Effects of SARS-CoV-2 infection on embryological outcomes in assisted reproductive technology during the Omicron epidemic.

BACKGROUND: The influence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection on assisted reproductive technology (ART) has received increasing attention. It has been reported that the SARS-CoV-2 RiboNucleic Acid (RNA) cannot be detected in follicular fluid and granulosa cells. However, the detection rate of SARS-CoV-2 RNA in immature oocytes and blastocysts has still unknown. Moreover, the effect of SARS-CoV-2 infection on embryological outcomes in ART during the Omicron epidemic is limited. METHODS: A prospective study was performed to explore the detection rate of viral RNA in biological specimens from patients who tested positive for SARS-CoV-2 RNA and the effects of SARS-CoV-2 infection on embryological outcomes. A total of 211 patients underwent transvaginal oocyte retrieval at the Third Affiliated Hospital of Guangzhou Medical University between December 13, 2022 and December 30, 2022. Prior to transvaginal oocyte retrieval, 61 individuals tested positive for SARS-CoV-2 RNA within 24 h. Follicular fluid was preserved during oocyte retrieval. Granular cells were collected after degranulation (Intracytoplasmic sperm injection only). Immature oocytes were collected at the end of the ICSI. Unavailable blastocysts were collected on day 6 (D6). The TIANLONG SARS-CoV-2 RT-PCR-Kit was used to detect SARS-CoV-2 RNA in all samples. The COVID-19 and Non COVID-19 groups were contrasted in the following areas: fertilization rate, 2PN rate, Day 3 (D3) available embryos rate, D3 good-quality embryos rate, blastocyst formation rate, good-quality blastocyst formation rate. RESULTS: All samples were negative except for an immature oocytes sample that was positive for SARS-CoV-2 viral RNA with a detection rate of 6.67%. Whether in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI), the rate of fertilization, 2PN, D3 available embryos, D3 good-quality embryos, blastocyst formation, good-quality blastocyst formation was not significantly negative different between the COVID-19 and the Non COVID-19 groups. Our findings were validated by an overview of the embryological outcome from the cycles before SARS- Cov-2 infection from the same patient. CONCLUSIONS: Except for immature oocytes, none of the follicular fluid, granulosa cells, or blastocysts samples contained viral RNA. In addition, SARS-CoV-2 infection had no detrimental effects on the embryological outcomes of ART.

Kallistatin prevents ovarian hyperstimulation syndrome by regulating vascular leakage.

Angiogenesis and increased permeability are essential pathological basis for the development of ovarian hyperstimulation syndrome (OHSS). Kallistatin (KS) is an endogenous anti-inflammatory and anti-angiogenic factor that participates in a variety of diseases, but its role in OHSS remains unknown. In this study, treating a human ovarian granulosa-like tumour cell line KGN and human primary granulosa cells (PGCs) with human chorionic gonadotropin (hCG) reduced the expression of KS, but increased the expression of VEGF. Furthermore, we found that KS could attenuate the protein level of VEGF in both KGN cells and human PGCs. More interestingly, we observed that exogenous supplementation of KS significantly inhibited a series of signs of OHSS in mice, including weight gain, ovarian enlargement, increased vascular permeability and up-regulation of VEGF expression. In addition, KS was proved to be safe on mice ovulation, progression of normal pregnancy and fetus development. Collectively, these findings demonstrated that KS treatment prevented OHSS, at least partially, through down-regulating VEGF expression. For the first time, these results highlight the potential preventive value of KS in OHSS.

Paper mill sludge-based carbon quantum dots as a specifically ratiometric fluorescent probe for the sensitive and selective detection of coptisine.

