PFKFB3-driven vascular smooth muscle cell glycolysis promotes vascular calcification via the altered FoxO3 and lactate production.

A link between increased glycolysis and vascular calcification has recently been reported, but it remains unclear how increased glycolysis contributes to vascular calcification. We therefore investigated the role of PFKFB3, a critical enzyme of glycolysis, in vascular calcification. We found that PFKFB3 expression was upregulated in calcified mouse VSMCs and arteries. We showed that expression of miR-26a-5p and miR-26b-5p in calcified mouse arteries was significantly decreased, and a negative correlation between Pfkfb3 mRNA expression and miR-26a-5p or miR-26b-5p was seen in these samples. Overexpression of miR-26a/b-5p significantly inhibited PFKFB3 expression in VSMCs. Intriguingly, pharmacological inhibition of PFKFB3 using PFK15 or knockdown of PFKFB3 ameliorated vascular calcification in vD3 -overloaded mice in vivo or attenuated high phosphate (Pi)-induced VSMC calcification in vitro. Consistently, knockdown of PFKFB3 significantly reduced glycolysis and osteogenic transdifferentiation of VSMCs, whereas overexpression of PFKFB3 in VSMCs induced the opposite effects. RNA-seq analysis and subsequent experiments revealed that silencing of PFKFB3 inhibited FoxO3 expression in VSMCs. Silencing of FoxO3 phenocopied the effects of PFKFB3 depletion on Ocn and Opg expression but not Alpl in VSMCs. Pyruvate or lactate supplementation, the product of glycolysis, reversed the PFKFB3 depletion-mediated effects on ALP activity and OPG protein expression in VSMCs. Our results reveal that blockade of PFKFB3-mediated glycolysis inhibits vascular calcification in vitro and in vivo. Mechanistically, we show that FoxO3 and lactate production are involved in PFKFB3-driven osteogenic transdifferentiation of VSMCs. PFKFB3 may be a promising therapeutic target for the treatment of vascular calcification.

Potential enhancement of post-stroke angiogenic response by targeting the oligomeric aggregation of p53 protein.

Tumor suppressor gene p53 and its aggregate have been found to be involved in many angiogenesis-related pathways. We explored the possible p53 aggregation formation mechanisms commonly occur after ischemic stroke, such as hypoxia and the presence of reactive oxygen species (ROS). The angiogenic pathways involving p53 mainly occur in nucleus or cytoplasm, with one exception that occurs in mitochondria. Considering the high mitochondrial density in brain and endothelial cells, we proposed that the cyclophilin D (CypD)-dependent vascular endothelial cell (VECs) necrosis pathway occurring in the mitochondria is one of the major factors that affects angiogenesis. Hence, targeting p53 aggregation, a key intermediate in the pathway, could be an alternative therapeutic target for post-stroke management.

The short-chain fatty acid butyrate accelerates vascular calcification via regulation of histone deacetylases and NF-κB signaling.

Recent studies have shown that short-chain fatty acids (SCFAs), primarily acetate, propionate and butyrate, play a crucial role in the pathogenesis of cardiovascular disease. Whether SCFAs regulate vascular calcification, a common pathological change in cardiovascular tissues, remains unclear. This study aimed to investigate the potential role of SCFAs in vascular calcification. Using cellular and animal models of vascular calcification, we showed that butyrate significantly enhanced high phosphate (Pi)-induced calcification and osteogenic transition of vascular smooth muscle cells (VSMC) in vitro, whereas acetate and propionate had no effects. Subsequent studies confirmed that butyrate significantly promoted high Pi-induced aortic ring calcification ex vivo and high dose vitamin D3 (vD3)-induced mouse vascular calcification in vivo. Mechanistically, butyrate significantly inhibited histone deacetylase (HDAC) expression in VSMCs, and a pan HDAC inhibitor Trichostatin A showed similar inductive effects on calcification and osteogenic transition of VSMCs to butyrate. In addition, the SCFA sensing receptors Gpr41 and Gpr109a were primarily expressed by VSMCs, and butyrate induced the rapid activation of NF-κB, Wnt and Akt signaling in VSMCs. Intriguingly, the NF-κB inhibitor SC75741 significantly attenuated butyrate-induced calcification and the osteogenic gene Msx2 expression in VSMCs. We showed that knockdown of Gpr41 but not Gpr109a attenuated butyrate-induced VSMC calcification. This study reveals that butyrate accelerates vascular calcification via its dual effects on HDAC inhibition and NF-κB activation. Our data provide novel insights into the role of microbe-host interaction in vascular calcification, and may have implications for the development of potential therapy for vascular calcification.

