Unraveling the connection between Hashimoto's Thyroiditis and non-alcoholic fatty liver disease: exploring the role of CD4+central memory T cells through integrated genetic approaches.

PURPOSE: Exploring the connection between Hashimoto's thyroiditis (HT) and non-alcoholic fatty liver disease (NAFLD) through integrated genetic approaches. METHODS: We utilized integrated genetic approaches, such as single-cell RNA sequencing (scRNA-seq) data analysis, Mendelian Randomization (MR), colocalization analysis, cell communication, and metabolic analyses, to investigate potential correlations between HT and NAFLD. RESULTS: Through the integrated analysis of scRNA-seq data from individuals with HT, NAFLD, and healthy controls, we observed an upregulation in the proportion of CD4+central memory (CD4+CM) T cells among T cells in both diseases. A total of 63 differentially expressed genes (DEGs) were identified in the CD4+CM cells after the differential analysis. By using MR, 8 DEGs (MAGI3, CSGALNACT1, CAMK4, GRIP1, TRAT1, IL7R, ERN1, and MB21D2) were identified to have a causal relationship with HT, and 4 DEGs (MAGI3, RCAN3, DOCK10, and SAMD12) had a causal relationship with NAFLD. MAGI3 was found to be causally linked to both HT and NAFLD. Therefore, MAGI3 was designated as the marker gene. Reverse MR and Steiger filtering showed no evidence of reverse causality. Colocalization analyses further indicated close links between MAGI3 and HT as well as NAFLD. Finally, based on the expression levels of MAGI3, we stratified CD4+CM cells into two subsets: MAGI3+CD4+CM cells and MAGI3-CD4+CM cells. Functional analyses revealed significant differences between the two subsets, potentially related to the progression of the two diseases. CONCLUSION: This study delves into the potential connections between HT and NAFLD through integrated genetic methods. Our research reveals an elevated proportion of CD4+CM cells within T cells in both HT and NAFLD. Through MR and colocalization analysis, we identify specific genes causally linked to HT and NAFLD, such as MAGI3. Ultimately, based on MAGI3 expression levels, we categorize CD4+CM cells into MAGI3+CD4+CM cells and MAGI3-CD4+CM cells, uncovering significant differences between them through functional analyses.

METTL3-mediated m6A modification of STEAP2 mRNA inhibits papillary thyroid cancer progress by blocking the Hedgehog signaling pathway and epithelial-to-mesenchymal transition.

Papillary thyroid cancer (PTC) is a common endocrine system malignancy all over the world. Aberrant expression of six transmembrane epithelial antigen of the prostate 2 (STEAP2) has been functionally associated with cancer progression in many cancers. Nevertheless, its biological function in PTC is still unclear. Here, we found that PTC tissues had preferentially downregulated STEAP2 as compared with noncancerous tissues. Low STEAP2 expression correlated with aggressive clinicopathological characteristics and dismal prognosis in patients with PTC. We performed gain- and loss-of-function experiments, including cell proliferation assay (Cell Counting Kit-8 assay), EdU (5-ethynyl-2'-deoxyuridine) and colony formation assays, transwell migration, and invasion assays, and constructed a nude mouse xenograft tumor model. The results demonstrated that STEAP2 overexpression inhibited PTC cell proliferation, migration, and invasion in vitro and inhibited lung metastasis and tumorigenicity in vivo. Conversely, silencing STEAP2 yielded the opposite results in vitro. Mechanistically, bioinformatics analysis combined with validation experiments identified STEAP2 as the downstream target of methyltransferase-like 3 (METTL3)-mediated N6-methyladenosine (m6A) modification. METTL3 stabilized STEAP2 mRNA and regulated STEAP2 expression positively in an m6A-dependent manner. We also showed that m6A-mediated STEAP2 mRNA translation initiation relied on a pathway dependent on the m6A reader protein YTHDF1. Rescue experiments revealed that silencing STEAP2 partially rescued the tumor-suppressive phenotype induced by METTL3 overexpression. Lastly, we verified that the METTL3-STEAP2 axis functions as an inhibitor in PTC by suppressing epithelial-mesenchymal transition and the Hedgehog signaling pathway. Taken together, these findings strongly suggest that METTL3-mediated STEAP2 m6A modification plays a critical tumor-suppressive role in PTC progression. The METTL3-STEAP2 axis may be a potential therapeutic molecular target against PTC.

