The impact of ischemic vascular stenosis on LIPU hyperthermia efficacy investigated Based on in vivo rabbit limb ischemia model.

Ischemic diseases due to arterial stenosis or occlusion are common and can have serious consequences if untreated. Therapeutic ultrasound like high-intensity focused ultrasound (HIFU) ablates tissues while low-intensity pulsed ultrasound (LIPU) promotes healing at relatively low temperatures. However, blood vessel cooling effect and reduced flow in ischemia impact temperature distribution and ultrasonic treatment efficacy. This work established a rabbit limb ischemia model by ligating the femoral artery, measuring vascular changes and temperature rise during LIPU exposures. Results showed the artery diameter was narrowed by 46.2% and the downstream velocity was reduced by 51.3% after ligation. Finite element simulations verified that the reduced flow velocity impaired heat dissipation, enhancing LIPU-induced heating. Simulation results also suggested the temperature rise was almost related linearly to vessel diameter but decayed exponentially with the increasing flow velocity. Findings indicate that the proposed model could be used as an effectively tool to model the heating effects in ischemic tissues during LIPU treatment. This research on relating varied ischemic flow to LIPU-induced thermal effects is significant for developing safe and efficacious clinical ultrasound hyperthermia treatment protocols for the patients with ischemic diseases.

Magnoflorine Ameliorates Collagen-Induced Arthritis by Suppressing the Inflammation Response via the NF-κB/MAPK Signaling Pathways.

Objective: Magnoflorine (Mag) has been reported to have anxiolytics, anti-cancer, and anti-inflammatory properties. In this study, we aim to investigate the effects of Mag on the rheumatoid arthritis (RA) and explore the underlying mechanism using a collagen-induced arthritis (CIA) mouse model and a lipopolysaccharide (LPS)-stimulated macrophage inflammation model. Methods: The in vivo effects of Mag on CIA were studied by inducing CIA in a mouse model using DBA/1J mice followed by treatment with vehicle, methotrexate (MTX, 1 mg/kg/d), and Mag (5 mg/kg/d, 10 mg/kg/d, and 20 mg/kg/d), and the in vitro effects of Mag on macrophages were examined by stimulation of RAW264.7 cells line and peritoneal macrophages (PMs) by LPS in the presence of different concentrations of Mag. Network pharmacology and molecular docking was then performed to predict the the binding ability between Mag and its targets. Inflammatory mediators were assayed by quantitative real-time PCR and enzyme linked immunosorbent assay (ELISA). Signaling pathway changes were subsequently determined by Western blotting and immunohistochemistry (IHC). Results: In vivo experiments demonstrated that Mag decreased arthritis severity scores, joints destruction, and macrophages infiltration into the synovial tissues of the CIA mice. Network pharmacology analysis revealed that Mag interacted with TNF-α, IL-6, IL-1ß, and MCP-1. Consistent with this, analysis of the serum, synovial tissue of the CIA mice, and the supernatant of the cultured RAW264.7 cells and PMs showed that Mag suppressed the expression of TNF-α, IL-6, IL-1ß, MCP-1, iNOS, and IFN-ß. Furthermore, Mag attenuated the phosphorylation of p65, IκBα, ERK, JNK, and p38 MAPKs in the synovial tissues of the CIA mice and LPS-stimulated RAW 264.7 cells. Conclusion: Mag may exert anti-arthritic and anti-inflammatory effects by inhibiting the activation of NF-κB and MAPK signaling pathways.

Dose-dependent effects of tetramethylpyrazine on the characteristics of human umbilical cord mesenchymal stem cells for stroke therapy.

