Smooth muscle cell-specific matrix metalloproteinase 3 deletion reduces osteogenic transformation and medial artery calcification.

AIMS: Vascular calcification is highly prevalent in atherosclerosis, diabetes, and chronic kidney disease. It is associated with increased morbidity and mortality in patients with cardiovascular disease. Matrix metalloproteinase 3 (MMP-3), also known as stromelysin-1, is part of the large matrix metalloproteinase family. It can degrade extracellular matrix components of the arterial wall including elastin, which plays a central role in medial calcification. In this study, we sought to determine the role of MMP-3 in medial calcification. METHODS AND RESULTS: We found that MMP-3 was increased in rodent models of medial calcification as well as in vascular smooth muscle cells (SMCs) cultured in a phosphate calcification medium. It was also highly expressed in calcified tibial arteries in patients with peripheral arterial disease (PAD). Knockdown and inhibition of MMP-3 suppressed phosphate-induced SMC osteogenic transformation and calcification, whereas the addition of a recombinant MMP-3 protein facilitated SMC calcification. In an ex vivo organ culture model and a rodent model of medial calcification induced by vitamin D3, we found that MMP-3 deficiency significantly suppressed medial calcification in the aorta. We further found that medial calcification and osteogenic transformation were significantly reduced in SMC-specific MMP-3-deficient mice, suggesting that MMP-3 in SMCs is an important factor in this process. CONCLUSION: These findings suggest that MMP-3 expression in vascular SMCs is an important regulator of medial calcification and that targeting MMP-3 could provide a therapeutic strategy to reduce it and address its consequences in patients with PAD.

Hop2 interacts with the transcription factor CEBPα and suppresses adipocyte differentiation.

CCAAT enhancer binding protein (CEBP) transcription factors (TFs) are known to promote adipocyte differentiation; however, suppressors of CEBP TFs have not been reported thus far. Here, we find that homologous chromosome pairing protein 2 (Hop2) functions as an inhibitor for the TF CEBPα. We found that Hop2 mRNA is highly and specifically expressed in adipose tissue, and that ectopic Hop2 expression suppresses reporter activity induced by CEBP as revealed by DNA transfection. Recombinant and ectopically expressed Hop2 was shown to interact with CEBPα in pull-down and coimmunoprecipitation assays, and interaction between endogenous Hop2 and CEBPα was observed in the nuclei of 3T3 preadipocytes and adipocytes by immunofluorescence and coimmunoprecipitation of nuclear extracts. In addition, Hop2 stable overexpression in 3T3 preadipocytes inhibited adipocyte differentiation and adipocyte marker gene expression. These in vitro data suggest that Hop2 inhibits adipogenesis by suppressing CEBP-mediated transactivation. Consistent with a negative role for Hop2 in adipogenesis, ablation of Hop2 (Hop2-/-) in mice led to increased body weight, adipose volume, adipocyte size, and adipogenic marker gene expression. Adipogenic differentiation of isolated adipose-derived mesenchymal stem cells showed a greater number of lipid droplet-containing colonies formed in Hop2-/- adipose-derived mesenchymal stem cell cultures than in wt controls, which is associated with the increased expression of adipogenic marker genes. Finally, chromatin immunoprecipitation revealed a higher binding activity of endogenous CEBPα to peroxisome proliferator-activated receptor γ, a master adipogenic TF, and a known CEBPα target gene. Therefore, our study identifies for the first time that Hop2 is an intrinsic suppressor of CEBPα and thus adipogenesis in adipocytes.

Recovery of limb perfusion and function after hindlimb ischemia is impaired by arterial calcification.