PALMD regulates aortic valve calcification via altered glycolysis and NF-κB-mediated inflammation.

Recent genome-wide association and transcriptome-wide association studies have identified an association between the PALMD locus, encoding palmdelphin, a protein involved in myoblast differentiation, and calcific aortic valve disease (CAVD). Nevertheless, the function and underlying mechanisms of PALMD in CAVD remain unclear. We herein investigated whether and how PALMD affects the pathogenesis of CAVD using clinical samples from CAVD patients and a human valve interstitial cell (hVIC) in vitro calcification model. We showed that PALMD was upregulated in calcified regions of human aortic valves and calcified hVICs. Furthermore, silencing of PALMD reduced hVIC in vitro calcification, osteogenic differentiation, and apoptosis, whereas overexpression of PALMD had the opposite effect. RNA-Seq of PALMD-depleted hVICs revealed that silencing of PALMD reduced glycolysis and nuclear factor-κB (NF-κB)-mediated inflammation in hVICs and attenuated tumor necrosis factor α-induced monocyte adhesion to hVICs. Having established the role of PALMD in hVIC glycolysis, we examined whether glycolysis itself could regulate hVIC osteogenic differentiation and inflammation. Intriguingly, the inhibition of PFKFB3-mediated glycolysis significantly attenuated osteogenic differentiation and inflammation of hVICs. However, silencing of PFKFB3 inhibited PALMD-induced hVIC inflammation, but not osteogenic differentiation. Finally, we showed that the overexpression of PALMD enhanced hVIC osteogenic differentiation and inflammation, as opposed to glycolysis, through the activation of NF-κB. The present study demonstrates that the genome-wide association- and transcriptome-wide association-identified CAVD risk gene PALMD may promote CAVD development through regulation of glycolysis and NF-κB-mediated inflammation. We propose that targeting PALMD-mediated glycolysis may represent a novel therapeutic strategy for treating CAVD.

17ß-estradiol promotes angiogenesis through non-genomic activation of Smad1 signaling in endometriosis.

17ß-estradiol (E2) plays a key role in endometriosis through regulation of angiogenesis. Smad1 has been reported to be up-regulated in patients with endometriosis. However, the role of Smad1 in E2-mediated angiogenesis during the development of endometriosis remains to be determined. This study aimed to explore the role of Smad1 in E2-mediated angiogenesis during endometriosis and its underlying mechanisms. Immunofluorescence staining and Western blotting were performed to examine the expression of p-Smad1 in ectopic and control endometrium. Western blotting was used to examine activation of Smad1 signaling in NMECs, EMECs and HUVECs. Tube formation assay was performed to examine the effect of E2 on angiogenesis. Cell proliferation and migration was determined using in real-time by xCELLigence RTCA DP instrument. We found that the expression of p-Smad1 was significantly up-regulated in ectopic endometrium and ectopic intima microvascular endothelial cells. E2 non-genomically stimulated phosphorylation of Smad1 in HUVECs. c-Src and p44/42 MAPK(ERK1/2) signaling pathways are required for E2's induction on Smad1 phosphorylation. Moreover, caveolae is involved in E2-induced Smad1 phosphorylation in vascular endothelial cells. E2 promoted tube formation of vascular endothelial cells through c-Src/ERK1/2/Smad1 signaling pathway. Knockdown of Smad1 expression attenuated E2-induced proliferation and migration of HUVECs. In conclusion, E2 promotes proliferation, migration and tube formation of HUVECs through c-Src/ERK1/2/Smad1 signaling pathway. Our data shed new lights on the mechanisms through which E2 contributes to endometriosis, and may provide novel strategies to treat endometriosis.

Regulatory Role of Sex Hormones in Cardiovascular Calcification.

