and VEGF-R2 using surface plasmon resonance. We validated our methods by reproducing the known affinities between VEGF-A_165a:VEGFR1 and VEGF-A_165a:VEGFR2, 1.0 pM and 10 pM respectively, and validated the known affinity VEGF-A_<em>121</em>:VEGFR2 as K_D = 0.66 nM. We found that VEGF-A_<em>121</em> also binds VEGFR1 with an affinity K_D = 3.7 nM. We further demonstrated that the anti-angiogenic variant, VEGF-A_165b selectively prefers VEGFR2 binding at an affinity = 0.67 pM while binding VEGFR1 with a weaker affinity-K_D = 1.4 nM. These results suggest that the - A_165b anti-angiogenic variant would preferentially bind VEGFR2. These discoveries offer a new paradigm for understanding VEGF-A, while further stressing the need to take care in differentiating the splice variants in all future VEGF-A studies.

BACKGROUND

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) isoforms, particularly the diffusible VEGF-<em>121</em>, could play a major role in the response of recurrent glioblastoma (GB) to anti-angiogenetic treatment with bevacizumab. We hypothesized that circulating VEGF-<em>121</em> may reduce the amount of bevacizumab available to target the heavier isoforms of VEGF, which are the most clinically relevant.

METHODS

We assessed the plasma level of VEGF-<em>121</em> in a brain xenograft model, in human healthy controls, and in patients suffering from recurrent GB before and after bevacizumab treatment. Data were matched with patients' clinical outcome.

RESULTS

In athymic rats with U87MG brain xenografts, the level of plasma VEGF-<em>121</em> relates with tumor volume and it significantly decreases after iv infusion of bevacizumab. Patients with recurrent GB show higher plasma VEGF-<em>121</em> than healthy controls (p = 0.0002) and treatment with bevacizumab remarkably reduced the expression of VEGF-<em>121</em> in plasma of these patients (p = 0.0002). Higher plasma level of VEGF-<em>121</em> was significantly associated to worse PFS and OS (p = 0.0295 and p = 0.0246, respectively).

CONCLUSIONS

Quantitative analysis of VEGF-<em>121</em> isoform in the plasma of patients with recurrent GB could be a promising predictor of response to anti-angiogenetic treatment.

BACKGROUND

Activation of endogenous stem cell mobilization can contribute to myocardial regeneration after ischemic injury. This study aimed to evaluate the possible role of Avemar or Echinacea extracts in inducing mobilization and homing of CD34(+) stem cells in relation to the inflammatory and hematopoietic cytokines in rats suffering from acute myocardial infarction (AMI).

METHODS

AMI was developed by two consecutive subcutaneous injections of isoprenaline (85 mg/kg). AMI rats were either post-treated or pre- and post-treated daily with oral doses of Avemar (<em>121</em> mg/kg) or Echinacea (130 mg/kg). In whole blood, the number of CD34(+) cells was measured by flow cytometry and their homing to the myocardium was immunohistochemically assessed. Serum creatine kinase, <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em>, interleukin-8 and granulocyte macrophage colony stimulating <em>factor</em> were determined on days 1, 7 and 14 after AMI. Sections of the myocardium were histopathologically assessed.

RESULTS

Rats pre- and post-treated with Avemar or Echinacea exhibited substantial increases in the number of circulating CD34(+) cells, peaking on the first day after AMI to approximately 13-fold and 15-fold, respectively, with a decline in their level on day 7 followed by a significant increase on day 14 compared to their corresponding AMI levels. Only post-treatment with Echinacea caused a time-dependent increase in circulating CD34(+) cells on days 7 and 14. Such increases in circulating CD34(+) cells were accompanied by increased homing to myocardial tissue 14 days after AMI. Interestingly, pre- and post-treatment with Avemar or Echinacea substantially increased serum creatine kinase on day 1, normalized its activity on day 7 and, on continued treatment, only Echinacea markedly increased its activity on day 14 compared to the corresponding AMI values. Moreover, both treatments modified differently the elevated serum vascular endothelial growth factor and the lowered granulocyte macrophage colony stimulating factor levels of the AMI group but did not affect the level of interleukin-8. These results were supported histopathologically by reduced inflammatory reactions and enhanced neovascularization.

