Effects of nitric oxide donors on vascular endothelial growth factor gene induction

To study the expression level of TGFβ1 and VEGF gene in patients with acute myeloid leukemia (AML) and its clinical prognostic value. Seventy-eight AML patients treated in our hospital from July 2016 to September 2018 were selected. After isolation of bone marrow mononuclear cells from the patients, the levels of TGFβ1 and VEGF genes were detected by RT-PCR, and the correlation of TGFβ1 with VEGF genes and clinical characteristics of AML patients was analyzed. OS and EFS of the patients were evaluated by Kaplan-Meier, and Cox risk ratio model was used to analyze the prognostic risk factors of AML patients. The relative expression level of TGFβ1 gene in AML patients was 0.32±0.04, which was significantly lower than that in control group (P<005). The relative expression level of vascular endothelial growth factor(VEGF) gene in the patients was 2.65±0.15, which was significantly higher than that in the control group (P<0.05). The levels of TGFβ1 and VEGF genes significantly correlated with leukocyte count, hemoglobin, platelet and peripheral blast levels in AML patients (P<0.05). The level of TGFβ1 in AML patients with complete remission was higher than that in patients with partial remission or non-remission (P<0.05). The level of TGFβ1 in AML patients with partial remission was significantly higher than that in patients with non-remission (P<0.05). The level of VEGF in AML patients with complete remission was lower than at in patients with partial remission or non-remission (P<0.05). The level of VEGF in AML patients with partial remission was significantly lower than that in patients with non-remission (P<0.05). Kaplan-Meier survival analysis showed that OS and DFS in AML patients with high expression of TGFβ1 were better than those in patients with low expression of TGFβ1 (P<0.05), OS and DFS in AML patients with low expression of VEGF were better than those in patients with high expression of VEGF (P<0.05). Multivariate Cox regression analysis showed that platelet, TGFβ1 and VEGF gene were independent influencing factors of OS (P<0.05). Leukocyte, TGFβ1 and VEGF gene were independent influencing factors of DFS (P<0.05). Decreased expression of TGFβ1 and increased expression of VEGF gene in AML patients closely relate to the poor prognosis of AML patients, which can provide reference for improving clinical efficacy of AML patients.

Vascular endothelial growth factor (VEGF) is a potent and specific mitogen for vascular endothelial cells and promotes neovascularization in vivo. To determine whether interleukin-1 beta (IL-1 beta), which is present in atherosclerotic lesions, induces VEGF gene expression in vascular smooth muscle cells, we performed RNA blot analysis on rat aortic smooth muscle cells (RASMC) with a rat VEGF cDNA probe. IL-1 beta increased VEGF mRNA levels in RASMC in a time- and dose-dependent manner. As little as 0.1 ng/ml IL-1 beta increased VEGF mRNA levels by 2-fold and 10 ng/ml IL-1 beta increased VEGF mRNA by 4-fold. We also measured the half-life of VEGF mRNA and performed nuclear run-on experiments before and after addition of IL-1 beta to see if IL-1 beta increased VEGF mRNA levels by stabilizing the mRNA or by increasing its rate of transcription. The normal, 2-h half-life of VEGF mRNA in RASMC was lengthened to 3.2 h (60%) by IL-1 beta, and IL-1 beta increased the rate of VEGF gene transcription by 2.1-fold. In immunoblot experiments with an antibody specific for VEGF, we found that IL-1 beta increased VEGF protein levels in RASMC by 3.3-fold. Together these data indicate that IL-1 beta induces VEGF gene expression in smooth muscle cells. This IL-1 beta-induced expression of VEGF may accelerate the progression of atherosclerotic lesions by promoting the development of new blood vessels.