Coptisine (COP), one of the bioactive components in Rhizoma Coptidis, has many pharmacological effects. Meanwhile, the determination of COP is essential in pharmacological and clinical applications. Herein, we prepared carbon quantum dots (CQDs) by one-step oil-thermal method using paper mill sludge (PMS) as precursor, and developed a ratiometric fluorescence method for the determination of COP. The structural and optical properties of PMS-CQDs were evaluated through high-resolution transmission electron microscopy (HRTEM), Fourier-transform infrared (FTIR) spectroscopy, X-ray photoelectron spectroscopy (XPS), X-ray powder diffraction (XRD), ultraviolet-visible (UV-vis), fluorescence, zeta potential and fluorescence lifetime experiments. Fluorescence intensity ratio at 550 nm and 425 nm (I550 /I425 ) was recorded as an index for quantitative detection of COP. The detection concentration of COP ranges from 0.1 to 50 µM in good linear correlation (R2  = 0.9974) with a limit of detection of 0.028 µM (3σ/k). The quenching mechanism was deduced to be inner filter effect and static quenching. The ratiometric fluorescent probe showed impressive selectivity and sensitivity towards COP, and was successfully applied to the detection of COP in human urine with expected recoveries (95.22-111.00%) and relative standard deviations (0.46-2.95%), indicating that our developed method has a great application prospect in actual sample detection.

Deoxyribonuclease 1-like 3 may be a potential prognostic biomarker associated with immune infiltration in colon cancer.

Colon adenocarcinoma (COAD) is a common cancer of the digestive system. It's high morbidity and mortality make it one of the leading causes of cancer deaths. In this study, we studied the microenvironment of colon cancer to find new diagnostic markers and immunotherapy targets for colon cancer. Tumor purity of colon cancer samples in TCGA database were obtained by ESTIMATE algorithm. Then, we analyzed the association of Immune, Stromal, and Estimate scores with tumor prognosis and clinicopathological features. By comparing the gene expression profiles between tumor and normal samples, the high and low immune score groups, 117 intersecting differentially expressed genes (DEGs) were obtained. The function, molecular pathway, and prognostic value of these 117 DEGs pointed toward the importance of deoxyribonuclease 1-like 3 (DNASE1L3). Validation results from multiple databases showed low expression of DNASE1L3 in colon cancer. A single GSEA and correlation analysis of immune cells indicated that DNASE1L3 was closely related to immunity. The low expression of DNASE1L3 in colon cancer samples was measured with qRT-PCR. The scratch and cell proliferation experiments suggested that DNASE1L3 may affect cell migration. Therefore, we concluded that DNASE1L3 might be a biomarker associated with prognosis and immune infiltration in colon cancer.

Effect of the time for embryo transfer from oocyte retrieval on clinical outcomes in freeze-all cycles: a retrospective cohort study.

PURPOSE: To identify the optimal time for the frozen embryo transfer (FET) after oocyte retrieval in freeze-all cycles. METHODS: A retrospective analysis of 977 patients was performed. Implantation, clinical pregnancy and live birth rates were analyzed. RESULTS: No significant difference was found between the first FET performed in the first menstrual cycle group and performed within the subsequent menstrual cycle group in terms of implantation, pregnancy and live birth rates. To rule out the effect of endometrial thickness, a hierarchical analysis was performed. There were no differences between groups for pregnancy, multiple pregnancy and live birth rates for all ranges of endometrial thickness. CONCLUSIONS: The first FET should be performed once the endometrial thickness has been prepared well rather than delaying until the subsequent menstrual cycles.

Activation of AMPK/MnSOD signaling mediates anti-apoptotic effect of hepatitis B virus in hepatoma cells.

AIM: To investigate the anti-apoptotic capability of the hepatitis B virus (HBV) in the HepG2 hepatoma cell line and the underlying mechanisms. METHODS: Cell viability and apoptosis were measured by MTT assay and flow cytometry, respectively. Targeted knockdown of manganese superoxide dismutase (MnSOD), AMP-activated protein kinase (AMPK) and hepatitis B virus X protein (HBx) genes as well as AMPK agonist AICAR and antagonist compound C were employed to determine the correlations of expression of these genes. RESULTS: HBV markedly protected the hepatoma cells from growth suppression and cell death in the condition of serum deprivation. A decrease of superoxide anion production accompanied with an increase of MnSOD expression and activity was found in HepG2.215 cells. Moreover, AMPK activation contributed to the up-regulation of MnSOD. HBx protein was identified to induce the expression of AMPK and MnSOD. CONCLUSION: Our results suggest that HBV suppresses mitochondrial superoxide level and exerts an anti-apoptotic effect by activating AMPK/MnSOD signaling pathway, which may provide a novel pharmacological strategy to prevent HCC.