Sex differences in cardiovascular disease (CVD), including aortic stenosis, atherosclerosis and cardiovascular calcification, are well documented. High levels of testosterone, the primary male sex hormone, are associated with increased risk of cardiovascular calcification, whilst estrogen, the primary female sex hormone, is considered cardioprotective. Current understanding of sexual dimorphism in cardiovascular calcification is still very limited. This review assesses the evidence that the actions of sex hormones influence the development of cardiovascular calcification. We address the current question of whether sex hormones could play a role in the sexual dimorphism seen in cardiovascular calcification, by discussing potential mechanisms of actions of sex hormones and evidence in pre-clinical research. More advanced investigations and understanding of sex hormones in calcification could provide a better translational outcome for those suffering with cardiovascular calcification.

Selective autophagy of intracellular organelles: recent research advances.

Macroautophagy (hereafter called autophagy) is a highly conserved physiological process that degrades over-abundant or damaged organelles, large protein aggregates and invading pathogens via the lysosomal system (the vacuole in plants and yeast). Autophagy is generally induced by stress, such as oxygen-, energy- or amino acid-deprivation, irradiation, drugs, etc. In addition to non-selective bulk degradation, autophagy also occurs in a selective manner, recycling specific organelles, such as mitochondria, peroxisomes, ribosomes, endoplasmic reticulum (ER), lysosomes, nuclei, proteasomes and lipid droplets (LDs). This capability makes selective autophagy a major process in maintaining cellular homeostasis. The dysfunction of selective autophagy is implicated in neurodegenerative diseases (NDDs), tumorigenesis, metabolic disorders, heart failure, etc. Considering the importance of selective autophagy in cell biology, we systemically review the recent advances in our understanding of this process and its regulatory mechanisms. We emphasize the 'cargo-ligand-receptor' model in selective autophagy for specific organelles or cellular components in yeast and mammals, with a focus on mitophagy and ER-phagy, which are finely described as types of selective autophagy. Additionally, we highlight unanswered questions in the field, helping readers focus on the research blind spots that need to be broken.

Zinc ameliorates human aortic valve calcification through GPR39 mediated ERK1/2 signalling pathway.

AIMS: Calcific aortic valve disease (CAVD) is the most common heart valve disease in the Western world. It has been reported that zinc is accumulated in calcified human aortic valves. However, whether zinc directly regulates CAVD is yet to be elucidated. The present study sought to determine the potential role of zinc in the pathogenesis of CAVD. METHODS AND RESULTS: Using a combination of a human valve interstitial cell (hVIC) calcification model, human aortic valve tissues, and blood samples, we report that 20 µM zinc supplementation attenuates hVIC in vitro calcification, and that this is mediated through inhibition of apoptosis and osteogenic differentiation via the zinc-sensing receptor GPR39-dependent ERK1/2 signalling pathway. Furthermore, we report that GPR39 protein expression is dramatically reduced in calcified human aortic valves, and there is a significant reduction in zinc serum levels in patients with CAVD. Moreover, we reveal that 20 µM zinc treatment prevents the reduction of GPR39 observed in calcified hVICs. We also show that the zinc transporter ZIP13 and ZIP14 are significantly increased in hVICs in response to zinc treatment. Knockdown of ZIP13 or ZIP14 significantly inhibited hVIC in vitro calcification and osteogenic differentiation. CONCLUSIONS: Together, these findings suggest that zinc is a novel inhibitor of CAVD, and report that zinc transporter ZIP13 and ZIP14 are important regulators of hVIC in vitro calcification and osteogenic differentiation. Zinc supplementation may offer a potential therapeutic strategy for CAVD.

Hdac9 inhibits medial artery calcification through down-regulation of Osterix.