Umbilical cord mesenchymal stem cells (ucMSCs) may serve as a new source for cell therapy in stroke patients; however, the poor efficiency of viability, migration, and differentiation limit the application of ucMSCs. This study determined the dose-dependent effects of tetramethylpyrazine (TMP) on the characteristics of ucMSCs in vitro. The effect on proliferation was determined with Cell Counting kit-8 assays. Cell migration was analyzed with Transwell assays and western blot analysis. Differentiation of ucMSCs was evaluated according to markers and the expression of relevant proteins and genes. Secretion capacity was detected by ELISA analysis. TMP protected ucMSCs against H2O2 induced-oxidative damage but had no influence on ucMSC activity at a low concentration. Furthermore, ucMSC migration was improved by TMP via the SDF-1/CXCR4 axis. The observed effects were dose dependent. At a high dose, however, TMP induced the differentiation of ucMSCs into neuron-like cells that expressed neuron-specific markers. In addition, the secretion of cytokines was significantly increased by TMP. Therefore, TMP pre-treatment of ucMSCs may be an effective strategy to enhance the efficiency of ucMSC transplantation in stroke therapy.

CK19 stabilizes CFTR at the cell surface by limiting its endocytic pathway degradation.

Protein interactions that stabilize the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) at the apical membranes of epithelial cells have not yet been fully elucidated. We identified keratin 19 (CK19 or K19) as a novel CFTR-interacting protein. CK19 overexpression stabilized both wild-type (WT)-CFTR and Lumacaftor (VX-809)-rescued F508del-CFTR (where F508del is the deletion of the phenylalanine residue at position 508) at the plasma membrane (PM), promoting Cl- secretion across human bronchial epithelial (HBE) cells. CK19 prevention of Rab7A-mediated lysosomal degradation was a key mechanism in apical CFTR stabilization. Unexpectedly, CK19 expression was decreased by ∼40% in primary HBE cells from homogenous F508del patients with CF relative to non-CF controls. CK19 also positively regulated multidrug resistance-associated protein 4 expression at the PM, suggesting that this keratin may regulate the apical expression of other ATP-binding cassette proteins as well as CFTR.-Hou, X., Wu, Q., Rajagopalan, C., Zhang, C., Bouhamdan, M., Wei, H., Chen, X., Zaman, K., Li, C., Sun, X., Chen, S., Frizzell, R. A., Sun, F. CK19 stabilizes CFTR at the cell surface by limiting its endocytic pathway degradation.

Oroxylin A inhibits Kaposi's sarcoma-associated herpes virus (KSHV) vIL-6-mediated lymphatic reprogramming of vascular endothelial cells through modulating PPARγ/Prox1 axis.

BACKGROUND AND PURPOSE: Kaposi's sarcoma-associated herpes virus (KSHV) vIL-6 is sufficient to induce lymphatic reprogramming of vascular endothelial cells, which is a key event in Kaposi's sarcoma (KS) development. This study was aimed to investigate the effect of Chinese herb oroxylin A on lymphatic reprogramming and neovascularization by KSHV vIL-6 in vitro and in vivo. METHODS: The lymphatic-phenotype endothelial cell line was generated by lentiviral KSHV vIL-6 infection. Cell viability and apoptosis were determined by MTT assay or flow cytometry with annexin V/propidium iodide staining. Migration, invasion, and neovascularization of the vIL-6-expressing lymphatic-phenotype endothelial cells were determined by wound healing assay, transwell chamber assay, microtubule formation assay, and chick chorioallantoic membrane assay, respectively. Quantitative polymerase chain reaction and Western blot analysis were used to test the expression of Prox1, VEGFR3, podoplanin, LYVE-1, and PPARγ in cells. Co-localization of Prox1 and PPARγ was determined by immunofluorescence. Ubiquitination of Prox1 was detected by in vivo ubiquitination assay. RESULTS: The lymphatic-phenotype endothelial cell line expressing KSHV vIL-6 was successfully generated. Oroxylin A induced cellular invasion abrogation, apoptosis induction, and neovascularization inhibition of the vIL-6-expressing endothelial cells. Mechanically, oroxylin A elevated PPARγ expression, which in turn interacted with and facilitated Prox1 to undergo ubiquitinational degradation, and subsequently leads to VEGFR3, LYVE-1, and podoplanin reduction. CONCLUSION: Through modulating PPARγ/Prox1 axis, oroxylin A inhibits lymphatic reprogramming and neovascularization of KSHV vIL-6. Thus, oroxylin A may serve as a candidate for the treatment of KS as well as other aggressive angiomas.