Circular RNA_0057209 Acts as ceRNA to Inhibit Thyroid Cancer Progression by Promoting the STK4-Mediated Hippo Pathway via Sponging MicroRNA-183.

Thyroid cancer is the most common malignancy of the endocrine system, and its outcome remains unsatisfactory. In recent years, circular RNAs (circRNAs) have emerged as crucial regulators in cancers. In the current study, we aimed to investigate whether and how circRNA_0057209 functioned in thyroid cancer. Initial results revealed that circRNA_0057209 and STK4 were both reduced, while miR-183 was up-regulated in thyroid cancer tissues and cells. Experiments including RNA pull-down and RIP assays further identified that upregulation of circRNA_0057209 augmented the expression of STK4, a target gene of miR-183, by competitively-binding to miR-183. Furthermore, functional experiments provided evidence that overexpression of circRNA_0057209 not only inhibited the proliferative, migratory, and invasive properties of thyroid cancer cells while facilitating their apoptosis but also delayed tumor growth. Conversely, upregulation of miR-183 or silencing of STK4 reversed the changes induced by circRNA_0057209. Meanwhile, mechanistic experimentation demonstrated that circRNA_0057209 promoted STK4 expression by sponging miR-183, while STK4 enhanced YAP phosphorylation to mediate the Hippo pathway, thereby suppressing tumor progression. Altogether, our findings indicated that circRNA_0057209 may serve as a competing endogenous RNA of miR-183 to increase STK4 expression, thus inhibiting the development of thyroid cancer.

METTL3-Induced miR-222-3p Upregulation Inhibits STK4 and Promotes the Malignant Behaviors of Thyroid Carcinoma Cells.

CONTEXT: Abnormally high expression of N6-methyladenosine (m6A) methyltransferase-like 3 (METTL3) has been implied to accompany thyroid carcinoma (TC) development. OBJECTIVE: This study aimed to explore the protumorigenic role and downstream signaling axis of METTL3 in TC. METHODS: This study was conducted at the Sun Yat-Sen Memorial Hospital Sun Yat-Sen University. METTL3 and miR-222-3p were overexpressed or downregulated in TC cells. Tumor and adjacent normal tissues were collected from 80 patients (19 men and 60 women, aged 30-70 years) with a pathological diagnosis of TC from January 2012 to January 2015. Cells were classified and subjected to different treatments. The expression of METTL3 was validated in TC tissues and cell lines. In functional studies, METTL3 and miR-222-3p were overexpressed or downregulated in TC cells to evaluate their effects on malignant behaviors, which were subsequently verified by xenografts in nude mice. RESULTS: The expression of METTL3 was elevated in TC, correlating with poor prognosis of TC patients. Heightened METTL3 expression accelerated malignant behaviors of TC cells. Mechanistically, METTL3 stimulated miR-222-3p expression by mediating the m6A modification of pri-miR-222-3p. miR-222-3p targeted and inversely regulated serine/threonine stress kinase 4 (STK4). Knockdown of METTL3 augmented STK4 expression by downregulating miR-222-3p, thereby suppressing the malignant behaviors of TC cells as well as tumor growth and lung metastasis in nude mice. CONCLUSION: Silencing METTL3 suppresses miR-222-3p expression and thus stimulates STK4 expression, thereby repressing the malignancy and metastasis of TC.

ß-Catenin/LEF-1 transcription complex is responsible for the transcriptional activation of LINC01278.

BACKGROUND: Our previous study shows that LINC01278 inhibits the malignant proliferation and invasion of papillary thyroid carcinoma (PTC) cells by regulating the miR-376c-3p/DNM3 axis. However, the regulation mechanism of LINC01278 expression in PTC cells is still unclear. METHODS: The luciferase reporter and ChIP assays were used to confirm the binding of LEF-1 to the putative promoter site of LINC01278 gene. The RNA immunoprecipitation and RNA pulldown were used to determine the enrichment of LINC01278 in ß-catenin protein. The proteasome inhibitors (MG132) was used for detecting the ß-catenin ubiquitination-proteasome degradation. Wnt/ß-catenin specific agonists (LiCI), inhibitors (WiKI4) and TOP/FOP-flash reporter assay were used for detecting the activation of Wnt/ß-catenin signal. Western blot was used to detected the expression of target proteins. RESULTS: The online PROMO algorithm determines a putative LEF-1 binding site on LINC01278 promoter, the LEF-1 binds to the putative promoter site of LINC01278 gene, and ß-catenin enhances the binding of LEF-1 to the LINC01278 gene promoter. Furthermore, LINC01278 negatively regulated the protein accumulation of ß-catenin in the cytoplasm, into nucleus, and ultimately inhibited the transcription of downstream target genes activated by Wnt/ß-catenin signal. The results of RNA immunoprecipitation and RNA pulldown proved the direct binding of LINC01278 to ß-catenin protein. In addition, the combination of LINC01278 and ß-catenin promotes the ß-catenin ubiquitination-proteasome degradation. CONCLUSION: In summary, we found the transcriptional activation of LINC01278 by the ß-catenin/LEF-1 transcription factor, and the negative feedback regulation of LINC01278 onß-catenin signal.