BACKGROUNDS: Medial artery calcification (MAC) significantly contributes to the increased cardiovascular death in patients with chronic kidney disease (CKD). Previous genome-wide association studies have shown that various genetic variants of the histone deacetylase Hdac9 are associated with cardiovascular disease, but the role of Hdac9 in MAC under CKD conditions remains unclear. METHODS: High phosphate-induced vascular smooth muscle cell (VSMC) calcification and MAC in mice administered with vitamin D3 (vD) were used in the present study. Alizarin red staining, calcium quantitative assay, qPCR, western blotting and histology were performed. RESULTS: Hdac9 expression was significantly down-regulated during high phosphate-induced vascular smooth muscle cell (VSMC) calcification and MAC in mice administered with vitamin D3 (vD). Furthermore, high phosphate treatment inhibited phosphorylation of Akt, and pharmacological inhibition of Akt signaling reduced Hdac9 expression in cultured VSMCs. Knockdown of Hdac9 significantly enhanced calcium deposition in VSMCs. Conversely, adenovirus mediated-overexpression of Hdac9 inhibited high phosphate induced VSMC in vitro calcification. Our subsequent mechanistic studies revealed that the anti-calcific effect of Hdac9 was mediated through down-regulation of osteoblast-specific transcription factor Osterix. CONCLUSION: These data suggest that Hdac9 is a novel inhibitor of MAC and may represent a potential therapeutic target for MAC in CKD patients.

Estrogen-Mediated Gaseous Signaling Molecules in Cardiovascular Disease.

Gender difference is well recognized as a key risk factor for cardiovascular disease (CVD). Estrogen, the primary female sex hormone, improves cardiovascular functions through receptor (ERα, ERß, or G protein-coupled estrogen receptor)-initiated genomic or non-genomic mechanisms. Gaseous signaling molecules, including nitric oxide (NO), hydrogen sulfide (H2S), and carbon monoxide (CO), are important regulators of cardiovascular function. Recent studies have demonstrated that estrogen regulates the production of these signaling molecules in cardiovascular cells to exert its cardiovascular protective effects. We discuss current understanding of gaseous signaling molecules in cardiovascular disease (CVD), the underlying mechanisms through which estrogen exerts cardiovascular protective effects by regulating these molecules, and how these findings can be translated to improve the health of postmenopausal women.

F0F1 ATP synthase regulates extracellular calcium influx in human neutrophils by interacting with Cav2.3 and modulates neutrophil accumulation in the lipopolysaccharide-challenged lung.

BACKGROUND: Neutrophils form the first line of innate host defense against invading microorganisms. We previously showed that F0F1 ATP synthase (F-ATPase), which is widely known as mitochondrial respiratory chain complex V, is expressed in the plasma membrane of human neutrophils and is involved in regulating cell migration. Whether F-ATPase performs cellular functions through other pathways remains unknown. METHODS: Blue native polyacrylamide gel electrophoresis followed by nano-ESI-LC MS/MS identification and bioinformatic analysis were used to identify protein complexes containing F-ATPase. Then, the identified protein complexes containing F-ATPase were verified by immunoblotting, immunofluorescence colocalization, immunoprecipitation, real-time RT-PCR and agarose gel electrophoresis. Immunoblotting, flow cytometry and a LPS-induced mouse lung injury model were used to assess the effects of the F-ATPase-containing protein complex in vitro and in vivo. RESULTS: We found that the voltage-gated calcium channel (VGCC) α2δ-1 subunit is a binding partner of cell surface F-ATPase in human neutrophils. Further investigation found that the physical connection between the two proteins may exist between the F1 part (α and ß subunits) of F-ATPase and the α2 part of VGCC α2δ-1. Real-time RT-PCR and PCR analyses showed that Cav2.3 (R-type) is the primary type of VGCC expressed in human neutrophils. Research on the F-ATPase/Cav2.3 functional complex indicated that it can regulate extracellular Ca2+ influx, thereby modulating ERK1/2 phosphorylation and reactive oxygen species production, which are typical features of neutrophil activation. In addition, the inhibition of F-ATPase can reduce neutrophil accumulation in the lungs of mice that were intratracheally instilled with lipopolysaccharide, suggesting that the inhibition of F-ATPase may prevent neutrophilic inflammation-induced tissue damage. CONCLUSIONS: In this study, we identified a mechanism by which neutrophil activity is modulated, with simultaneous regulation of neutrophil-mediated pulmonary damage. These results show that surface F-ATPase of neutrophils is a potential innate immune therapeutic target.

17ß-Estradiol nongenomically induces vascular endothelial H2S release by promoting phosphorylation of cystathionine γ-lyase.