Ultrasound-Enhanced Protective Effect of Tetramethylpyrazine via the ROS/HIF-1A Signaling Pathway in an in Vitro Cerebral Ischemia/Reperfusion Injury Model.

Reactive oxygen species-induced oxidative stress is an important pathophysiological process during cerebral ischemia/reperfusion (I/R) injury. It has been reported that the protective effect of tetramethylpyrazine (TMP) against cerebral I/R injury can be significantly improved by its combination with ultrasound exposure. However, the molecular mechanisms and signaling pathways underlying the synergistic protective effect remain unclear. In the present work, the damage induced by I/R injury was modeled by glutamate-induced toxicity to pheochromocytoma (PC12) cells. The ultrasound-enhanced protective effect of TMP was systemically investigated by measuring variations in cell viability, cell migration and levels of intracellular reactive oxygen species, the oxidative stress-related protein glutathione, apoptosis-related proteins (caspase-8, -9 and -3), as well as expression of related genes (hypoxia-inducible factor-1a, p53, murine double minute2). The results suggest that the ultrasound-enhanced protective effect of TMP against cerebral I/R injury might act via the reactive oxygen species/hypoxia-inducible factor-1a signaling pathway, and an appropriate ultrasound intensity should be selected to achieve an optimal synergistic neuroprotective effect.

Growth arrest-specific protein 6 (Gas6) as a noninvasive biomarker for early detection of diabetic nephropathy.

BACKGROUND: To investigate the diagnostic level of cystatin C and growth arrest-specific gene 6 (Gas6) levels in elderly type 2 diabetic patients with different degrees of diabetic nephropathy (DN). METHODS: Four hundred and eighty-two old people, including 130 healthy controls, 130 normoalbuminuric diabetic patients, 122 with microalbuminuria, and 100 with macroalbuminuria, were recruited. Plasma Gas6 and serum cystatin C levels were measured. RESULTS: Plasma Gas6 concentration was significantly lower in diabetic patients with microalbuminuria or macroalbuminuria, as compared with diabetic subjects with normoalbuminuria; while cystatin C was significantly higher. Gas6 was inversely correlated with BMI, WHR, and HbA1c, while cystatin C was inversely correlated with urea nitrogen and creatinine. Multivariate logistic regression analysis showed that, after adjusted for established diabetes risk factors, higher plasma Gas6 was significantly associated with a decreased risk of DN, while higher serum cystatin C was significantly associated with an increased risk. Receiver operating characteristic curve analysis showed that Gas6 was better than cystatin C as a biomarker for early diagnosis and detection of DN, with a cutoff value of 9.435 ng/mL (86.1% sensitivity and 84.6% specificity). CONCLUSION: Compared to cystatin C, Gas6 may be potentially a better noninvasive diagnostic biomarker for early detection of DN.

PGRMC1-dependent autophagy by hyperoside induces apoptosis and sensitizes ovarian cancer cells to cisplatin treatment.

Cisplatin treatment some times leads to chemoresistance, which is now acknowledged partially due to the inductive expression of progesterone receptor membrane component (PGRMC)1 in ovarian cancer cells. PGRMC1 enhances autophagy, activates cytochrome p450, and inveigles signaling pathways to promote cell survival and reduce the effect of drug treatments. In this study, we give first line evidence that hyperoside inhibits cell viability, triggers autophagy and apoptosis in ovarian cancer cell lines. Mechanistically, PGRMC1-dependent autophagy was utilized by hyperoside to induce apoptotic cell death. Hyperoside induced the conversion of LC3B-I to LC3B-II and the formation of autophagosomes in ovarian cancer cells. Notably, PGRMC1 colocolized with LC3B­II, and PGRMC1 overexpression enhanced hyperoside-induced autophagy and apoptosis, while PGRMC1 knockdown abrogated the action. Additionally, AKT signaling and Bcl-2 family were also involved in the hyperoside-induced autophagy and apoptosis. Importantly, in cisplatin-resistant ovarian cancer cells where PGRMC1 was overexpressed, hyperoside sensitized the cells to cisplatin treatment. Together these findings indicate hyperoside functions as a complementary therapy for ovarian cancer patients receiving platinum-based therapy.

