Vascular smooth muscle-specific LRRC8A knockout ameliorates angiotensin II-induced cerebrovascular remodeling by inhibiting the WNK1/FOXO3a/MMP signaling pathway.

Hypertensive cerebrovascular remodeling involves the enlargement of vascular smooth muscle cells (VSMCs), which activates volume-regulated Cl- channels (VRCCs). The leucine-rich repeat-containing family 8 A (LRRC8A) has been shown to be the molecular identity of VRCCs. However, its role in vascular remodeling during hypertension is unclear. In this study, we used vascular smooth muscle-specific LRRC8A knockout (CKO) mice and an angiotensin II (Ang II)-induced hypertension model. The results showed that cerebrovascular remodeling during hypertension was ameliorated in CKO mice, and extracellular matrix (ECM) deposition was reduced. Based on the RNA-sequencing analysis of aortic tissues, the level of matrix metalloproteinases (MMPs), such as MMP-9 and MMP-14, were reduced in CKO mice with hypertension, which was further verified in vivo by qPCR and immunofluorescence analysis. Knockdown of LRRC8A in VSMCs inhibited the Ang II-induced upregulation of collagen I, fibronectin, and matrix metalloproteinases (MMPs), and overexpression of LRRC8A had the opposite effect. Further experiments revealed an interaction between with-no-lysine (K)-1 (WNK1), which is a "Cl--sensitive kinase", and Forkhead transcription factor O3a (FOXO3a), which is a transcription factor that regulates MMP expression. Ang II induced the phosphorylation of WNK1 and downstream FOXO3a, which then increased the expression of MMP-2 and MMP-9. This process was inhibited or potentiated when LRRC8A was knocked down or overexpressed, respectively. Overall, these results demonstrate that LRRC8A knockout in vascular smooth muscle protects against cerebrovascular remodeling during hypertension by reducing ECM deposition and inhibiting the WNK1/FOXO3a/MMP signaling pathway, demonstrating that LRRC8A is a potential therapeutic target for vascular remodeling-associated diseases such as stroke.

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.

Elevated peripheral blood lymphocyte-to-monocyte ratio predicts a favorable prognosis in the patients with metastatic nasopharyngeal carcinoma.

INTRODUCTION: Patients with metastatic nasopharyngeal carcinoma (NPC) have variable survival outcomes. We have previously shown that an elevated peripheral blood lymphocyte-to-monocyte ratio (LMR) is associated with an increased metastatic risk in patients with primary NPC. The present study aimed to investigate the prognostic value of pretreatment LMR in a large cohort of metastatic NPC patients. METHODS: Clinical data of 672 patients with metastatic NPC diagnosed between January 2003 and December 2009 were analyzed. The peripheral lymphocyte and monocyte counts were retrieved, and LMR was calculated. Receiver operating characteristic (ROC) curve analysis and univariate and multivariate COX proportional hazards analyses were performed to evaluate the association of LMR with overall survival (OS). RESULTS: Univariate analysis revealed that an elevated absolute lymphocyte count (≥1.390×10(9)/L) and LMR (≥2.475) as well as a decreased monocyte count (<0.665×10(9)/L) were significantly associated with prolonged OS. Multivariate Cox proportional hazard analysis showed that LMR (hazard ratio [HR]=0.50, 95% confidence interval [CI]=0.41-0.60, P<0.001), absolute lymphocyte count (HR=0.77, 95% CI=0.64-0.93, P=0.007), and monocyte count (HR=1.98, 95% CI=1.63-2.41, P<0.001) were independent prognostic factors. By stratification analyses, only LMR remained a significant predictor of prognosis. CONCLUSION: We identified pretreatment LMR as an independent prognostic factor for patients with metastatic NPC. Independent validation of our findings is needed.

Antifatigue Effects of Ethanol Extracts and Polysaccharides Isolated from Abelmoschus esculentus.

BACKGROUND: The aim of this study is to determine the antifatigue active fraction from Abelmoschus esculentus. The in vivo antifatigue effects of ethanol extracts and polysaccharides from A. esculentus fruit have been determined. The polysaccharides of A. esculentus were determined as the best effective fractions of antifatigue effects. MATERIALS AND METHODS: About 360 Kunming male mice were randomly divided into nine subgroups: normal control subgroup, model subgroup, positive subgroup and the ethanol extracts of A. esculentus with high dose (3.2 g/kg) subgroup, medium dose (1.6 g/kg) subgroup and low dose (0.8 g/kg) subgroup, the polysaccharides of high dose (3.2 g/kg) subgroup, medium dose (1.6 g/kg) subgroup, and the low dose (0.8 g/kg) subgroup. The antifatigue effects of ethanol extracts and polysaccharides form A. esculentus were measured by comparing body weight, food intake, swimming time, liver glycogen, serum urea, blood lactic acid as well as visceral parameter in mice. RESULTS: Compared with the model subgroup, other subgroups significantly prolonged swimming time, and high dose polysaccharides administration was the most effective (P < 0.01). High dose polysaccharides significantly increased liver glycogen, serum lactic acid, and serum urea (P < 0.01) in mice. In contrast with model group, the high dose polysaccharides administration could also significantly elevated the parameters of testicles and epididymis (P < 0.01). The study established that the ethanol extracts and polysaccharides of A. esculentus both have antifatigue effects. CONCLUSIONS: The results demonstrated that both the ethanol extracts and polysaccharides of A. esculentus have antifatigue effects. The high dosage polysaccharides have significant antifatigue properties. The results will provide the basis for further development and utilization of this plant. SUMMARY: The high dosage polysaccharides have restoration ability on kidney yang deficiency mice.The high dosage polysaccharides have significant effects of relieving body fatigue of mice.The polysaccharide of Abelmoschus esculentus showed better antifatigue effects than the ethanol extracts. Abbreviations used: A. esculentus: Abelmoschus esculentus, BUN: Blood urine nitrogen, LD: Lactic Acid dehydrogenase, AE: Abelmoschus esculentus ethanol extracts, AP: Abelmoschus esculentus polysaccharides, LAC: Lactic acid content.