LncRNA-HCG18 regulates the viability, apoptosis, migration, invasion and epithelial-mesenchymal transition of papillary thyroid cancer cells via regulating the miR-106a-5p/PPP2R2A axis.

The incidence of papillary thyroid cancer (PTC) has experienced a rapid increase in recent years. Long non-coding RNA-homo sapiens HLA complex group (HCG) 18 plays a regulatory role in cancers, but its role in PTC remained unknown. This study determined the expressions of HCG18, microRNA (miR)-106a-5p, and protein phosphatase 2 regulatory subunit B alpha (PPP2R2A) in PTC tissues and cells by qRT-PCR. ENCORI predicted the targeting relation between HCG18 and miR-106a-5p. TargetScan v7.2 predicted the targeting relation between miR-106a-5p and PPP2R2A. Dual-luciferase reporter assay was performed to validate the two targeting relations. The viability, migration, and invasion of PTC cells were detected by Cell Counting Kit-8, wound healing assay, and Transwell assay, respectively. The expressions of matrix metalloproteinase 2 (MMP-2), MMP-9, E-cadherin, N-cadherin, and Vimentin in TPC-1 and MDA-T68 cells were assessed by qRT-PCR and Western blot. It was found that HCG18 was down-regulated in PTC. Overexpressing HCG18 suppressed viability, migration, and invasion, promoted apoptosis, and inhibited miR-106a-5p expression in PTC cells. HCG18 interacted with miR-106a-5p, the expression of which was upregulated in PTC. Upregulating miR-106a-5p expression by lentivirus infection promoted viability, migration and invasion and inhibited apoptosis of PTC cells, reversed the effect of HCG18 on the biological behaviors of PTC cells, and promoted the expressions of MMP-2, MMP-9, E-cadherin, and Vimentin and downregulated E-cadherin expression in PTC cells. PPP2R2A, a direct target of miR-106a-5p, was downregulated in PTC, and HCG18 promoted PPP2R2A expression in PTC cells by sponging miR-106a-5p. Furthermore, PPP2R2A reversed the effects of miR-106a-5p on PTC cells. In conclusion, HCG18 suppressed viability, migration, invasion and epithelial-mesenchymal transition and promoted apoptosis of PTC cells via regulating the miR-106a-5p/PPP2R2A axis.

Comprehensive circular RNA profiling reveals the regulatory role of circRNA_0007694 in papillary thyroid carcinoma.

PURPOSE: The present study aimed to identify differentially expressed circRNAs in thyroid cancer and verify their potential functions. METHODS: Next-generation sequencing was used to identify differentially expressed circRNAs between papillary thyroid carcinoma (PTC) tissues and paired pericarcinomatous tissues. Polymerase chain reaction and Sanger sequencing methods successfully identified hsa_circ_0007694. A hsa_circ_0007694 over-expression vector was prepared to determine the effect of this circRNA on proliferation, migration, invasion, apoptosis, and the cell cycle in PTC cells. An in vivo animal assay was conducted by injecting PTC cells into the chests of mice. Further, RNA-seq was performed, followed by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, to verify the regulatory mechanism of hsa_circ_0007694. Western blotting was used to verify the genes thought to be involved in the hsa_circ_0007694 regulatory pathways based on KEGG analysis. RESULTS: We identified a circRNA, hsa_circ_0007694 that was down-regulated in PTC tissues compared to pericarcinomatous tissues. Over-expression of hsa_circ_0007694 promoted apoptosis and inhibited proliferation, migration, and invasion in PTC cells in vitro, and decreased tumor growth in vivo. Transcriptome sequence analysis suggested 129 differentially expressed genes between PTC tissue and paired pericarcinomatous tissue. KEGG analysis and western blotting indicated that the PI3K/AKT/mTOR and Wnt signaling networks are most likely to be related to hsa_circ_0007694 in thyroid cancer. CONCLUSION: The circRNA hsa_circ_0007694 is down-regulated in PTC and is therefore a potential therapeutic target.