An Antagomir to MicroRNA-106b-5p Ameliorates Cerebral Ischemia and Reperfusion Injury in Rats Via Inhibiting Apoptosis and Oxidative Stress.

We previously observed that microRNA miR-106b-5p significantly increased in serum of patients with acute ischemic stroke. The present study was to determine whether miR-106b-5p antagomir can protect against cerebral ischemia/reperfusion (I/R) injury and elucidate its underlying mechanisms. Middle cerebral artery occlusion (MCAO) was operated on male Sprague Dawley rats. MiR-106b-5p antagomir significantly decreased neurological deficit scores, infarct volumes, and neuronal injury. Furthermore, miR-106b-5p antagomir markedly reduced malondialdehyde (MDA) content, restored superoxide dismutase (SOD) activity, increased the expression of myeloid cell leukemia-1 (Mcl-1) and B cell lymphoma-2 (Bcl-2), and decreased the expression of Bax in the ischemic cortex. In PC12 cells, miR-106b-5p inhibitor increased the Mcl-1 and Bcl-2 expression, which provided protection against glutamate-induced apoptosis and oxidative damage, as evidenced by decreased lactate dehydrogenase (LDH) release, and enhanced SOD activity. Notably, luciferase reported assay proved Mcl-1 was the target gene of miR-106b-5p. In conclusion, our data indicates that the neuroprotective effects of miR-106b-5p antagomir on cerebral I/R injury are associated with its inhibition of apoptosis and oxidative stress, suggesting a potential therapeutic target for ischemic stroke.

Electroacupuncture alleviates cerebral ischemia and reperfusion injury via modulation of the ERK1/2 signaling pathway.

Electroacupuncture (EA) has anti-oxidative and anti-inflammatory actions, but whether the neuroprotective effect of EA against cerebral ischemia-reperfusion (I/R) injury involves modulation of the extracellular regulated kinase 1/2 (ERK1/2) signaling pathway is unclear. Middle cerebral artery occlusion (MCAO) was performed in Sprague-Dawley rats for 2 hours followed by reperfusion for 24 hours. A 30-minute period of EA stimulation was applied to both Baihui (DU20) and Dazhui (DU14) acupoints in each rat (10 mm EA penetration depth, continuous wave with a frequency of 3 Hz, and a current intensity of 1-3 mA) when reperfusion was initiated. EA significantly reduced infarct volume, alleviated neuronal injury, and improved neurological function in rats with MCAO. Furthermore, high mRNA expression of Bax and low mRNA expression of Bcl-2 induced by MCAO was prevented by EA. EA substantially restored total glutathione reductase (GR), glutathione (GSH) and glutathione peroxidase (GSH-Px) levels. Additionally, Nrf2 and glutamylcysteine synthetase (GCS) expression levels were markedly increased by EA. Interestingly, the neuroprotective effects of EA were attenuated when ERK1/2 activity was blocked by PD98059 (a specific MEK inhibitor). Collectively, our findings indicate that activation of the ERK1/2 signaling pathway contributes to the neuroprotective effects of EA. Our study provides a better understanding of the regulatory mechanisms underlying the therapeutic effectiveness of EA.

Pretreatments with injured microenvironmental signals altered the characteristics of human umbilical cord mesenchymal stem cells.