Contrast-induced nephropathy (CIN) is associated with increased mortality and morbidity in patients undergoing coronary angiography (CAG) and percutaneous coronary intervention (PCI). We aimed to assess the latest evidence on the preventive effects of nitric oxide (NO) donors in CIN patients undergoing CAG/PCI. We conducted a comprehensive systematic review and meta-analysis of RCTs from PubMed, Web of Science, Scopus, Embase, and Cochrane searches until May 5th, 2024. Dichotomous data were pooled using risk ratio (RR), and continuous data were pooled using mean difference (MD), both with a 95% confidence interval (CI), using (R version 4.3). Our analysis included 13 RCTs encompassing 3,550 patients. NO donors were significantly associated with a decreased incidence of CIN compared to placebo either as an oral administration (RR: 0.33 with 95% CI [0.26, 0.42], P < 0.01) or IV infusions (RR: 0.56 with 95% CI [0.40, 0.78], P < 0.01). Moreover, NO donors were significantly associated with decreased serum creatinine levels compared to placebo either as an oral administration (MD: - 0.07 with 95% CI [- 0.10, - 0.04], P < 0.01) or IV infusions (MD: - 0.07 with 95% CI [- 0.09, - 0.04], P < 0.01). In terms of safety, NO donors were significantly associated with a decreased incidence of major adverse cardiac events (MACE) compared to placebo as an oral administration (RR: 0.64 with 95% CI [0.45, 0.89], P < 0.01). However, there was no significant difference between NO donors as IV infusions and placebo in MACE (RR: 0.68 with 95% CI [0.38, 1.21], P = 0.18). Finally, NO donors were significantly associated with a decreased incidence of all-cause mortality compared to placebo as an oral administration (RR: 0.58 with 95% CI [0.36, 0.94], P = 0.03). Nevertheless, there was no statistically significant difference in all-cause mortality between IV infusions of NO donors and placebo (RR: 1.84 with 95% CI [0.40, 8.52], P = 0.44). NO donors as adjunct therapy are associated with reduced incidence of CIN and decreased serum creatinine levels, either as an oral or IV administration. They were also associated with reduced incidence of MACE, all-cause mortality, and recurrent myocardial infarction as an oral administration, which makes this simple, low-cost intervention an important therapeutic option in patients undergoing CAG/PCI.

Background: Contrast-induced nephropathy (CIN) is associated with increased mortality and morbidity in patients undergoing coronary angiography (CAG) and percutaneous coronary intervention (PCI). We aimed to assess the latest evidence on the preventive effects of nitric oxide (NO) donors in CIN in patients undergoing CAG/PCI. Methods: We conducted a systematic review and meta-analysis of RCTs from PubMed, Web of Science, Scopus, Embase, and Cochrane searches until May 5th, 2024. Dichotomous data were pooled using risk ratio (RR), and continuous data were pooled using mean difference (MD), both with a 95% confidence interval (CI), using (R version 4.3). Results: Our analysis included 13 RCTs encompassing 3,550 patients. NO donors were significantly associated with a decreased incidence of CIN compared to placebo either as an oral administration (RR: 0.33 with 95% CI [0.26, 0.42], P&lt; 0.01) or IV infusions (RR: 0.56 with 95% CI [0.40, 0.78], P&lt; 0.01). Moreover, NO donors were significantly associated with decreased serum creatinine levels compared to placebo either as an oral administration (MD: -0.07 with 95% CI [-0.10, -0.04], P&lt; 0.01) or IV infusions (MD: -0.07 with 95% CI [-0.09, -0.04], P&lt; 0.01). In terms of safety, NO donors were significantly associated with a decreased incidence of MACE compared to placebo as an oral administration (RR: 0.64 with 95% CI [0.45, 0.89], P&lt; 0.01). However, there was no significant difference between NO donors as IV infusions and placebo in MACE (RR: 0.68 with 95% CI [0.38, 1.21], P= 0.18). Finally, NO donors were significantly associated with a decreased incidence of all-cause mortality compared to placebo as an oral administration (RR: 0.58 with 95% CI [0.36, 0.94], P= 0.03). However, there was no significant difference between NO donors as IV infusions and placebo in all-cause mortality (RR: 1.84 with 95% CI [0.40, 8.52], P= 0.44). Conclusion: NO donors as an adjunct therapy are associated with reduced incidence of CIN, and decreased serum creatinine levels either as an oral or IV administration. Also, it was associated with decreased incidence of MACE and all-cause mortality as an oral administration which make this simple low-cost intervention an important therapeutic option in patients undergoing CAG/PCI.