Medial artery calcification results from deposition of calcium hydroxyapatite crystals on elastin layers, and osteogenic changes in vascular smooth muscle cells. It is highly prevalent in patients with chronic kidney disease, diabetes, and peripheral artery disease (PAD), and when identified in lower extremity vessels, it is associated with increased amputation rates. This study aims to evaluate the effects of medial calcification on perfusion and functional recovery after hindlimb ischemia in rats. Medial artery calcification and acute limb ischemia were induced by vitamin D3 (VitD3 ) injection and femoral artery ligation in rats. VitD3 injection robustly induced calcification in the medial layer of femoral arteries in vivo. Laser Doppler perfusion imaging revealed that perfusion decreased and then partially recovered after hindlimb ischemia in vehicle-injected rats. In contrast, VitD3 -injected rats showed markedly impaired recovery of perfusion following limb ischemia. Accordingly, rats with medial calcification showed worse ischemia scores and delayed functional recovery compared with controls. Immunohistochemical and histological staining did not show differences in capillary density or muscle morphology between VitD3 - and vehicle-injected rats at 28 days after femoral artery ligation. The evaluation of cardiac and hemodynamic parameters showed that arterial stiffness was increased while cardiac function was preserved in VitD3 -injected rats. These findings suggest that medial calcification may contribute to impaired perfusion in PAD by altering vascular compliance, however, the specific mechanisms remain poorly understood. Reducing or slowing the progression of arterial calcification in patients with PAD may improve clinical outcomes.

Hop2 Interacts with ATF4 to Promote Osteoblast Differentiation.

Activating transcription factor 4 (ATF4) is a member of the basic leucine zipper (bZip) transcription factor family required for the terminal differentiation of osteoblasts. Despite its critical importance as one of the three main osteoblast differentiation transcription factors, regulators of osteoblast terminal maturation remain poorly defined. Here we report the identification of homologous pairing protein 2 (Hop2) as a dimerization partner of ATF4 in osteoblasts via the yeast two-hybrid system. Deletional mapping revealed that the Zip domain of Hop2 is necessary and sufficient to bind ATF4 and to enhance ATF4-dependent transcription. Ectopic Hop2 expression in preosteoblasts increased endogenous ATF4 protein content and accelerated osteoblast differentiation. Mice lacking Hop2 (Hop2-/- ) have a normal stature but exhibit an osteopenic phenotype similar to the one observed in Atf4-/- mice, albeit milder, which is associated with decreased Osteocalcin mRNA expression and reduced type I collagen synthesis. Compound heterozygous mice (Atf4+/- :Hop2+/- ) display identical skeletal defects to those found in Hop2-/- mice. These results indicate that Hop2 plays a previous unknown role as a determinant of osteoblast maturation via its regulation of ATF4 transcriptional activity. Our work for the first time reveals a function of Hop2 beyond its role in guiding the alignment of homologous chromosomes. © 2019 American Society for Bone and Mineral Research.

Medial artery calcification increases neointimal hyperplasia after balloon injury.

Arterial calcification predicts accelerated restenosis after angioplasty and stenting. We studied the effects of calcification on neointimal hyperplasia after balloon injury in the rat carotid. Arterial calcification was induced by subcutaneous injection of vitamin D3 or by adventitial application of calcium chloride. After balloon catheter injury, neointimal hyperplasia was significantly increased in rats with medial calcification compared with controls. Neointimal cell proliferation in calcified arteries as assessed by proliferating cell nuclear antigen (PCNA) staining was also higher. In calcified arteries, bone morphogenetic protein 2 (BMP-2)levels were increased at the time of injury suggesting a possible explanation for the altered responses. In vascular smooth muscle cells (SMCs) grown under calcifying conditions , stimulation with BMP-2 significantly increased cell proliferation, however, this did not occur in those grown under non-calcifying conditions. These data suggest that neointimal hyperplasia is accelerated in calcified arteries and that this may be due in part to increased BMP-2 expression in medial SMCs. Treatments aimed at inhibiting restenosis in calcified arteries may differ from those that work in uncalcified vessels.

Enhancing Chlorobenzene Biodegradation by Delftia tsuruhatensis Using a Water-Silicone Oil Biphasic System.