Estrogen exerts its cardiovascular protective role at least in part by regulating endothelial hydrogen sulfide (H2S) release, but the underlying mechanisms remain to be fully elucidated. Estrogen exerts genomic effects, i.e. those involving direct binding of the estrogen receptor (ER) to gene promoters in the nucleus, and nongenomic effects, mediated by interactions of the ER with other proteins. Here, using human umbilical vein endothelial cells (HUVECs), immunological detection, MS-based analyses, and cGMP and H2S assays, we show that 17ß-estradiol (E2) rapidly enhances endothelial H2S release in a nongenomic manner. We found that E2 induces phosphorylation of cystathionine γ-lyase (CSE), the key enzyme in vascular endothelial H2S generation. Mechanistically, E2 enhanced the interaction of membrane ERα with the Gα subunit Gαi-2/3, which then transactivated particulate guanylate cyclase-A (pGC-A) to produce cGMP, thereby activating protein kinase G type I (PKG-I). We also found that PKG-Iß, but not PKG-Iα, interacts with CSE, leading to its phosphorylation, and rapidly induces endothelial H2S release. Furthermore, we report that silencing of either CSE or pGC-A in mice attenuates E2-induced aorta vasodilation. These results provide detailed mechanistic insights into estrogen's nongenomic effects on vascular endothelial H2S release and advance our current understanding of the protective activities of estrogen in the cardiovascular system.

ENPP1 in the Regulation of Mineralization and Beyond.

ENPP1 is well known for its role in regulating skeletal and soft tissue mineralization. It primarily exerts its function through the generation of pyrophosphate, a key inhibitor of hydroxyapatite formation. Several previous studies have suggested that ENPP1 also contributes to a range of human diseases including diabetes, cancer, cardiovascular disease, and osteoarthritis. In this review, we summarize the pathological roles of ENPP1 in mineralization and these soft tissue disorders. We also discuss the underlying mechanisms through which ENPP1 exerts its pathological effects. A fuller understanding of the pathways through which ENPP1 acts may help to develop novel therapeutic strategies for these commonly diagnosed morbidities.

Extragonadal Effects of Follicle-Stimulating Hormone on Osteoporosis and Cardiovascular Disease in Women during Menopausal Transition.

The risk of osteoporosis and cardiovascular disease increases significantly in postmenopausal women. Until recently, the underlying mechanisms have been primarily attributed to estrogen decline following menopause. However, follicle-stimulating hormone (FSH) levels rise sharply during menopausal transition and are maintained at elevated levels for many years. FSH receptor has been detected in various extragonadal sites, including osteoclasts and endothelial cells. Recent advances suggest FSH may contribute to postmenopausal osteoporosis and cardiovascular disease. Here, we review the key actions through which FSH contributes to the risk of osteoporosis and cardiovascular disease in women as they transition through menopause. Advancing our understanding of the precise mechanisms through which FSH promotes osteoporosis and cardiovascular disease may provide new opportunities for improving health-span for postmenopausal women.

Functional Characterization of the Osteoarthritis Genetic Risk Residing at ALDH1A2 Identifies rs12915901 as a Key Target Variant.

OBJECTIVE: To identify the functional single-nucleotide polymorphisms (SNPs) and mechanisms conferring increased risk of hand osteoarthritis (OA) at the ALDH1A2 locus, which is a retinoic acid regulatory gene. METHODS: Tissue samples from 247 patients with knee, hip, or hand OA who had undergone joint surgery were included. RNA-sequencing analysis was used to investigate differential expression of ALDH1A2 and other retinoic acid signaling pathway genes in cartilage. Expression of ALDH1A2 in joint tissues obtained from multiple sites was quantified using quantitative reverse transcription-polymerase chain reaction. Allelic expression imbalance (AEI) was measured by pyrosequencing. The consequences of ALDH1A2 depletion by RNA interference were assessed in primary human chondrocytes. In silico and in vitro analyses were used to pinpoint which, among 62 highly correlated SNPs, could account for the association at the locus. RESULTS: ALDH1A2 expression was observed across multiple joint tissue samples, including osteochondral tissue from the hand. The expression of ALDH1A2 and of several retinoic acid signaling genes was different in diseased cartilage compared to non-diseased cartilage, with ALDH1A2 showing lower levels in OA cartilage. Experimental depletion of ALDH1A2 resulted in changes in the expression levels of a number of chondrogenic markers, including SOX9. In addition, reduced expression of the OA risk-conferring allele was witnessed in a number of joint tissues, with the strongest effect in cartilage. The intronic SNP rs12915901 recapitulated the AEI observed in patient tissues, while the Ets transcription factors were identified as potential mediators of this effect. CONCLUSION: The ALDH1A2 locus seems to increase the risk of hand OA through decreased expression of ALDH1A2 in joint tissues, with the effect dependent on rs12915901. These findings indicate a mechanism that may now be targeted to modulate OA risk.