A novel promoter mutation (HBB: c.-75G>T) was identified as a cause of ß(+)-thalassemia.

We report a novel ß-globin gene promoter mutation in a Chinese family identified using fluorescence resolution melting curve analysis and gene sequencing. The proband, who showed the phenotype of ß-thalassemia intermedia (ß-TI), was found to be a compound heterozygote for the novel mutation -25 (G>T) (HBB: c.-75G>T) and a codon 17 (HBB: c.52A>T) mutation. Moreover, conservation analysis using phyloP and phastCons indicated that the mutated base in the proband was conserved. This novel point mutation on the ß-globin gene is in close proximity to the conserved ATAA sequence located at position -25 relative to the mRNA Cap site. We performed a further comparative analysis of the clinical phenotypes and hematological parameters in this pedigree and found that the father was a carrier of the novel point mutation and showed low levels of hemoglobin (Hb), mean corpuscular volume (MCV) and mean corpuscular Hb (MCH). Thus, the available evidence suggests that this novel mutation, -25, results in ß(+)-thalassemia (ß(+)-thal).

Plasminogen kringle 5 induces endothelial cell apoptosis by triggering a voltage-dependent anion channel 1 (VDAC1) positive feedback loop.

Human plasminogen kringle 5 (K5) is known to display its potent anti-angiogenesis effect through inducing endothelial cell (EC) apoptosis, and the voltage-dependent anion channel 1 (VDAC1) has been identified as a receptor of K5. However, the exact role and underlying mechanisms of VDAC1 in K5-induced EC apoptosis remain elusive. In the current study, we showed that K5 increased the protein level of VDAC1, which initiated the mitochondrial apoptosis pathway of ECs. Our findings also showed that K5 inhibited the ubiquitin-dependent degradation of VDAC1 by promoting the phosphorylation of VDAC1, possibly at Ser-12 and Thr-107. The phosphorylated VDAC1 was attenuated by the AKT agonist, glycogen synthase kinase (GSK) 3ß inhibitor, and siRNA, suggesting that K5 increased VDAC1 phosphorylation via the AKT-GSK3ß pathway. Furthermore, K5 promoted cell surface translocation of VDAC1, and binding between K5 and VDAC1 was observed on the plasma membrane. HKI protein blocked the impact of K5 on the AKT-GSK3ß pathway by competitively inhibiting the interaction of K5 and cell surface VDAC1. Moreover, K5-induced EC apoptosis was suppressed by VDAC1 antibody. These data show for the first time that K5-induced EC apoptosis is mediated by the positive feedback loop of "VDAC1-AKT-GSK3ß-VDAC1," which may provide new perspectives on the mechanisms of K5-induced apoptosis.

Proapoptotic PEDF functional peptides inhibit prostate tumor growth--a mechanistic study.

PEDF inhibits tumor growth via anti-angiogenic activity; however, the direct effect of PEDF on prostate carcinoma and its functional epitope as well as the underlying mechanism regulating the pathway from extracellular receptors to nuclear transcription factors has not been fully elucidated. This study investigates the ability and mechanism by which the functional PEDF peptides PEDF34 and PEDF44 suppress tumor growth. The results showed that death receptor pathway was activated by PEDF34 through up-regulation of FasL and activation of caspase-8 in both xenograft tumor tissues and PC-3 cells. FasL knockdown by siRNA or JNK-p inhibition attenuated apoptosis induced by PEDF34. NF-κB and PPARγ are crucial transcription factors for FasL expression. PEDF34 up-regulated PPARγ but did not affect NF-κB. PEDF34-induced up-regulation of FasL was abolished by siRNA-mediated PPARγ knockdown or using PPARγ inhibitor GW9662, whereas inhibition of NF-κB by the inhibitor PDTC or by siRNA had no effect. Furthermore, activation of JNK is necessary for PEDF34-induced up-regulation of FasL. PEDF34 has stronger hydropathicity and more interactions with laminin receptor than PEDF44. Blocking the laminin receptor abolished the up-regulation of FasL and PPARγ by PEDF34. Moreover, PEDF34 uses a similar mechanism to induce apoptosis in both endothelial and cancer cells. This study provides evidence that PEDF34, not PEDF44, serves as the proapoptotic epitope and exerts proapoptotic activity in both cancer and endothelial cells through activation of the extrinsic death receptor pathway. The dual anti-tumor and anti-angiogenic activities of PEDF34 suggest that it may be a promising agent for the treatment of prostate cancer.