[Variants of exon 4 and its flanking region of LPL Gene in patients with hyperlipidemia].

To elucidate abnormalities of LPL gene in hyperlipidemia in the Chinese population in Guangdong,genomic DNA was extracted from leukocyte of 258 patients with primary hyperlipidemia.A segment of LPL gene including exon 4 and its flanking sequences was analyzed by PCR-SSCP. The PCR products with abnormal SSCP pattern were cloned and sequenced. As a C-->T transition mutation at-6 bp of intron 3 was found in two Chinese with hyperlipidemia and the mutation was not found in 252 normolipidemic controls,the C-->T transition in intron 3 may be related to hyperlipedemia.

[High-dose glucose induces human retinal endothelial cell apoptosis].

OBJECTIVE: To examine the direct effect of high glucose levels on primary cultured human retinal capillary endothelial cells (HRCEC). METHODS: HRCECs were isolated from donated eyes and cultured for 6 days in the media containing 5 or 25 mmol/L glucose. The cell viability was determined by trypan blue exclusion assay and cell cycle analyzed by flow cytometry, with the cell apoptosis assayed by TUNEL method. RESULTS: The cell viability was significantly decreased after exposure to 25 mmol/L glucose, and the number of apoptotic cells determined by flow cytometry and TUNEL was significantly increased in response to high-dose glucose treatment. CONCLUSION: High-dose glucose induces apoptosis in HRCEC, which may contribute to the development of diabetic retinopathy.

[A deletion mutant of plasminogen kringle 5 inhibits retinal capillary endothelial cell proliferation].

OBJECTIVE: To obtain purified deletion mutant of plasminogen kringle 5 (K5) using gene mutation and genetic recombination methods and assess its anti-angiogenic activity in vitro. METHODS: A deletion mutant of K5 was obtained by deleting 15 amino acids from K5 while retaining all the 3 disulfide bonds. This K5 mutant (Mut1) was expressed in E. coli and affinity purified. The inhibition effect of K5 Mut1 on primary retinal capillary endothelial cells and pericytes from the same origin was assessed by MTT assay. RESULTS: The K5 Mut1 inhibited the proliferation of primary retinal capillary endothelial cells in a concentration-dependent manner, with an apparent half-inhibition concentration (EC(50)) of approximately 35 nmol/L, which was 2-fold more potent than intact K5. In the same concentration range, this peptide did not inhibit pericytes from the same origin, suggesting an endothelial cell-specific inhibition. CONCLUSION: This K5 deletion mutant is a more potent angiogenic inhibitor than K5 and may have therapeutic potential in the treatment of such disorders with abnormal neovascularization as diabetic retinopathy, age-related macular degeneration and solid tumor.

Elevated apolipoprotein A-I levels are associated with favorable prognosis in metastatic nasopharyngeal carcinoma.

The survival outcomes of patients with metastatic nasopharyngeal carcinoma (NPC) differ significantly between individuals. Serum lipids and lipoproteins have been reported to be associated with prognosis in some cancers, but it has not been studied in metastatic NPC. The aim of this study was to evaluate whether serum lipid and lipoproteins could predict the prognosis of metastatic NPC patients. Eight hundred and seven patients with metastatic NPC were analyzed retrospectively, and the values of serum lipids and lipoproteins at baseline were retrieved. Receiver operating characteristic curve analysis and univariate and multivariate Cox proportional hazards analyses were used to evaluate the associations of serum lipids and lipoproteins with overall survival (OS). Univariate analysis revealed that higher values of baseline cholesterol (≥4.655 mmol/L), baseline high-density lipoprotein cholesterol (≥0.965 mmol/L), and baseline apolipoprotein A-I (ApoA-I) (≥1.065 g/L) were significantly associated with superior OS (p < 0.001), respectively. Multivariate Cox proportional hazard analysis showed that higher ApoA-I level (vs. lower ApoA-I level, HR 0.64, 95 % CI 0.52-0.80, p < 0.001) was an independent protective factor against progression. In addition, higher body mass index, earlier N stages, single lesion, and absence of liver metastasis were also revealed to be independent protective factors. In conclusion, the elevated baseline ApoA-I level may predict those patients likely to have a favorable OS.