Effects of nitric oxide donors on basal and K +-evoked release of [formula omitted]noradrenaline from rat cerebral cortex synaptosomes

We analyzed the effect of nitric oxide (NO) on oxygen-dependent cytotoxic responses mediated by neutrophils against unopsonized erythrocytes using three NO donors: S-nitrosoglutathione (GSNO), S-nitroso-N-acetylpenicillamine (SNAP), and sodium nitroprusside (SNP). Neutrophils were treated with these compounds for 1-2 min at 37 degrees C and cytotoxicity was then triggered in the presence of NO donors by precipitating immune complexes, aggregated IgG, the chemotactic peptide FMLP, or opsonized zymosan. GSNO induced, in all cases, a marked increase in cytotoxic responses, while SNAP moderately increased cytotoxicity triggered by immune complexes, aggregated IgG, or Z, opsonized zymosen, without modifying those responses induced by FMLP. By contrast, SNP dramatically suppressed cytotoxicity triggered by all of the stimuli assessed. The enhancing effects mediated by GSNO and SNAP did not depend on the stimulation of guanylyl cyclase and were prevented by the NO scavengers hemoglobin and PTIO (2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl 3-oxide). The inhibitory activity of SNP, on the other hand, was not prevented by NO scavengers, suggesting that it cannot be ascribed to the release of NO. In another set of experiments, neutrophils were pretreated with GSNO or SNAP for different times. Then cells were washed to remove NO donors from the culture medium, and cytotoxicity was triggered by different stimuli. It was found that neutrophils must be pretreated with NO donors for at least 4 h to increase cytotoxic responses, and pretreatment for longer periods (i.e., 8 or 18 h) further increased cytotoxicity. Not only cytotoxic responses, but also the production of O2- and H2O2, and the release of myeloperoxidase were increased under these conditions.

To study the expression level of TGFβ1 and VEGF gene in patients with acute myeloid leukemia (AML) and its clinical prognostic value. Seventy-eight AML patients treated in our hospital from July 2016 to September 2018 were selected. After isolation of bone marrow mononuclear cells from the patients, the levels of TGFβ1 and VEGF genes were detected by RT-PCR, and the correlation of TGFβ1 with VEGF genes and clinical characteristics of AML patients was analyzed. OS and EFS of the patients were evaluated by Kaplan-Meier, and Cox risk ratio model was used to analyze the prognostic risk factors of AML patients. The relative expression level of TGFβ1 gene in AML patients was 0.32±0.04, which was significantly lower than that in control group (P<005). The relative expression level of vascular endothelial growth factor(VEGF) gene in the patients was 2.65±0.15, which was significantly higher than that in the control group (P<0.05). The levels of TGFβ1 and VEGF genes significantly correlated with leukocyte count, hemoglobin, platelet and peripheral blast levels in AML patients (P<0.05). The level of TGFβ1 in AML patients with complete remission was higher than that in patients with partial remission or non-remission (P<0.05). The level of TGFβ1 in AML patients with partial remission was significantly higher than that in patients with non-remission (P<0.05). The level of VEGF in AML patients with complete remission was lower than at in patients with partial remission or non-remission (P<0.05). The level of VEGF in AML patients with partial remission was significantly lower than that in patients with non-remission (P<0.05). Kaplan-Meier survival analysis showed that OS and DFS in AML patients with high expression of TGFβ1 were better than those in patients with low expression of TGFβ1 (P<0.05), OS and DFS in AML patients with low expression of VEGF were better than those in patients with high expression of VEGF (P<0.05). Multivariate Cox regression analysis showed that platelet, TGFβ1 and VEGF gene were independent influencing factors of OS (P<0.05). Leukocyte, TGFβ1 and VEGF gene were independent influencing factors of DFS (P<0.05). Decreased expression of TGFβ1 and increased expression of VEGF gene in AML patients closely relate to the poor prognosis of AML patients, which can provide reference for improving clinical efficacy of AML patients.

VEGF gene sequence variation defines VEGF gene expression status and angiogenic activity in non-small cell lung cancer

The aim of this study was to investigate the role of nitric oxide (NO), and the expression of endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF) genes in developing hearts at embryonic day 13.5 of embryos from diabetic mice. The protein and mRNA expression levels of eNOS and VEGF were significantly altered in the developing hearts of embryos from diabetic mice. The NO level was significantly decreased, whereas the VEGF concentration was significantly increased in the developing hearts of the embryos from diabetic mice. In vitro study showed a significant reduction in eNOS expression and cell proliferation in cardiac myoblast cells exposed to high glucose concentrations. Further, high glucose induced apoptosis in myoblast cells. Ultrastructural changes characteristics of apoptosis, including cell blebbing, aggregation of ribosomes and vacuoles in the cytoplasm were also evident in myoblast cells exposed to high glucose. It is suggested that hyperglycemia alters the expression of eNOS and VEGF genes that are involved in the regulation of cell growth and vasculogenesis, thereby contributing to the cardiac malformations seen in embryos from diabetic mice.