Inhibition of arterial medial calcification and bone mineralization by extracellular nucleotides: The same functional effect mediated by different cellular mechanisms.

Arterial medial calcification (AMC) is thought to share some outward similarities to skeletal mineralization and has been associated with the transdifferentiation of vascular smooth muscle cells (VSMCs) to an osteoblast-like phenotype. ATP and UTP have previously been shown to inhibit bone mineralization. This investigation compared the effects of extracellular nucleotides on calcification in VSMCs with those seen in osteoblasts. ATP, UTP and the ubiquitous mineralization inhibitor, pyrophosphate (PPi ), dose dependently inhibited VSMC calcification by ≤85%. Culture of VSMCs in calcifying conditions was associated with an increase in apoptosis; treatment with ATP, UTP, and PPi reduced apoptosis to levels seen in non-calcifying cells. Extracellular nucleotides had no effect on osteoblast viability. Basal alkaline phosphatase (TNAP) activity was over 100-fold higher in osteoblasts than VSMCs. ATP and UTP reduced osteoblast TNAP activity (≤50%) but stimulated VSMC TNAP activity (≤88%). The effects of extracellular nucleotides on VSMC calcification, cell viability and TNAP activity were unchanged by deletion or inhibition of the P2Y2 receptor. Conversely, the actions of ATP/UTP on bone mineralization and TNAP activity were attenuated in osteoblasts lacking the P2Y2 receptor. Ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) hydrolyses ATP and UTP to produce PPi . In both VSMCs and osteoblasts, deletion of NPP1 blunted the inhibitory effects of extracellular nucleotides suggesting involvement of P2 receptor independent pathways. Our results show that although the overall functional effect of extracellular nucleotides on AMC and bone mineralization is similar there are clear differences in the cellular mechanisms mediating these actions.

Isolation and Characterization of Primary Rat Valve Interstitial Cells: A New Model to Study Aortic Valve Calcification.

Calcific aortic valve disease (CAVD) is characterized by the progressive thickening of the aortic valve leaflets. It is a condition frequently found in the elderly and end-stage renal disease (ESRD) patients, who commonly suffer from hyperphosphatemia and hypercalcemia. At present, there are no medication therapies that can stop its progression. The mechanisms that underlie this pathological process remain unclear. The aortic valve leaflet is composed of a thin layer of valve endothelial cells (VECs) on the outer surfaces of the aortic cusps, with valve interstitial cells (VICs) sandwiched between the VECs. The use of a rat model enables the in vitro study of ectopic calcification based on the in vivo physiopathological serum phosphate (Pi) and calcium (Ca) levels of patients who suffer from hyperphosphatemia and hypercalcemia. The described protocol details the isolation of a pure rat VIC population as shown by the expression of VIC markers: alpha-smooth muscle actin (α-SMA) vimentin and tissue growth factor beta (TGFß) 1 and 2, and the absence of cluster of differentiation (CD) 31, a VEC marker. By expanding these VICs, biochemical, genetic, and imaging studies can be performed to study and unravel the key mediators underpinning CAVD.

End stage renal disease-induced hypercalcemia may promote aortic valve calcification via Annexin VI enrichment of valve interstitial cell derived-matrix vesicles.