In this study, a water-silicone oil biphasic system was developed to enhance the biodegradation of monochlorobenzene (CB) by Delftia tsuruhatensis LW26. Compared to the single phase, the biphasic system with a suitable silicone oil fraction (v/v) of 20% allowed a 2.5-fold increase in the maximum tolerated CB concentration. The CB inhibition on D. tsuruhatensis LW26 was reduced in the presence of silicone oil, and the electron transport system activity was maintained at high levels even under high CB stress. Adhesion of cells to the water-oil interface at the water side was observed using confocal laser scanning microscopy. Nearly 75% of cells accumulated on the interface, implying that another interfacial substrate uptake pathway prevailed besides that initiated by cells in the aqueous phase. The 8-fold increase in cell surface hydrophobicity upon the addition of 20% (v/v) silicone oil showed that silicone oil modified the surface characteristics of D. tsuruhatensis LW26. The protein/polysaccharide ratio of extracellular polymeric substances (EPS) from D. tsuruhatensis LW26 presented a 3-fold enhancement. These results suggested that silicone oil induced the increase in the protein content of EPS and rendered cells hydrophobic. The resulting hydrophobic cells could adhere on the water-oil interface, improving the mass transfer by direct CB uptake from silicone oil.

Inhibition of endo-lysosomal function exacerbates vascular calcification.

Vascular calcification is a pathologic response to mineral imbalances and is prevalent in atherosclerosis, diabetes mellitus, and chronic kidney disease. When located in the media, it is highly associated with increased cardiovascular morbidity and mortality, particularly in patients on dialysis. Vascular calcification is tightly regulated and controlled by a series of endogenous factors. In the present study, we assess the effects of lysosomal and endosomal inhibition on calcification in vascular smooth muscle cells (VSMCs) and aortic rings. We observed that lysosomal function was increased in VSMCs cultured in calcification medium containing 3.5 mM inorganic phosphate (Pi) and 3 mM calcium (Ca2+) for 7 days. We also found that the lysosomal marker lysosome-associated membrane protein 2 was markedly increased and colocalized with osteogenic markers in calcified aortas from vitamin D3-treated rats. Interestingly, both the lysosomal inhibitor chloroquine and the endosomal inhibitor dynasore dose-dependently enhanced Pi + Ca2+-mediated VSMC calcification. Inhibition of lysosomal and endosomal function also promoted osteogenic transformation of VSMCs. Additionally, lysosome inhibition increased Pi-induced medial calcification of aortic rings ex vivo. These data suggest that the endosome-lysosome system may play a protective role in VSMC and medial artery calcification.

[Isolation and Identification of a Chlorobenzene-degrading Bacterium and Its Degradation Characteristics].

A bacterium strain LW26 which could utilize chlorobenzene (CB) as sole carbon and energy source was isolated from a biotrickling filter reactor treating CB-contaminated off-gas. Based on its morphological and physiological characteristics, as well as the analysis of 16S rRNA gene sequence and Biolog test, the strain LW26 was identified as Delftia tsuruhatensis. To our best knowledge, it is the first time that the strain Delftia tsuruhatensis was applied for CB purification. In this study, the effects of temperature, pH, initial CB concentration and Cl- concentration on the biodegradation were investigated. The results showed that the optimal temperature and pH for CB biodegradation were 25℃ and 7.0,respectively; the maximum CB tolerated concentration for LW26 was as high as 500 mg·L-1; when the concentration of Cl- was above 0.14 mol·L-1, the CB degradation was significantly restrained. The degrading process of the strain LW26 followed the Haldane kinetic model and the maximum specific growth rate and the maximum specific degradation rate were 0.42 h-1 and 2.53 h-1, respectively.GC-MS analysis of the metabolites revealed that CB was firstly converted to o-chlorophenol by strain LW26. Combined with the activity of catechol dioxygenase, it can be speculated that CB was finally mineralized to CO2, or converted to cell biomass after processes of ortho cleavage,dechlorination and oxidation.