Purpose: Gene therapy provides a useful method to enhance wound healing, but it has not been attempted in healing of wounds in flexor tendons in the hands, which inherently lack sufficient intrinsic healing capacity. To explore the potential of application of gene therapy to tendon wound healing, we genetically modified tenocytes with the platelet-derived growth factor-B (PDGF-B) and the vascular endothelial growth factor (VEGF) gene and investigated the expression of the genes for collagen production in an in vitro model of the proliferating tenocytes. Method: Tenocytes were obtained from cultures of rat intrasynovial tendons and randomly distributed to 34 dishes. The tenocytes in dishes in two experimental groups (n = 9, each group) were treated for 12 hours with the plasmid containing the PDGF-B cDNA or the VEGF cDNA and were then cultured for 5 days; tenocytes in the other eight dished received sham vector and the tenocytes in the control dishes (n = 8) did not receive the exogenous gene. Efficiency of the gene transfer was evaluated by detection of the presence of the transgene in the tenocytes by reverse transcription polymerase chain reactions (RT-PCR). Levels of expression of type I and III collagen, and TGF-β genes were determined by quantitative analysis of the products of RT-PCR. Results: Expression of the type I collagen gene was significantly increased by transfer of the exogenous PFDGF gene to the tenocytes (p < 0.001). Transfer of the PDGF-B gene did not affect the expression of TGF-β and the type III collagen genes significantly. Expression of TGF-β gene increased significantly in the cells treated with exogenous VEGF cDNA (p = 0.039). Expression of type I and III collagen genes by tenocytes was minimally affected by transfer of the VEGF gene to the tenocytes and was significantly weaker than that stimulated by PDGF-B gene therapy (p = 0.001). Efficient gene transfer was confirmed by the presence of the PDGF-B gene or the VEGF gene in the tenocytes receiving the transferred genes. Conclusions: Transfer of exogenous PDGF-B gene significantly enhances expression of the type I collagen gene of the tenocytes. Transfer of exogenous VEGF gene has very limited effects on promotion of collagen production in the proliferating tenocytes. In contrast, transfer of the VEGF gene significantly increases expression of the TGF-β gene. This study suggests that transfer of the PDGF-B gene may offer a novel way of effectively promoting healing of intrasynovial flexor tendons. VEGF gene therapy is not as beneficial as PDGF-B gene therapy to tendon healing and may increase activities of TGF-β that are associated with adhesion formations.

680. Application of Gene Therapy to Tendon Healing: Modification of Tenocytes with Exogenous PDGF and VEGF Genes to Promote Collagen Production

To evaluate the possibility and efficiency of nanoparticle as a new vector in vascular endothelial growth factor (VEGF) gene transference, and investigate the efficacy of direct gene transfer of nanoparticle with VEGF(165) gene into ischemic myocardium. Nanoparticle-VEGF (Np/VEGF) complex was prepared with poly (D, L-lactide-co-glycolide) (PLGA) loading VEGF(165) gene and the envelopment efficiency and size of the complex were determined. The Np/VEGF was transfected into the cultured myocardial cells, RT-PCR and ELISA were used to evaluate the transfection of VEGF. Suspension of Np/VEGF was injected into the myocardial tissue of 4 rabbits. 96 hours after operation myocardial tissue was obtained, made into sections, and observed with electron microscope. New Zealand White rabbits underwent thoracotomy followed by ligation of left anterior descending coronary artery to establish ischemic models. The New Zealand White rabbits were divided into 3 groups: Np/VEGF group (n = 12, nanoparticle with VEGF(165) were injected into the cordial myocardium), blank plasmid group (n = 12, injected with blank VEGF(165) plasmid), and control group (n = 8, injected with normal saline). Ultrasonography and immunohistochemistry with factor VIII related antigen were conducted to evaluate the cardiac function and the collateral circulation of the occluded artery. One month later the rabbits were killed to observe the vascularization of capillaries in the ischemic myocardium. The envelopment efficiency of the Np/VEGF complex thus prepared, 50 - 300 nm in size, were 1.87% y. RT-PCR and ELISA showed that VEGF gene had been successfully transfected into myocardial cells by the nanoparticles. A great number of nanoparticles were observed in the myocardial cytoplasm and nuclei. One month after operation, the ventricular wall motor amplitude of the Np/VEGF group was 1.87 mm +/- 0.32 mm, significantly larger than those of the blank plasmid group (1.59 mm +/- 0.24 mm, P < 0.05) and control group (0.93 mm +/- 0.40 mm, P < 0.05); and the left ventricular ejection fraction of the Np/VEGF group was 60% +/- 10%, significantly higher than those of the blank plasmid group (50% +/- 6%, P < 0.05) and control group (40% +/- 8%, P < 0.05). The capillary density at low power field (x 100) of the Np/VEGF group was 57 +/- 12, significantly higher than those of the VEGF group (41 +/- 14) and control group (24 +/- 8). Nanoparticle can act as a vector to transfect specific gene in vitro and in vivo. Direct gene transfer of nanoparticle with DNA encoding VEGF into the ischemic rabbit myocardium can increase capillary number; therefore it may be a novel therapeutic approach for myocardial ischemia.