Dual inhibition of plasminogen kringle 5 on angiogenesis and chemotaxis suppresses tumor metastasis by targeting HIF-1α pathway.

We had demonstrated that plasminogen kringle 5 (K5), a potent angiogenic inhibitor, inhibited retinal neovascularization and hepatocellular carcinoma growth by anti-angiogenesis. The current study investigated the effects and the underlying mechanisms of K5 on both tumor growth and spontaneous pulmonary metastasis in Lewis lung carcinoma (LLC) implanted mouse model. Similarly, K5 could decrease expression of VEGF in LLC cells and grafted tissues and suppress tumor angiogenesis and growth. K5 had no direct effect on proliferation and apoptosis of LLC. However, K5 could significantly inhibit SDF-1α-induced chemotaxis movement of LLC cells and resulted in a great reduction of surface metastatic nodules and micrometastases in the lungs of LLC tumor-bearing mice. K5 also decreased expression of chemokine (C-X-C motif) receptor 4 (CXCR4) in LLC cells and grafted tissues. Furthermore, K5 down-regulated SDF-1α expression in metastatic lung tissues of LLC-bearing mice. Therefore, K5 may suppress tumor pulmonary metastasis through inhibiting SDF-1α-CXCR4 chemotaxis movement and down-regulation of VEGF. Moreover, the role of hypoxia inducible factor-1α (HIF-1α), a crucial transcriptional factor for both VEGF and CXCR4 expression, was evaluated. The siRNA of HIF-1α attenuated expression of VEGF and CXCR4 and inhibited LLC migration. K5 decreased HIF-1α protein level and impaired nuclear HIF-1α accumulation. These results showed for the first time that K5 inhibits LLC growth and metastasis via the dual effects of anti-angiogenesis and suppression of tumor cell motility by targeting the pivotal molecule, HIF-1α.

[Reconstruction of the hemipelvis with saddle prosthesis after excision of malignant tumors around the pelvis and acetabulum: a report of 12 cases].

BACKGROUND & OBJECTIVE: Malignant tumors around the pelvis and acetabulum locate deeply with complex anatomic structure, meanwhile, the resection area involves the weight-loading alignment. Therefore, tumor resection plus acetabular joint reconstruction is a complicated operation. This study was to summarize our experience of tumor resection plus prosthesis reconstruction of the acetabular joint for this disease. METHODS: Clinical data of 12 patients with malignant tumors around the pelvis and acetabulum, treated with tumor resection plus prosthesis reconstruction of the acetabular joint from 1995 to 2006, were reviewed. The characteristics of the operating for this disease were analyzed in terms of preoperative preparation, operating strategy, prosthesis design, operating procedure, acetabular reconstruction, and postoperative rehabilitation. RESULTS: The patients were followed for 8-86 months, with a median of 46 months. Of the 4 patients with tumor relapse, 2 osteosarcoma patients died of lung metastasis at 15 months and 22 months after operation; 1 chondrosarcoma patient relapsed locally at 26 months after operation and died at 38 months after operation; 1 giant cell tumor patient relapsed locally at 13 months after operation and was treated by clearance of focal lesion, and survived tumor-freely till the end of follow-up. The other 9 patients still survived tumor-freely till the end of follow-up. The 1-year survival rate was 75.0%; the 3-and 5-year survival rates were 33.3%; the 7-year survival rate was 25.0%. According to Harris scoring criteria after total hip replacement, 3 patients scored 60-69, 5 scored 70-79, and 4 scored 80-90 in limb function. CONCLUSIONS: Pelvic tumor resection and prosthesis reconstruction of the acetabular joint has the characteristics of difficulty and high-risk. For bone tumors with relatively low malignancy, this surgical treatment is an ideal option.

Novel method for expression and purification of human pigment epithelium-derived factor with biological activities in Escherichia coli.

We developed a novel method for the expression and purification of recombinant human PEDF in Escherchia coli, and proved it to be simple, convenient, and cheap to obtain this protein with biological activity intact. Human PEDF gene, amplified by PCR from human retinal cNDA library, was cloned into the prokaryotic expression vector pET-22b(+). The recombinant pET-22b(+)/PEDF was expressed in E. coli strain BL21(DE3). The recombinant protein showed a molecular weight of about 50 kDa and was mainly in the form of inclusion bodies according to SDS-PAGE and Western blot analysis. The insoluble rPEDF was solublized from inclusion bodies by denaturation using 6 M urea, purified by His-tag affinity chromatography, and renatured to natural structure by dialysis in the presence of DTT. The rPEDF could cell-type-specifically inhibit HRCEC proliferation in a dose-dependent manner and induce HRCEC apoptosis.