Lysine-Specific Demethylase 1 Affects the Progression of Papillary Thyroid Carcinoma via HIF1α and microRNA-146a.

CONTEXT: Lysine-specific demethylase 1 (LSD1) stabilizes hypoxia-inducible factor 1α (HIF1α) to advance tumor progression, while HIF1α functions as a transcription factor to increase the expression of microRNA-146a (miR-146a). OBJECTIVE: We aim to investigate whether LSD1 affects the development of papillary thyroid carcinoma (PTC) via HIF1α and miR-146a. DESIGN: In vitro assays were performed with Nthy-ori 3-1, BHP5-16, BCPAP, K1, and BHP2-7 cell lines. In vivo assays were conducted with established xenograft tumors in nude mice. SETTING: This study was conducted at our lab. PATIENTS AND MATERIALS: PTC tissues and corresponding adjacent normal tissues were obtained from 45 patients hospitalized in Sun Yat-Sen Memorial Hospital. Assays were conducted using Nthy-ori 3-1, BHP5-16, BCPAP, K1, and BHP2-7 cell lines, as well as 50 male BALB/c nude mice. INTERVENTION: Cells were transfected with sh-LSD1, sh-GABPA, oe-LSD1, oe-HIF1α, miR-146a mimic, and miR-146a inhibitor. In addition, K1 cells expressing lv-oe-LSD1, lv-miR-146a inhibitor, lv-oe-LSD1 or miR-146a inhibitor were injected into the right side of the mice. LSD1 gene and protein expression patterns were analyzed in 45 clinical PTC tissue samples. MAIN OUTCOME MEASURE: Expression of LSD1, HIF1α, miR-146a, and GA-binding protein transcription factor alpha (GABPA), as well as their effects on PTC. RESULTS: LSD1 was highly expressed in clinical PTC tissues. LSD1 stabilized HIF1α and inhibited the degradation of its ubiquitin proteasome. HIF1α was enriched in the promoter region of miR-146a, an upregulated miRNA in PTC. HIF1α increased miR-146a expression to promote PTC progression in vitro, which was achieved by inhibiting GABPA, a target gene of miR-146a. LSD1 upregulated miR-146a to enhance the development and metastasis of PTC in nude mice. CONCLUSION: Our results show that LSD1 functions as an oncogene in PTC by upregulating HIF1α and miR-146a, elucidating an understanding of undefined mechanisms associated with tumor progression in PTC.

Long non-coding RNA TNRC6C-AS1 promotes methylation of STK4 to inhibit thyroid carcinoma cell apoptosis and autophagy via Hippo signalling pathway.

The role of long non-coding RNAs (lncRNAs) in thyroid carcinoma (TC), the most frequent endocrine malignancy, has been extensively examined. This study investigated effect of interaction among lncRNA TNRC6C-AS1, serine/threonine-protein kinase 4 (STK4) and Hippo signalling pathway on TC. Initially, lncRNA TNRC6C-AS1 expression in TC tissues was detected. To explore roles of lncRNA TNRC6C-AS1, STK4 and Hippo signalling pathway in TC progression, their expressions were altered. Interaction between lncRNA TNRC6C-AS1 and STK4, STK4 promoter methylation, or Hippo signalling pathway was verified. After that, a series of experiments were employed to evaluate in vitro ability of apoptosis, proliferation and autophagy of TC cells and in vivo tumorigenicity, and tumour growth of TC cells. lncRNA TNRC6C-AS1 was highly expressed while STK4 was poorly expressed in TC tissues. LncRNA TNRC6C-AS1 promoted the STK4 methylation and down-regulated STK4 expression, which further activated the Hippo signalling pathway. STK4 silencing was observed to promote the proliferation ability of TC cells, inhibit the apoptosis and autophagy abilities, as well as enhance the tumorigenicity and tumour growth. Moreover, the in vitro proliferation ability as well as the in vivo tumorigenicity and tumour growth of TC cells were inhibited after the blockade of Hippo signalling pathway, while the apoptosis and autophagy abilities were promoted. The results demonstrate that the lncRNA TNRC6C-AS1 increases STK4 promoter methylation to down-regulate STK4 expression, thereby promoting the development of TC through activation of Hippo signalling pathway. It highlights that lncRNA TNRC6C-AS1 may be a novel therapeutic target for the treatment of TC.