Pigment epithelial-derived factor (PEDF)-triggered lung cancer cell apoptosis relies on p53 protein-driven Fas ligand (Fas-L) up-regulation and Fas protein cell surface translocation.

Pigment epithelium-derived factor (PEDF), a potent antiangiogenesis agent, has recently attracted attention for targeting tumor cells in several types of tumors. However, less is known about the apoptosis-inducing effect of PEDF on human lung cancer cells and the underlying molecular events. Here we report that PEDF has a growth-suppressive and proapoptotic effect on lung cancer xenografts. Accordingly, in vitro, PEDF apparently induced apoptosis in A549 and Calu-3 cells, predominantly via the Fas-L/Fas death signaling pathway. Interestingly, A549 and Calu-3 cells are insensitive to the Fas-L/Fas apoptosis pathway because of the low level of cell surface Fas. Our results revealed that, in addition to the enhancement of Fas-L expression, PEDF increased the sensitivity of A549 and Calu-3 cells to Fas-L-mediated apoptosis by triggering the translocation of Fas protein to the plasma membrane in a p53- and FAP-1-dependent manner. Similarly, the up-regulation of Fas-L by PEDF was also mediated by p53. Furthermore, peroxisome proliferator-activated receptor γ was determined to be the upstream regulator of p53. Together, these findings uncover a novel mechanism of tumor cell apoptosis induced by PEDF and provide a potential therapeutic strategy for tumors that are insensitive to Fas-L/Fas-dependent apoptosis because of a low level of cell surface Fas.

High metastaticgastric and breast cancer cells consume oleic acid in an AMPK dependent manner.

Gastric cancer and breast cancer have a clear tendency toward metastasis and invasion to the microenvironment predominantly composed of adipocytes. Oleic acid is an abundant monounsaturated fatty acid that releases from adipocytes and impinges on different energy metabolism responses. The effect and underlying mechanisms of oleic acid on highly metastatic cancer cells are not completely understood. We reported that AMP-activated protein kinase (AMPK) was obviously activated in highly aggressive carcinoma cell lines treated by oleic acid, including gastric carcinoma HGC-27 and breast carcinoma MDA-MB-231 cell lines. AMPK enhanced the rates of fatty acid oxidation and ATP production and thus significantly promoted cancer growth and migration under serum deprivation. Inactivation of AMPK attenuated these activities of oleic acid. Oleic acid inhibited cancer cell growth and survival in low metastatic carcinoma cells, such as gastric carcinoma SGC7901 and breast carcinoma MCF-7 cell lines. Pharmacological activation of AMPK rescued the cell viability by maintained ATP levels by increasing fatty acid ß-oxidation. These results indicate that highly metastatic carcinoma cells could consume oleic acid to maintain malignancy in an AMPK-dependent manner. Our findings demonstrate the important contribution of fatty acid oxidation to cancer cell function.

Dual regulation of adipose triglyceride lipase by pigment epithelium-derived factor: a novel mechanistic insight into progressive obesity.

Both elevated plasma free fatty acids (FFA) and accumulating triglyceride in adipose tissue are observed in the process of obesity and insulin resistance. This contradictory phenomenon and its underlying mechanisms have not been thoroughly elucidated. Recent studies have demonstrated that pigment epithelium-derived factor (PEDF) contributes to elevated plasma FFA and insulin resistance in obese mice via the activation of adipose triglyceride lipase (ATGL). However, we found that PEDF downregulated adipose ATGL protein expression despite of enhancing lipolysis. Plasma PEDF and FFA were increased in associated with a progressive high-fat-diet, and those outcomes were also accompanied by fat accumulation and a reduction in adipose ATGL. Exogenous PEDF injection downregulated adipose ATGL protein expression and elevated plasma FFA, while endogenous PEDF neutralization significantly rescued the adipose ATGL reduction and also reduced plasma FFA in obese mice. PEDF reduced ATGL protein expression in a time- and dose-dependent manner in differentiated 3T3-L1 cells. Small interfering RNA-mediated PEDF knockdown and antibody-mediated PEDF blockage increased endogenous ATGL expression, and PEDF overexpression downregulated ATGL. PEDF resulted in a decreased half-life of ATGL and regulated ATGL degradation via ubiquitin-dependent proteasomal degradation pathway. PEDF stimulated lipolysis via ATGL using ATGL inhibitor bromoenol lactone, and PEDF also downregulated G0/G1 switch gene 2 (G0S2) expression, which is an endogenous inhibitor of ATGL activation. Overall, PEDF attenuated ATGL protein accumulation via proteasome-mediated degradation in adipocytes, and PEDF also promoted lipolysis by activating ATGL. Elevated PEDF may contribute to progressive obesity and insulin resistance via its dual regulation of ATGL.