OBJECTIVE: Human umbilical cord mesenchymal stem cells (hUCMSCs) have renoprotective effects but the influence of the microenvironment on characteristics of hUCMSCs has not been well studied. Here, we investigate the effects of injury conditions on properties of hUCMSCs. RESULTS: hUCMSCs were treated in vitro under conditions mimicking the injury microenvironment of acute kidney injury. Cells stimulated with factor-treated medium proliferated slowly at first but quickly afterwards their morphology subsequently changed from spindle to stellate shape. Increased number of cells with strong expression of thymine-1 (Thy-1) or α-smooth muscle actin (α-SMA) was detected at 1 or 2 weeks after stimulation. Hepatocyte growth factor (HGF) level markedly increased after culture for 6 h under hypoxia condition. The expressions of HGF and insulin growth factor-1 (IGF-1) were significantly up-regulated from 0.22 ± 0.03 to 0.9 ± 0.02 and 0.07 ± 0.03 to 0.19 ± 0.01 in H/R-treated hUCMSCs respectively. Co-culture with injured renal tubular epithelial cells significantly promoted the expression of HGF (1.19 ± 0.21) and IGF-1 (0.24 ± 0.03) in hUCMSCs. CONCLUSION: The characteristics of hUCMSCs change in response to inured conditions, which may enhance the efficacy of stem cell therapy and provide novel strategies in maximizing biological and functional properties of hUCMSCs.

Brevundimonas diminuta bacteremia in a man with myelodysplastic syndromes.

Brevundimonas diminuta are ubiquitous in the environment, but are infrequently isolated from clinical samples. Here we report a case of B. diminuta bacteremia in a man with myelodysplastic syndromes (MDS) at a teaching hospital in China and review the previously reported cases. The organism was confirmed by culture and 16s rRNA sequence analysis with highly sensitivity to broad-spectrum antibiotics. Our report and other cases demonstrated that the optimal therapeutic duration for B. diminuta infections in various situations remains to be established.

Identification of circulating microRNAs as potential biomarkers for detecting acute ischemic stroke.

MicroRNAs (miRNAs) are present in serum and have the potential to serve as disease biomarkers. As such, it is important to explore the clinical value of miRNAs in serum as biomarkers for ischemic stroke (IS) and cast light on the pathogenesis of IS. In this study, we screened differentially expressed serum miRNAs from IS and normal people by miRNA microarray analysis, and validated the expression of candidate miRNAs using quantitative reverse-transcriptase polymerase chain reaction assays. Furthermore, we performed gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway analyses to disclose functional enrichment of genes predicted to be regulated by the differentially expressed miRNAs. Notably, our results revealed that 115 miRNAs were differentially expressed in IS, among which miR-32-3p, miR-106-5p, and miR-532-5p were first found to be associated with IS. In addition, GO and KEGG pathway analyses showed that genes predicted to be regulated by differentially expressed miRNAs were significantly enriched in several related biological process and pathways, including axon guidance, glioma, MAPK signaling, mammalian target of rapamycin signaling, and ErbB-signaling pathway. In conclusion, we identified the changed expression pattern of miRNAs in IS. Serum miR-32-3p, miR-106-5p, miR-1246, and miR-532-5p may serve as potential diagnostic biomarkers for IS. Our results also demonstrate a novel role for miRNAs in the pathogenesis of IS.

Fas/FasL Complex Promotes Proliferation and Migration of Brain Endothelial Cells Via FADD-FLIP-TRAF-NF-κB Pathway.

Accumulating evidence indicates that factor associated with suicide ligand (FasL) is a bidirectional regulator. At higher dosage, soluble FasL induced the apoptosis of Fas-expressing brain endothelial cells. Reduced concentration of soluble FasL (sFasL), however, promoted the proliferation and migration of brain endothelial cells. The effect of sFasL on endothelial cells proliferation was completely abolished by silencing Fas expression using siRNA. Treating brain endothelial cells with low-dose sFasL led to increased secretion of VEGF and up-regulated expression of FLIP, TRAF, and NF-κB that played a crucial role in the proliferation of endothelial cells. Our results further underscore the potential stimulating role of Fas/FasL interaction in angiogenesis.

Ultrasound-enhanced protective effect of tetramethylpyrazine against cerebral ischemia/reperfusion injury.