Patients with end-stage renal disease (ESRD) have elevated circulating calcium (Ca) and phosphate (Pi), and exhibit accelerated progression of calcific aortic valve disease (CAVD). We hypothesized that matrix vesicles (MVs) initiate the calcification process in CAVD. Ca induced rat valve interstitial cells (VICs) calcification at 4.5 mM (16.4-fold; p < 0.05) whereas Pi treatment alone had no effect. Ca (2.7 mM) and Pi (2.5 mM) synergistically induced calcium deposition (10.8-fold; p < 0.001) in VICs. Ca treatment increased the mRNA of the osteogenic markers Msx2, Runx2, and Alpl (p < 0.01). MVs were harvested by ultracentrifugation from VICs cultured with control or calcification media (containing 2.7 mM Ca and 2.5 mM Pi) for 16 hr. Proteomics analysis revealed the marked enrichment of exosomal proteins, including CD9, CD63, LAMP-1, and LAMP-2 and a concomitant up-regulation of the Annexin family of calcium-binding proteins. Of particular note Annexin VI was shown to be enriched in calcifying VIC-derived MVs (51.9-fold; p < 0.05). Through bioinformatic analysis using Ingenuity Pathway Analysis (IPA), the up-regulation of canonical signaling pathways relevant to cardiovascular function were identified in calcifying VIC-derived MVs, including aldosterone, Rho kinase, and metal binding. Further studies using human calcified valve tissue revealed the co-localization of Annexin VI with areas of MVs in the extracellular matrix by transmission electron microscopy (TEM). Together these findings highlight a critical role for VIC-derived MVs in CAVD. Furthermore, we identify calcium as a key driver of aortic valve calcification, which may directly underpin the increased susceptibility of ESRD patients to accelerated development of CAVD.

Identification of characteristic TRB V usage in HBV-associated HCC by using differential expression profiling analysis.

Liver cancer is one of the most common cancers worldwide. CDR3 sequencing-based immune repertoire can be closely associated with cancer prognosis and development. Identifying the specific interaction between the TCR and cellular antigens is important for developing novel immunotherapeutic approaches for the treatment of cancer. The rearranged TCRß loci amplified using Vß- and Jß-specific primers by multi-PCR and sequenced using high-throughput sequencing (HTS) in liver cancers were compared with those of T cells from healthy adult peripheral blood and from adjacent liver tissue. The T-cell repertoires within each tumor show strong similarity to one another but are distinct from those of the circulating T-cell repertoire. In addition, our results demonstrate that there are significant differences in the T-cell repertoires of HCC (hepatocellular carcinoma), ICC (intrahepatic cholangiocarcinoma), and MHC (mixed hepatocellular and cholangiocellular carcinoma). Furthermore, we found that the highly expanded clone (HEC) ratio in blood samples from liver cancer patients differed significantly from those in the blood of healthy adults and hepatitis patients (p < 0.001). The above results suggest that comparison of the T-cell repertoires of tissue and blood could be used to distinguish liver cancer patients from healthy adults and from hepatitis patients. In the future, the diversity of CDR3 sequences in liver cancer may prove to be a useful and novel biomarker for detecting aggressive tumors with high invasive or metastatic capacity.

A protective role for FGF-23 in local defence against disrupted arterial wall integrity?

Increasing interest is focusing on the role of the FGF-23/Klotho axis in mediating vascular calcification. However, the underpinning mechanisms have yet to be fully elucidated. Murine VSMCs were cultured in calcifying medium for a 21 d period. FGF-23 mRNA expression was significantly up-regulated by 7d (1.63-fold; P<0.001), with a concomitant increase in protein expression. mRNA and protein expression of both FGFR1 and Klotho were confirmed. Increased FGF-23 and Klotho protein expression was also observed in the calcified media of Enpp1(-/-) mouse aortic tissue. Reduced calcium deposition was observed in calcifying VSMCs cultured with recombinant FGF-23 (10 ng/ml; 28.1% decrease; P<0.01). Calcifying VSMCs treated with PD173074, an inhibitor of FGFR1 and FGFR3, showed significantly increased calcification (50 nM; 87.8% increase; P<0.001). FGF-23 exposure induced phosphorylation of ERK1/2. Treatment with FGF-23 in combination with PD98059, an ERK1/2 inhibitor, significantly increased VSMC calcification (10 µM; 41.3% increase; P<0.01). Use of FGF-23 may represent a novel therapeutic strategy for inhibiting vascular calcification.