Intracellular pigment epithelium-derived factor contributes to triglyceride degradation.

Pigment epithelium-derived factor is well known as a secreted glycoprotein with multiple functions, such as anti-angiogenic, neuroprotective and anti-tumor activities. However, its intracellular role remains unknown. The present study was performed to demonstrate the intracellular function of pigment epithelium-derived factor on triglyceride degradation. Hepatic pigment epithelium-derived factor levels increased at the early stage and subsequently decreased after 16 weeks in high-fat-diet-fed mice compared to those in chow-fed mice. Similarly, oleic acid led to long-term downregulation of pigment epithelium-derived factor in HepG2 cells. Endogenous pigment epithelium-derived factor was an intracellular protein with cytoplasmic distribution in hepatocytes by immunostaining. Exogenous FITC-labeled pigment epithelium-derived factor could be absorbed into hepatocytes. Both signal peptide deletion and full-length pigment epithelium-derived factor transfection HeLa cells and hepatocytes promoted triglyceride degradation. Intracellular pigment epithelium-derived factor co-immunoprecipitated with adipose triglyceride lipase and promoted triglyceride degradation in an adipose triglyceride lipase-dependent manner. Additionally, pigment epithelium-derived factor bound to the C-terminal of adipose triglyceride lipase (aa268-504) and adipose triglyceride lipase-G0/G1 switch gene-2 complex simultaneously, which facilitated adipose triglyceride lipase-G0/G1 switch gene-2 translocation onto lipid droplet using bimolecular fluorescence complementation assay. Moreover, knockdown of endogenous pigment epithelium-derived factor in hepatocytes diminished triglyceride degradation. Taken together, these results indicate that hepatic pigment epithelium-derived factor was decreased in obese mice accompanied with hepatic steatosis. Intracellular pigment epithelium-derived factor binds to and facilitates adipose triglyceride lipase translocation onto lipid droplet, which promotes triglyceride degradation. These findings suggest that a decreased level of hepatic pigment epithelium-derived factor may contribute to hepatic steatosis in obesity.

The artemisinin derivative artesunate inhibits corneal neovascularization by inducing ROS-dependent apoptosis in vascular endothelial cells.

PURPOSE: Without therapeutic intervention, corneal neovascularization rapidly compromises visual acuity, and is a leading cause of blindness. Artesunate was reported to inhibit angiogenesis in tumors, although, the effects of artesunate on nontumor angiogenesis have not been investigated. This study was designed to investigate the effect of artesunate on corneal neovascularization and delineate its underlying mechanism of action. METHODS: Rats with alkali-burned corneas were treated with artesunate for 11 days. Corneal neovascularization was evaluated by measuring the length and area of corneal vasculature in the rats. Apoptotic cells were stained with AnnexinV and propidine iodide (PI), and measured with flow cytometry analysis. Apoptosis-related and p38 mitogen-activated protein kinases (p38MAPK) signaling were evaluated by Western blot analysis. RESULTS: Artesunate significantly inhibited corneal neovascularization and inflammation via specifically inducing apoptosis of vascular endothelial cells. In vascular endothelial cells, artesunate increased the Bax/Bcl-2 ratio, reduced mitochondrial membrane potential, stimulated release of cytochrome C, and cleavage of caspase 9 and 3, suggesting that the mitochondrial apoptotic pathway was involved. Artesunate activated p38MAPK, and specific p38MAPK inhibitors suppressed artesunate-induced apoptosis in endothelial cells. Reactive oxygen species (ROS) levels were increased by artesunate. N-acetyl-L-cysteine blocked p38MAPK activation and protected endothelial cells from artesunate-induced apoptosis. Ferrous salt increased ROS levels and elevated the cytotoxic effect of artesunate on endothelial cells, while the iron chelating agent deferoxamine decreased ROS levels and artesunate-induced apoptosis. Artesunate had no effect on expression of Fas, Fas Ligand, or caspase 8 cleavage. CONCLUSIONS: These results suggest that artesunate induces apoptosis of endothelial cells via an iron/ROS-dependent p38MAPK-mitochondrial pathway.