CONCLUSIONS

Avemar and Echinacea extracts can effectively induce mobilization and homing of CD34(+) stem cells to the myocardial tissue and thus may help in stem cell-based regeneration of the infarcted myocardium.

<AbstractText>Predicting the development of early allograft dysfunction (EAD) following liver transplantation (LT) remains challenging for transplant clinicians. The objectives of this study are to investigate the potential relationship between the protein profiles of pretransplant grafts and the onset of EAD, and then combine with clinical parameters to construct a mathematically predictive model.</AbstractText><AbstractText>Clinical data of <em>121</em> LT procedures from donation after circulatory death at the authors' centre were analysed. The expression levels of seven studied proteins were determined by immunohistochemistry. Another independent cohort of 37 subjects was designed for further validation of the predictive model.</AbstractText><AbstractText>With an incidence of 43.0% (52/<em>121</em>), EAD was linked to significantly increased risk of acute kidney injury and renal replacement therapy, as well as reduced 6-month patient and liver graft survival. Allograft weight and high intrahepatic <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) expression were identified as independent risk <em>factors</em> of EAD and survival outcomes. Liver grafts with high VEGF expression exhibited delayed functional recovery within the first postoperative week. The combination of VEGF overexpression and EAD yielded the highest frequency of renal dysfunction and the worst survival. Based on allograft weight and intrahepatic VEGF expression, an EAD Risk Assessment Model was developed. The incidence of EAD differed significantly between grafts with risk scores ≥-1.72 and <-1.72. The model functioned well in the validation cohort.</AbstractText><AbstractText>Pretransplant intrahepatic protein profiling contributes to the estimation of early graft performance and recipient outcomes following LT. The predictive model could allow for an accurate prediction of EAD.</AbstractText>

<p><div><b>BACKGROUND AND PURPOSE</b></div><em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> A (VEGF-A) is a key mediator of angiogenesis. A striking feature of the binding of a fluorescent analogue of VEGF₁₆₅ a to nanoluciferase-tagged VEGF receptor 2 (VEGFR2) in living cells is that the BRET signal is not sustained and declines over time. This may be secondary to receptor internalisation. Here, we have compared the binding of three fluorescent VEGF-A isoforms to VEGFR2 in cells and isolated membrane preparations.</p><p><div><b>EXPERIMENTAL APPROACH</b></div>Ligand-binding kinetics were monitored in both intact HEK293T cells and membranes (expressing nanoluciferase-tagged VEGFR2) using BRET between tagged receptor and fluorescent analogues of VEGF₁₆₅ a, VEGF₁₆₅ b, and VEGF_<em>121</em> a. VEGFR2 endocytosis in intact cells expressing VEGFR2 was monitored by following the appearance of fluorescent ligand-associated receptors in intracellular endosomes using automated quantitative imaging.</p><p><div><b>KEY RESULTS</b></div>Quantitative analysis of the effect of fluorescent VEGF-A isoforms on VEGFR2 endocytosis in cells demonstrated that they produce a rapid and potent translocation of ligand-bound VEGFR2 into intracellular endosomes. NanoBRET can be used to monitor the kinetics of the binding of fluorescent VEGF-A isoforms to VEGFR2. In isolated membrane preparations, ligand-binding association curves were maintained for the duration of the 90-min experiment. Measurement of the k_off at pH 6.0 in membrane preparations indicated shorter ligand residence times than those obtained at pH 7.4.</p><p><div><b>CONCLUSIONS AND IMPLICATIONS</b></div>These studies suggest that rapid VEGF-A isoform-induced receptor endocytosis shortens agonist residence times on the receptor (1/k_off ) as VEGFR2 moves from the plasma membrane to the intracellular endosomes.</p>

OBJECTIVE

To investigate the feasibility of establishing a murine hemangioma model with injection of recombinant adeo-associated virus mediated human <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em>-<em>121</em> (rAAV-hVEGF(<em>121</em>)) gene.

METHODS

rAAV-hVEGF(<em>121</em>) was constructed, identified and then implanted to the left back ear of each mouse (1.0 x 10(11)VG in 50 microl per mouse and 10 nude mice received the injection), the rights served as controls with an injection of the same volume of phosphate buffered solution (PBS). The skin color and swelling of left back ear were observed every other day. Histological examination was carried out after mice were sacrificed 2, 4, 6, 8, 12 weeks after injection.