In traditional Chinese medicine, Ligusticum wallichii (Chuan Xiong) and its bioactive ingredient, tetramethylpyrazine (TMP), have been used to treat cardiovascular diseases and to relieve various neurological symptoms, such as those associated with ischemic injury. In the present study, we investigated whether ultrasound (US) exposure could enhance the protective effect of TMP against cerebral ischemia/reperfusion (I/R) injury. Glutamate-induced toxicity to pheochromocytoma (PC12) cells was used to model I/R injury. TMP was paired with US to examine whether this combination could alleviate glutamate-induced cytotoxicity. The administration of TMP effectively protected cells against glutamate-induced apoptosis, which could be further enhanced by US-mediated sonoporation. The anti-apoptotic effect of TMP was associated with the inhibition of oxidative stress and a change in the levels of apoptosis-related proteins, Bcl-2 and Bax. Furthermore, TMP reduced the expression of proinflammatory cytokines such as TNF-α and IL-8, which likely also contributes to its cytoprotective effects. Taken together, our findings suggest that ultrasound-enhanced TMP treatment might be a promising therapeutic strategy for ischemic stroke. Further study is required to optimize ultrasound treatment parameters.

Tetramethylpyrazine promotes the proliferation and migration of brain endothelial cells.

The aim of the present study was to investigate the role of tetramethylpyrazine (TMP), one of the alkaloids isolated from the Chinese herb Chuanxiong, on the proliferation and migration of brain endothelial cells. A different dosage of TMP was employed to stimulate the mouse microvascular cell line bEnd.3 in vitro. TMP at lower concentrations (0.25 ng/ml), however not at high concentrations (100 ng/ml) could promote the proliferation and migration of endothelial cells, which was further enhanced if combined with soluble Fas ligand (sFasL). TMP alone, or combined with sFasL, increased the autocrine signaling of vascular endothelial growth factor (VEGF) by endothelial cells and TMP improved the expression of Fas on endothelial cells, which may explain the effect of the sFasL. These results provide insight into the underlying mechanisms of the effects of TMP on stroke and other vascular diseases.

Progesterone protects ovarian cancer cells from cisplatin-induced inhibitory effects through progesterone receptor membrane component 1/2 as well as AKT signaling.

Progesterone, also known as P4 (pregn-4-ene-3, 20-dione), is a C-21 steroid hormone involved in the female menstrual cycle, pregnancy (supports gestation) and embryogenesis of humans and other species. Despite the physiological effects, P4 is also effective for the treatment of numerous pathological states, such as multiple sclerosis, rheumatoid arthritis and systemic lupus erythematosus as well as cancer. Considering the hormone microenvironment of gynecological cancers, P4 should be particularly noted in ovarian cancer. The present study demonstrated that P4 protected the ovarian cancer cell line HO-8910 from cisplatin (CDDP)-induced cell cycle arrest and restored the cell migratory capability following treatment of CDDP. Mechanistically, both progesterone receptor membrane component 1 (PGRMC1) and the progesterone receptor (PGR) were decreased in the cells treated with CDDP plus P4, while the level of progesterone receptor membrane component 2 (PGRMC2) was significantly elevated. Reversely, in the HO-8910 cells treated with CDDP alone, levels of both PGRMC1 and PGR were increased while the level of PGRMC2 was decreased. In addition to the receptor expression profile, the PI3K/AKT signaling pathway was also involved in the action of P4 in the CDDP-resistant HO-8910 cells, and a chemical inhibitor for PI3K, LY294002, significantly abolished the anti-apoptotic effect of P4. Consequently, the addition of a PI3K inhibitor to CDDP-based chemotherapy may have a more beneficial application for ovarian cancer therapy.

Association among polymorphisms in EGFR gene exons, lifestyle and risk of gastric cancer with gender differences in Chinese Han subjects.