Linc01278 inhibits the development of papillary thyroid carcinoma by regulating miR-376c-3p/DNM3 axis.

PURPOSE: Papillary thyroid carcinoma (PTC) is the most common endocrine malignancy, and its incidence has continuously increased in recent years. Therefore, it is essential to develop more useful therapeutic strategies. METHODS: We collected 56 pairs of PTC tissues and adjacent normal tissues and determined the expression patterns of linc01278, miR-376c-3p and DNM3. In addition, we analyzed the relationship between linc01278 expression and pathological information of PTC patients. Furthermore, the effects of linc01278, miR-376c-3p and DNM3 overexpression on proliferation, clonality, apoptosis, migration and invasion of PTC cell lines TPC1 and BCPAP were evaluated. The dual luciferase reporter assay was used to confirm the direct interaction between miR-376c-3p and linc01278. RESULTS: Linc01278 and DNM3 were remarkably down-regulated in PTC tissues and cell lines, whereas miR-376c-3p was significantly up-regulated. In addition, lower linc01278 expression was associated with increased tumor size, lymph node metastasis and higher clinical stage. Linc01278 inhibited cell proliferation of PTC cells by inducing apoptosis, and demonstrated attenuating effects on migration and invasion abilities of PTC cells by regulating the EMT process. More importantly, dual luciferase reporter experiments demonstrated the direct interaction between miR-376c-3p and linc01278, which revealed that DNM3 was a novel target of miR-376c-3p. The miR-376c-3p mimic significantly promoted the proliferation, migration and invasion of PTC cells, and inhibited cell apoptosis. Overexpression of DNM3 abolished the effects of the miR-376c-3p mimic on PTC cells. DNM3 expression was negatively correlated with miR-376c-3p expression, but was positively correlated with linc01278 expression. CONCLUSION: Overall, we found that linc01278 can act as a competing endogenous RNA (ceRNA) to sponge miR-376c-3p, thereby positively regulating DNM3 expression and ultimately acting as a tumor suppressor gene in PTC progression.

Oncogenic Ras/squamous cell carcinoma antigen signaling pathway activation promotes invasiveness and lymph node metastases in papillary thyroid carcinoma.

Papillary thyroid carcinoma (PTC) is a type of cancer with one of the fastest increasing incidences worldwide. However, the therapeutic choices for PTC patients are limited and it is critical to further understand the molecular pathology underlying this disease. Squamous cell carcinoma antigens (SCCAs) are overexpressed in many tumors and participate in tumorigenesis. However, their roles in PTC are incompletely understood. Therefore, this study investigated the role of SCCA in PTC, evaluating its expression, its clinical implications and prognostic significance in PTC patient samples, as well as its function in vitro and in vivo, using a thyroid cancer cell line in which SCCA levels have been knocked down or overexpressed. In this study, SCCA expression levels were measured by immunohistochemistry (IHC) in non­cancerous and tumor tissues. Kaplan­Meier analyses assessed the survival in PTC patients. MTT assay, western blot analysis, invasion assay and xenograft tumor assay were used to calculate cell proliferation, migration, invasion and tumor growth. Our results showed that SCCA was overexpressed in PTC tissues and was correlated with the clinical stage of PTC. Patients with high SCCA expression had lower overall survival (OS), disease­free survival (DFS), lymph node recurrence­free survival (LNRFS), and distant recurrence­free survival (DRFS), compared to patients expressing low level of the SCCA protein. SCCA knockdown suppressed thyroid cancer cell proliferation, invasion and reduced xenograft tumor growth, whereas SCCA overexpression increased cell proliferation, invasion and xenograft tumor growth. Mechanistically, the activation of Ras increased SCCA expression, and SCCA expression was positively correlated with Ras levels in the PTC tissues. In conclusion, SCCA protein is overexpressed in PTC and may represent a predictive prognostic factor for PTC patients. Furthermore, activation of the Ras/SCCA pathway plays an important role in promoting tumor growth, invasion and metastasis in PTC.

Activation of the ROCK1/MMP-9 pathway is associated with the invasion and poor prognosis in papillary thyroid carcinoma.