RESULTS

The rAAV-hVEGF(<em>121</em>) was correctly constructed and confirmed by restriction endonuclease analysis, polymerase chain reaction and DNA sequencing analysis. The skin of left back ear became red 2 weeks after injection and gradually exhibited a red lump which was at its utmost 12 weeks after injection. Such phenomena were not observed in right back ear. Histological examinations showed aggregates of <em>endothelial</em> cells by 2 weeks and at 8 weeks the swollen tissue contained many cysts filled with a mass of red cells. CD-34 staining suggested most of the newly-formed cells were <em>endothelial</em> cells.

CONCLUSIONS

A hemangioma model was established in mice with injection of recombinant rAAV-hVEGF(<em>121</em>) gene.

OBJECTIVE

To study the expression and the mechanism of miR-155in the villi of patients with unexplained recurrent spontaneous abortion (URSA).

METHODS

The expression of miR-155 in the villi of 36 cases with URSA (URSA group) and 25 women with normal early pregnancy (control group) were detected by stem-loop real-time reverse transcription (RT) qPCR.Expression of hypoxia inducible factor-1 (HIF-1α), vascular endothelial cell growth factor (VEGF) and micro lymphatic vessel density (MVD) in the villi of were measured by immnohistochemical staining among two groups.

RESULTS

(1)miR-155 expression:the mean miR-155 expression were 1.456 (0.489, 2.459) in URSA group and 2.833 (1.740, 3.794) in control group, which reached statistical difference (P < 0.05). The mean expression of miR-155 of 1.683 (0.902, 2.459) in URSA group with abortion times ( ≤ 3) was significantly higher than 1.229 (0.489, 1.719) in URSA group with more than 4 times abortion (P < 0.05). (2) Indexes :the expression of HIF-1α, VEGF and MVD value were 121 ± 12, 134 ± 12, 36 ± 6 in URSA group and 99 ± 10, 109 ± 10, 28 ± 4 in control group, which reached statistical difference (P < 0.01). The expression of HIF-1α, VEGF and MVD value of 119 ± 12, 134 ± 12, 35 ± 5 in URSA group with less than 3 times abortion was significantly lower than 128 ± 12, 138 ± 12, 43 ± 6 in URSA group with more than 4 times abortion (P < 0.01).

CONCLUSIONS

The expression of miR-155 and HIF-1α is topically stimulated by oxygen signal.HIF-1α adjusts the transcription and translation of VEGF, which together involved in placental trophoblast invasion and placental angiogenesis. The low expression of miR-155 could interfere with expression of HIF-1α and VEGF, which might be involved in villous vascular dysplasia in URSA.

OBJECTIVE

To investigate the feasibility of vascular graft material polytetrafluoroethylene (PTFE) as gene carrier of vascular endothelial growth factor (VEGF).

METHODS

The PTFE strips were soaked in pCDI-hVEGF(121) solution,marked with EB solution, and then observed under ultraviolet light. The PTFE strips carrying pCDI-hVEGF(121) were soaked in normal saline and the value of optical density at 260 nm on different time was measured, then the controlled release curve was made. The PTFE strips carrying pCDI-hVEGF(121) were implanted into the left thigh muscles of rabbits and the PTFE strips without VEGF gene were implanted into the right. The expression of VEGF(121) mRNA versus beta-actin mRNA in muscles around vascular graft materials was evaluated by reverse transcriptase polymerase chain reaction(RT-PCR). The expression of VEGF(121) protein in muscle was determined by Western blot method.

RESULTS

The fluorescence on PTFE strips carrying pCDI-hVEGF(121) was observed under ultraviolet light. The controlled release curve demonstrated that the gene release was fast during 0.5 -4 h, then slowed down afterwards,and lasted for 72 h. RT-PCR and Western blot showed VEGF(121)/beta-actin mRNA ratios were 1.053+/-0.356,1. 718+/-0.404, 2.021+/-0.303, 1.872+/-0.231, 0.986+/-0.254, 0.340+/-0.116 and VEGF(121)protein expression levels were 0.328+/-0.088, 1.019+/-0.105, 2.249 +/-0.203, 2.036+/-0.079, 1.670+/-0.132, 0.636+/-0.107 at 1, 7, 14, 28, 42, and 56 days after implantation, respectively.