SERPINA3K induces apoptosis in human colorectal cancer cells via activating the Fas/FasL/caspase-8 signaling pathway.

SERPINA3K, also known as kallikrein-binding protein (KBP), is a serine proteinase inhibitor with anti-inflammatory and anti-angiogenic activities. Our previous studies showed that SERPINA3K inhibited proliferation in a dose-dependent manner and induced apoptosis of endothelial cells but had no influence on SGC-7901 gastric carcinoma cells or HepG2 hepatocarcinoma cells. However, it is unknown whether SERPINA3K has a direct impact on other carcinoma cells and which mechanisms are involved. In this study, we report for the first time that SERPINA3K not only decreased cell viability but also induced apoptosis in the colorectal carcinoma cell lines SW480 and HT-29. SERPINA3K-induced apoptosis of SW480 and HT-29 was rescued by interference with Fas ligand (FasL) small hairpin RNA. Moreover, SERPINA3K increased the expression of FasL and activated caspase-8. Peroxisome proliferator-activated receptor γ (PPARγ), a transcription factor of FasL, was also upregulated by SERPINA3K in a dose-dependent manner. The upregulation effect of FasL induced by SERPINA3K was reversed after interference with PPARγ small interfering RNA. These results demonstrated that SERPINA3K-induced SW480 and HT-29 cell apoptosis was mediated by the PPARγ/Fas/FasL signaling pathway. Therefore, our study provides additional insight into the direct anti-tumor function by inducing tumor cell apoptosis of SERPINA3K in colorectal tumors.

Acidic/neutral amino acid residues substitution in NH2 terminal of plasminogen kringle 5 exerts enhanced effects on corneal neovascularization.

PURPOSE: Recent results showed that plasminogen kringle 5 (K5) has improved inhibitory effect on human umbilical vein endothelial cells (HUVECs) viability when 5 acidic amino acids in NH2 terminal outside kringle domain were replaced by 5 serine residues (mutant K5, mK5). This study was designed to identify the enhanced antiangiogenic activity of mK5 in corneal neovascularization (CNV). METHODS: Alkali burn-induced CNV was induced and treated with K5 and mK5 for 11 days. CNV and inflammation were evaluated by the CNV area and the inflammatory index, respectively. At the end of treatment, the corneas were removed for terminal deoxynucleotidyl transferase dUTP nick end labeling detection and immunohistochemistry. The effects of mK5 and K5 on HUVECs apoptosis were tested by MTT, BrdU, and flow cytometry. The expression levels of pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor (VEGF) were detected by Western blot. RESULTS: In a rat model of CNV induced by alkali, topical treatment with mK5 significantly decreased the neovascular area and inflammation compared with the wild-type K5-treated group. Meanwhile, mK5 and K5 specifically inhibited the HUVECs proliferation and induced vascular endothelial cell apoptosis in vitro and in vivo, and mK5 exerted higher apoptosis induction. Toward the mechanism of action, both mK5 and K5 significantly upregulated the expression of PEDF and mildly downregulated the expression of VEGF. The elevation of PEDF/VEGF ratio induced by mK5 was higher than that by K5. CONCLUSIONS: These findings suggest that mK5 has more effective therapeutic potential in CNV than wild-type K5.

Kallikrein-binding protein inhibits LPS-induced TNF-α by upregulating SOCS3 expression.