Objective To explore the association of vascular endothelial growth factor (VEGF) gene rs2010963 G/C single nucleotide polymorphism (SNP) and rs833061 T/C SNP with susceptibility to polycystic ovary syndrome (PCOS). Methods The genotypes of VEGF gene rs2010963G/C SNP and rs833061T/C SNP were performed by polymerase chain reaction-ligase detection reaction (PCR-LDR) method in 118 PCOS patients (PCOS group) and 130 healthy controls (control group). Results The frequencies of G allele and C allele of the VEGF gene rs2010963 G/C SNP in the control were 60.8%, 39.25% and 69.9%, 30.1% in PCOS group, respectively. There was a significant difference in the allele distribution of the rs2010963 G/C SNP between the two groups (P=0.033). The GG, GC and CC genotype frequencies of VEGF gene rs2010963 G/C SNP were 36.2%, 49.2%, 14.6% in control group and 50.9%, 38.1%, 11.0% in PCOS group, respectively. The women carrying GC, GC+CC genotype had a lower risk of PCOS compared with the women with the GG genotype. The OR was 0.55 (95% CI=0.32-0.95) and 0.55 (95% CI=0.33-0.91), respectively. No significant difference was observed in the frequency of the genotype and allele of VEGF gene rs833061 T/C SNP between the two groups (P>0.05). VEGF gene rs2010963G/C SNP and rs833061T/C SNP were combined for analysis using 2LD software. Results showed that linkage disequilibrium exists between these two SNPs (D'=0.82). The haplotype of these two SNPs was analyzed using EH software. The result revealed that the GT was the most frequent haplotype. Conclusion The finding in our pilot study suggests that the VEGF gene rs2010963 G/C SNP is significantly associated with the risk of PCOS. The women carrying the C allele (GC+CC genotype) had a lower risk of PCOS compared with the women with GG genotype. Key words: Vascular endothelial growth factor (VEGF); Single nucleotide polymorphism (SNP); Polycystic ovary syndrome (PCOS); Genetic susceptibility

Tendon healing in vitro: Modification of tenocytes with exogenous vascular endothelial growth factor gene increases expression of transforming growth factor β but minimally affects expression of collagen genes

The effect of nitric oxide (NO) donors on survival of conidia, germination and growth of the opportunistic pathogenic fungus Aspergillus fumigatus was investigated. Most efficient was sodium nitrite in an acidic milieu (pH 4.5). At a concentration of 5 mmol/L it killed all resting conidia in buffer within 16 h. S-Nitroso derivatives of thiols (cysteine, N-acetylcysteine and N-acetylpenicillamine) at the same concentration killed about 30-50% of spores within 24 h. The NO scavenger, oxyhemoglobin, abolished these effects. S-Nitrosoglutathione had no fungicidal effect and promoted germination. Sodium nitrite and S-nitroso-N-acetylcysteine inhibited germination of conidia in various media from concentration of 0.5 mmol/L and stopped it at concentrations of 1.4-2.9 mmol/L. In media with glucose and casein hydrolyzate or sodium nitrate as nitrogen source, growth inhibition by sodium nitrite (0.5-2 mmol/L) was only weak and mostly transient. In general, the used strain A. fumigatus seems to be less sensitive to nitric oxide donors than dimorphic pathogenic fungi. Thus, nitric oxide is probably not a major effector molecule in killing phagocytized elements of this fungus by host's immunocytes.