CONCLUSIONS

The PTFE graft can be used as carrier of VEGF gene and VEGF gene can be transferred to skeletal muscle by this method.

Despite major advances in understanding angiogenesis over the last few years, the ability to induce angiogenesis in ischemic wounds or larger tissue-engineering constructs remains elusive. Serious risks and limited control over dose, duration, and localization of <em>growth</em> <em>factor</em> delivery make materials-based approaches viable alternatives. In an effort to minimize passive diffusion and control the release profile of delivered <em>growth</em> <em>factors</em>, matrix properties have been engineered with regard to pore size, <em>growth</em> <em>factor</em> affinity or stable <em>growth</em> <em>factor</em> binding. Recently, fibrin or biomimetic hydrogels have been engineered towards the covalent immobilization of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF). Most of the studies pertaining to VEGF delivery by fibrin gel constructs have focused on characterizing release profiles, receptor activation, and the angiogenic response in vitro and in vivo. Herein we demonstrate that gels containing covalently-linked VEGF (α₂PI_1-8-VEGF_<em>121</em>), compared to diffusible VEGF, elicit stronger and longer-lasting angiogenic responses in subcutaneous implants of mice. This superior angiogenic response was due to both the sustained release and significant retention of bioactivity (80%) of the delivered engineered VEGF over a 12-day period. To the best of our knowledge, this is the first report to characterize long-term matrix liberated α₂PI_1-8-VEGF_<em>121</em> bioactivity, important for future efforts in angiogenesis research.

Background: Extreme panting under heat stress threatens dairy cattle milk production. Previous research has revealed that the gas exchange-mediated respiratory drive in critically ill dairy cattle with low O₂ saturation induces panting. Vascular endothelial growth factor (VEGF) signaling may play important roles in immunosuppression and oxidative stress during severe respiratory stress responses in heat-stressed cattle. The objectives of this study were to transcriptomically analyze mRNA expression mediating heat-induced respiratory stress-associated panting, evaluate gas exchange, screen hub genes, and verify the expression of proteins encoded by differentially expressed genes in lymphocyte pathways.

Results: Jersey cattle were naturally heat-exposed. Physiological data were collected for response evaluation, and blood was collected for gas exchange and gene expression assays at 06:00, 10:00 and 14:00 continuously for 1 week. Lymphocytes were isolated from whole-blood samples for mRNA-seq and expression analysis of key pathway genes/proteins. The cattle respiration rates differed with time, averaging 51 bpm at 06:00, 76 bpm at 10:00, and 121 bpm at 14:00 (p < 0.05). Gas exchange analysis showed that both pH and pCO₂ differed with time: they were 7.41 and 41 mmHg at 06:00, 7.45 and 37.5 mmHg at 10:00, and 7.49 and 33 mmHg at 14:00, respectively (p < 0.01). Sixteen heat-related differentially expressed genes (DEGs; 13 upregulated and 3 downregulated) were screened between 212 DEGs and 1370 heat stress-affected genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) hub gene functional analysis annotated eleven genes to signal transduction, six genes to the immune response, and five genes to the endocrine response, including both prostaglandin-endoperoxide synthase 2 (PTGS2) and VEGF. Gene Ontology (GO) functional enrichment analysis revealed that oxygen regulation was associated with the phosphorus metabolic process, response to oxygen levels, response to decreased oxygen levels, response to hypoxia and cytokine activity terms. The main signaling pathways were the VEGF, hypoxia inducible factor-1(HIF-1), cytokine-cytokine receptor interaction and TNF pathways. Four genes involved Integrin beta 3 (ITBG3), PTGS2, VEGF, and myosin light chain 9 (MYL9) among the 16 genes related to immunosuppression, oxidative stress, and endocrine dysfunction were identified as participants in the VEGF signaling pathway and oxygenation.

Conclusion: These findings help elucidate the underlying immune and oxygen regulation mechanisms associated with the VEGF signaling pathway in heat-stressed dairy cattle.

Keywords: Dairy cattle; Heat stress; Immune; Lymphocytes; Vascular endothelial growth factor.