Rho-associated protein kinase 1 (ROCK1), a serine/threonine kinase, has previously been shown to be over-expressed in various types of human malignant tumors and to play an important role in cancer development and progression. Although ROCK1 has gained growing prominence as an important protein kinase in cancer biology, its potential as a predictive biomarker and a therapeutic target in papillary thyroid carcinoma (PTC) remains unknown. In the present study, ROCK1 expression was examined in 356 formalin-fixed, paraffin-embedded papillary thyroid carcinoma tissues using immunohistochemistry, and its clinical implications and prognostic significance were analyzed. Our results showed that ROCK1 expression was significantly increased in PTC compared with normal tissues, and was significantly associated with tumor size, lymphatic metastasis, distant organ metastasis, extrathyroid invasion, vascular invasion and tumor, node and metastasis (TNM) stage. Patients with strong ROCK1 expression had lower overall survival, disease-free survival, lymph node recurrence-free survival and distant recurrence-free survival rates than those with weak expression. Furthermore, overexpression of ROCK1 in papillary thyroid carcinoma cells was found to increase their invasiveness. Silencing ROCK1 by siRNA, however, caused an inhibition of cell invasion. Knockdown of ROCK1 decreased the volume and weight of the xenograft tumors, while overexpression of ROCK1 showed a proliferative tendency with significantly greater tumor volume and weight in vivo. Moreover, the upregulation of ROCK1 increased the expression of MMP-9, and levels of MMP-9 positively correlated with the ROCK1 levels in PTC tissues, implicating that MMP-9 may be involved in the mechanism of ROCK1 in the development and progression of PTC. These data suggest that ROCK1 might be a potential prognostic marker and therapeutic target for the treatment of PTC.

A Carbon Nanoparticle Lymphatic Tracer Protected Parathyroid Glands During Radical Thyroidectomy for Papillary Thyroid Non-Microcarcinoma.

The study assessed the role of an activated carbon nanoparticle lymphatic tracer in reducing unintentional damage to the parathyroid glands during thyroidectomy for papillary thyroid non-microcarcinoma diagnosed intraoperatively by cryosections. A total of 103 patients with papillary thyroid non-microcarcinomas diagnosed by intraoperative cryosection were randomly assigned to receive routine radical thyroidectomy or radical thyroidectomy following administration of activated carbon nanoparticle lymphatic tracer to the contralateral thyroid, at the department of Thyroid Surgery, Sun Yat-sen Memorial Hospital (Guangzhou, China), between January 2012 and May 2013. The success of level VI lymphadenectomy and postoperative parathyroid function were compared. Administration of the activated carbon nanoparticle lymphatic tracer did not affect the frequency of recovered lymph nodes containing metastases; however, it did significantly reduce the incidence of permanent and transient hypoparathyroidism from 2 to 0 and 18 to 6, and reduced the mean recovery time for transient hypoparathyroidism from 57.0 days to 22.3 days. Administration of activated carbon nanoparticles to the contralateral thyroid after intraoperative cryosections did not contribute to lymphadenectomy for papillary thyroid non-microcarcinoma, but significantly protected parathyroid functions. This approach could decrease the morbidity of radical thyroidectomy and the occurrence of hypoparathyroidism.

CHI3L1 overexpression is associated with metastasis and is an indicator of poor prognosis in papillary thyroid carcinoma.

OBJECTIVE: In this study, we examined the relationships between the expression level of CHI3L1 and the clinicopathological characteristics of papillary thyroid carcinoma. METHODS: A total of 322 tissue samples from patients with papillary thyroid carcinoma were collected, and the CHI3L1 expression levels in tumor tissues, matched adjacent noncancerous tissues were detected using immunohistochemistry (IHC) and qRT-PCR. The relationships between CHI3L1 expression levels and the clinical characteristics were evaluated. RESULTS: CHI3L1 expression was significantly increased in papillary thyroid carcinoma compared with matched adjacent noncancerous tissues (P< 0.001), tumor tissues with lymph node metastasis (LNM) compared with tumor tissues without LNM (P< 0.001) and tumor tissues with distant organ metastasis (DOM) compared with tumor tissues without DOM (P< 0.01). CHI3L1 expression was significantly associated with tumor size (P= 0.0001), lymph node metastasis (P< 0.0001), distant organ metastasis (P< 0.0001), extrathyroid invasion (P= 0.0022), vascular invasion (P= 0.0004) and TNM stage (P= 0.0001). CHI3L1 overexpression in papillary thyroid carcinoma tissues correlates with the tumor malignant potential (P< 0.01). More importantly, Cox multifactor analysis indicated that patients with high CHI3L1 expression have lower overall survival, disease-free survival, lymph node recurrence-free survival, and distant recurrence free survival rates than those with low expression (P< 0.05). And our findings were further validated by online Oncomine database. CONCLUSIONS: CHI3L1 is associated with tumor metastasis and might be a prognostic biomarker for papillary thyroid carcinoma.