BACKGROUND: The epidermal growth factor receptor (EGFR) gene plays a key role in tumor survival, invasion, angiogenesis, and metastatic spread. Recent studies showed that gastric cancer (GC) was associated with polymorphisms of the EGFR gene and environmental influences, such as lifestyle factors. In this study, seven known SNPs in EGFR exons were investigated in a high-risk Chinese population in Jiangsu province to test whether genetic variants of EGFR exons and lifestyle are associated with an increased risk of GC. METHODOLOGY/PRINCIPAL FINDINGS: A hospital-based case-control study was performed in Jiangsu province. The results showed that smoking, drinking and preference for salty food were significantly associated with the risk of GC. The differences of lifestyle between males and females might be as the reason of higher incidence rates in males than those in females. Seven exon SNPs were genotyped rs2227983,rs2072454,rs17337023,rs1050171,rs1140475, rs2293347, and rs28384375. It was noted that the variant rs2072454 T allele and TT genotype were significantly associated with an increased risk of GC. Interestingly, our result suggested the ACAGCA haplotype might be associated with decreased risk of GC. However, no significant association was examined between the other six SNPs and the risk of GC both in the total population and the age-matching population even with gender differences. CONCLUSIONS: Smoking, drinking and preference for salty food were significantly associated with the risk of GC in Jiangsu province with gender differences. Although only one SNP (rs2072454) was significantly associated with an increased risk of GC, combined the six EGFR exon SNPs together may be useful for predicting the risk of GC.

Functional epidermal growth factor gene polymorphisms and risk of gastric cancer.

Epidermal growth factor (EGF) is critical in cancer progression and various genotypes of the EGF gene have been reported to be correlated with susceptibility to gastric cancer; however, the results are unclear. The aim of this study was to explore the associations of functional EGF gene polymorphisms and risk of gastric cancer in a Chinese population. We genotyped seven functional single-nucleotide polymorphisms (SNPs) of the EGF gene [rs3756261, rs11568835 and rs4444903 in the promoter region; rs11568943, rs2237051 and rs11569017 in the non-synonymous exon region and rs3733625 in the 3' untranslated region (UTR)] in a hospital-based case-control study, including 387 gastric cancer cases and 392 healthy controls by polymerase chain reaction-ligation detection reaction (PCR-LDR) methods. We found that individuals carrying the AG/GG genotype of rs2237051 (Ile to Met) in the exon region had an increased risk of gastric cancer (adjusted OR=1.577; 95% CI=1.163-2.138), compared with the wild-type homozygous AA genotype. The heterozygous AG/GG genotype of rs3733625 in the 3'UTR demonstrated a protective effect (adjusted OR=0.690; 95% CI=0.501-0.951), compared with the homozygous AA genotype. In addition, the effects of the two SNPs were more evident in intestinal gastric cancer (P<0.05); however, this was not case for the diffuse type (P>0.05). These findings indicate that a change in amino acids from isoleucine to methionine of rs2237051 and rs3733625 in the 3'UTR may contribute to the etiology of intestinal gastric cancer in the Chinese population.

Enhancement effect of ultrasound-induced microbubble cavitation on branched polyethylenimine-mediated VEGF(165) transfection with varied N/P ratio.

One isoform of the vascular endothelial growth factor, VEGF(165), has been reported to be a dominant mediator and regulator of angiogenic process, which plays an important role in treating cardiovascular diseases and chronically ischemic wounds. Branched polyethylenimine (bPEI) has been widely used as a non-viral delivery vector for gene therapy. Although bPEI-mediated DNA transfection efficiency can be raised by increasing the PEI nitrogen:DNA phosphate (N/P) ratio, cytotoxicity increases as well. In this study, the enhancement effect of microbubble inertial cavitation (IC) on bPEI-mediated VEGF(165) transfection was investigated, in an effort to optimize transfection efficiency using low N/P ratios. HEK 293T cells, mixed with bPEI:VEGF(165) complexes, were exposed to 1-MHz ultrasound pulses. The results show that: (1) IC activity induced by microbubble destruction can be quantified as an IC "dose" (ICD) and will increase with increasing acoustic driving pressure; (2) larger sonoporation pores can be generated by increasing ICD; (3) the transfection efficiency can be enhanced by increasing ICD until reaching a saturation level; and (4) microbubble IC activity has less cytotoxicity than bPEI, although a combinatorial effect of microbubble IC activity and bPEI could be observed on cell viability. The results suggest that, with appropriate ultrasound parameters, it is possible to optimize bPEI-mediated VEGF transfection efficiency using relatively low N/P ratios by employing ultrasound-induced microbubble inertial cavitation.