Kallikrein-binding protein (KBP) was previously identified as a serpin family member with specific inhibitory effect on tissue kallikrein and angiogenesis, while there is little knowledge about the effects on inflammation. The aim of this study is to investigate whether KBP can suppress LPS-induced inflammatory process. Our results showed that both recombinant KBP and KBP overexpression inhibited LPS-stimulated TNF-α transcription and translation in macrophage cell line RAW264.7 and primary macrophages. Furthermore, KBP treatment protected mice from endotoxin shock and repressed serum TNF-α production, increasing survival rate of mice from 10% to 50% when compared to LPS alone. Moreover, qPCR and Western blot analysis demonstrated that both suppressor of cytokine signaling 3 (SOCS3) transcription and translation were induced by KBP treatment in the present of LPS. RNA interference assay and luciferase assay showed that SOCS3 was responsible for the down-regulation of TNF-α by KBP, rather than NF-κB subunit p65 and ß-catenin. Therefore, we demonstrated that KBP suppressed LPS-induced TNF-α production via upregulating SOCS3 expression. These results present the protective effects of KBP on LPS-induced inflammation and provide novel information for the anti-inflammation mechanism.

Pigment epithelium-derived factor gene loaded in cRGD-PEG-PEI suppresses colorectal cancer growth by targeting endothelial cells.

Pigment epithelium-derived factor (PEDF) recombinant protein has been investigated in many kinds of solid tumors due to its potent antiangiogenic activity. However, the complexity of protein purification, instability of recombinant protein and requirement of repeated injections are obstacles for the recombinant PEDF therapy for solid tumors. We successfully synthesized polyethyleneglycol-polyetherimide (PEG-PEI) and cRGD-PEG-PEI which was coupled with a cyclic RGD peptide, a special ligand for integrin αvß3 receptor, as the vehicle for PEDF gene therapy in this study. In vitro, the competitive binding assay showed that cRGD contributed to the enhanced gene transfection efficiency of PEG-PEI in human umbilical vein endothelial cells (HUVECs). PEDF gene delivered by cRGD-PEG-PEI apparently suppressed growth of tumor with a 67.4% reduction and decreased microvessel density in nude mice bearing SW620 human colorectal xenografts. Accordingly, SW620 tumors from cRGD-PEG-PEI/PEDF-pcDNA3.1 (+)-treated mice expressed more PEDF than that of the control groups. Our study demonstrated that cRGD-PEG-PEI transported the PEDF gene into endothelia cells more efficiently than PEG-PEI, resulting in more effective inhibitory effects on tumor growth by anti-angiogenesis. Therefore, for the first time, we have explored an effective non-viral vehicle for PEDF gene therapy by targeting endothelial cells.

Plasminogen K5 activates mitochondrial apoptosis pathway in endothelial cells by regulating Bak and Bcl-x(L) subcellular distribution.

Plasminogen Kringle 5(K5) is a proteolytic fragment of plasminogen, which displays potent anti-angiogenic activities. K5 has been shown to induce apoptosis in proliferating endothelial cells; however the exact mechanism has not been well explored. The present study was designed to elucidate the possible molecular mechanism of K5-induced endothelial cell apoptosis. Our results showed that K5 inhibited basic fibroblast growth factors activated in human umbilical vein endothelial cells (HUVECs), indicating proliferation in a dose-dependent manner and induced endothelial cell death via apoptosis. K5 exposure activated caspase 7, 8 and 9. These results suggested that both the intrinsic mitochondrial apoptosis pathway and extrinsic pathway might be involved in K5-induced apoptosis. K5 reduced mitochondrial membrane potential (MMP) of HUVECs, demonstrating mitochondrial depolarization in HUVECs. K5 increased the ratio of Bak to Bcl-x(L) on mitochondria, decreased the ratio in cytosol, and had no effect on the total amounts of these proteins. K5 also did not effect on Bax/Bcl-2 distribution. K5 increased the ratio of Bak to Bcl-x(L) on mitochondrial that resulted in mitochondrial depolarization, cytochrome c release and consequently the cleavage of caspase 9. These results suggested that K5 induces endothelial cell apoptosis at least in part via activating mitochondrial apoptosis pathway. The regulation of K5 on Bak and Bcl-x(L) distribution may play an important role in endothelial cell apoptosis. These results provide further insight into the anti-angiogenesis roles of K5 in angiogenesis-related ocular diseases and solid tumors.