A new murine model of stress-induced complex atherosclerotic lesions.

The primary purpose of this investigation was to determine whether ApoE(-/-) mice, when subjected to chronic stress, exhibit lesions characteristic of human vulnerable plaque and, if so, to determine the time course of such changes. We found that the lesions were remarkably similar to human vulnerable plaque, and that the time course of lesion progression raised interesting insights into the process of plaque development. Lard-fed mixed-background ApoE(-/-) mice exposed to chronic stress develop lesions with large necrotic core, thin fibrous cap and a high degree of inflammation. Neovascularization and intraplaque hemorrhage are observed in over 80% of stressed animals at 20 weeks of age. Previously described models report a prevalence of only 13% for neovascularization observed at a much later time point, between 36 and 60 weeks of age. Thus, our new stress-induced model of advanced atherosclerotic plaque provides an improvement over what is currently available. This model offers a tool to further investigate progression of plaque phenotype to a more vulnerable phenotype in humans. Our findings also suggest a possible use of this stress-induced model to determine whether therapeutic interventions have effects not only on plaque burden, but also, and importantly, on plaque vulnerability.

Aging-induced collateral dysfunction: impaired responsiveness of collaterals and susceptibility to apoptosis via dysfunctional eNOS signaling.

Despite positive animal studies, clinical angiogenesis trials have been disappointing, possibly due to risk factors present in humans but usually unexplored in animals. We recently demonstrated aging causes impaired collateral remodeling and collateral dropout; here, we investigate potential mechanisms responsible for these findings. Four-, 10-, and 18-month-C57BL/6J mice were subjected to femoral artery ligation; flow was measured using laser Doppler perfusion imaging. Endothelial nitric oxide synthase (eNOS) and phosphorylated eNOS were measured in calf muscle. Apoptosis was assessed in endothelial (EC) and smooth muscle (SMC) cells isolated from young and old mice. Angiogenesis was measured using a Matrigel plug assay. Lethally irradiated young and old mice received bone marrow cells (BMC) from either young or old donors and were subjected to femoral artery ligation (FAL). BMC mobilization and homing were assessed. Flow recovery was impaired and less eNOS and phosphorylated eNOS was present in older vs. young mice (p < 0.001 and p = 0.015, respectively). ECs and SMCs from older mice were more sensitive to an apoptotic stimulus, but were rescued by NO-enhancing drugs. In older mice, angiogenesis (Matrigel plug assay) was impaired, as was mobilization and homing of BM progenitor cells following FAL. Although both mobilization and homing improved when older mice received BMC transplantation from young donors, flow recovery failed to improve. Aging impairs BMC mobilization and homing, collateral responsiveness to angiogenic stimuli, and increases EC and SMC susceptibility to apoptosis via dysfunctional eNOS signaling. The latter could contribute to impaired remodeling and collateral dropout. These finding identify potential obstacles to therapeutic interventions in elderly patients.

Gender differences affect blood flow recovery in a mouse model of hindlimb ischemia.

Blood flow restoration to ischemic tissue is affected by various risk factors. The aim of this study was to examine gender effects on arteriogenesis and angiogenesis in a mouse ischemic hindlimb model. C57BL/6J mice were subjected to unilateral hindlimb ischemia. Flow recovery was less and hindlimb use impairment was greater in females. No gender difference in vessel number was found at baseline, although 7 days postsurgery females had fewer α-smooth muscle actin-positive vessels in the midpoint of the adductor region. Females had higher hindlimb vascular resistance, were less responsive to vasodilators, and were more sensitive to vasoconstrictors postligation. Western blotting showed that females had higher baseline levels of vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) in the calf, while 7 days postligation males had higher levels of VEGF, eNOS, and phosphorylated vasodilator stimulated phosphoprotein. Females had less angiogenesis in a Matrigel plug assay and less endothelial cell proliferation in vitro. Females have impaired recovery of flow, a finding presumably caused by multiple factors including decreased collateral remodeling, less angiogenesis, impaired vasodilator response, and increased vasoconstrictor activity; our results also suggest the possibility that new collateral formation, from capillaries, is impaired in females.

Metallothionein enhances angiogenesis and arteriogenesis by modulating smooth muscle cell and macrophage function.

OBJECTIVE: In a previous study we identified metallothionein (MT) as a candidate gene potentially influencing collaterogenesis. In this investigation, we determined the effect of MT on collaterogenesis and examined the mechanisms contributing to the effects we found. METHODS AND RESULTS: Collateral blood flow recovery was assessed using laser Doppler perfusion imaging, and angiogenesis was measured using a Matrigel plug assay. Smooth muscle cells were isolated from MT knockout (KO) mice for functional assays. Gene expression of matrix metalloproteinase-9, platelet-derived growth factor, vascular endothelial growth factor, and Fat cadherin in smooth muscle cells was measured by real-time polymerase chain reaction, and protein levels of vascular endothelial growth factor and matrix metalloproteinase-9 were determined using enzyme-linked immunosorbent assay and Western blot. CD11b(+) macrophages were tested for invasiveness using a real-time impedance assay. Both flow recovery and angiogenesis were impaired in MT KO mice. Proliferation, migration, and invasion were decreased in MT KO smooth muscle cells, and matrix metalloproteinase-9, platelet-derived growth factor, and vascular endothelial growth factor expression were also decreased, whereas FAT-1 cadherin expression was elevated. MT KO CD11b(+) cells were more invasive than wild-type cells. CONCLUSIONS: MT plays an important role in collateral flow recovery and angiogenesis, an activity that appears to be mediated, in part, by the effects of MT on the functionality of 3 cell types essential for these processes: endothelial cells, smooth muscle cells, and macrophages.

Combination of stromal-derived factor-1alpha and vascular endothelial growth factor gene-modified endothelial progenitor cells is more effective for ischemic neovascularization.

BACKGROUND: Recruitment and entrapment of bone marrow-derived endothelial progenitor cells (EPCs) is important in vascular endothelial growth factor (VEGF)-induced angiogenesis. EPC mobilization and differentiation are modulated by stromal-derived factor-1alpha (SDF-1alpha/CXCL12), another important chemokine. In this study, we investigated the hypothesis that SDF-1alpha and VEGF might act synergistically on EPC-mediated vasculogenesis. METHODS: EPCs were isolated and cultured from human peripheral blood, then transduced with retroviral vectors pBabe containing human VEGF(165) complimentary DNA (Td/V-EPCs) and pBabe wild-type (Td/p-EPCs). EPC migration activity was investigated with a modified Boyden chamber assay. EPC apoptosis induced by serum starvation was studied by annexin V assays. The combined effect of local administration of SDF-1alpha and Td/V-EPC transplantation on neovascularization was investigated in a murine model of hind limb ischemia. RESULTS: Over-expression of hVEGF(165) increased SDF-1alpha-mediated EPC migration. SDF-1alpha-mediated migration was significantly increased when EPCs were modified with VEGF (Td/V-EPCs) vs when VEGF was not present (Td/p-EPCs) or when VEGF alone was present (Td/V-EPCs; 196.8 +/- 15.2, 81.2 +/- 9.8, and 67.4 +/- 7.4/mm(2), respectively P < .001). SDF-1alpha combined with VEGF reduced serum starvation-induced apoptosis of EPCs more than SDF-1alpha or VEGF alone (P < .001). To determine the effect of this combination in vivo, SDF-1alpha was locally injected alone into the ischemic hind limb muscle of nude mice or combined with systemically injected Td/V-EPCs. The SDF-1alpha plus VEGF group showed significantly increased local accumulation of EPCs, blood-flow recovery, and capillary density compared with the other groups. The ratio of ischemic/normal blood flow in Td/V-EPCs plus SDF-1alpha group was significantly higher (P < .01), as was capillary density (capillaries/mm(2)), an index of neovascularization (Td/V-EPCs plus SDF-1alpha group, 863 +/- 31; no treatment, 395 +/-13; SDF-1alpha, 520 +/- 29; Td/p-EPCs, 448 +/- 28; Td/p-EPCs plus SDF-1alpha, 620 +/- 29; Td/V-EPCs, 570 +/- 30; P < .01). To investigate a possible mechanistic basis, we showed that VEGF up-regulated the receptor for SDF-1alpha, CXCR4, on EPCs in vitro. CONCLUSION: The combination of SDF-1alpha and VEGF greatly increases EPC-mediated angiogenesis. The use VEGF and SDF-1alpha together, rather than alone, will be a novel and efficient angiogenesis strategy to provide therapeutic neovascularization.