Dorsomorphin homologue 1, a highly selective small-molecule bone morphogenetic protein inhibitor, suppresses medial artery calcification.

BACKGROUND: Medial artery calcification develops in diabetes, chronic kidney disease, and as part of the aging process. It is associated with increased morbidity and mortality in vascular patients. Bone morphogenetic proteins (BMPs) have previously been implicated in the initiation and progression of vascular calcification. We thus evaluated whether dorsomorphin homologue 1 (DMH1), a highly selective BMP inhibitor, could attenuate vascular calcification in vitro and in an organ culture model of medial calcification. METHODS: Confluent human aortic smooth muscle cells (SMCs) were cultured in calcification medium containing 3.0 mM inorganic phosphate (Pi) for 7 days with or without DMH1. Medial calcification was assessed using an aortic organ culture model. Calcification was visualized by alizarin red S staining, and calcium concentration was assessed by an o-cresolphthalein complexone calcium assay. Osteogenic cell and vascular SMC markers were determined by Western blot, quantitative reverse transcription polymerase chain reaction, and immunohistochemical staining. RESULTS: DMH1 reduced Pi-induced calcium deposition in human SMCs. It also antagonized human recombinant BMP2-induced calcium accumulation. Western blot further revealed that DMH1 was able to block Pi-mediated upregulation of the osteoblast markers osterix and alkaline phosphatase and downregulation of the SMC markers smooth muscle myosin heavy chain and SM22α as well as p-Smad1/5/8, suggesting that DMH1 may regulate SMC osteogenic differentiation through the BMP/Smad1/5/8 signaling pathway. Finally, using an ex vivo aortic ring organ culture model, we observed that DMH1 reduces Pi-induced aortic medial calcification. CONCLUSIONS: The selective BMP inhibitor DMH1 can inhibit calcium accumulation in vascular SMCs and arterial segments exposed to elevated phosphate levels. Such small molecules may have clinical utility in reducing medial artery calcification in our population of vascular patients.

Transforming growth factor ß suppresses osteoblast differentiation via the vimentin activating transcription factor 4 (ATF4) axis.

ATF4 is an osteoblast-enriched transcription factor of the leucine zipper family. We recently identified that vimentin, a leucine zipper-containing intermediate filament protein, suppresses ATF4-dependent osteocalcin (Ocn) transcription and osteoblast differentiation. Here we show that TGFß inhibits ATF4-dependent activation of Ocn by up-regulation of vimentin expression. Osteoblasts lacking Atf4 (Atf4(-/-)) were less sensitive than wild-type (WT) cells to the inhibition by TGFß on alkaline phosphatase activity, Ocn transcription and mineralization. Importantly, the anabolic effect of a monoclonal antibody neutralizing active TGFß ligands on bone in WT mice was blunted in Atf4(-/-) mice. These data establish that ATF4 is required for TGFß-related suppression of Ocn transcription and osteoblast differentiation in vitro and in vivo. Interestingly, TGFß did not directly regulate the expression of ATF4; instead, it enhanced the expression of vimentin, a negative regulator of ATF4, at the post-transcriptional level. Accordingly, knockdown of endogenous vimentin in 2T3 osteoblasts abolished the inhibition of Ocn transcription by TGFß, confirming an indirect mechanism by which TGFß acts through vimentin to suppress ATF4-dependent Ocn activation. Furthermore, inhibition of PI3K/Akt/mTOR signaling, but not canonical Smad signaling, downstream of TGFß, blocked TGFß-induced synthesis of vimentin, and inhibited ATF4-dependent Ocn transcription in osteoblasts. Thus, our study identifies that TGFß stimulates vimentin production via PI3K-Akt-mTOR signaling, which leads to suppression of ATF4-dependent Ocn transcription and osteoblast differentiation.

Molecular cloning and expression analysis of porcine ghrelin o-acyltransferase.

The peptide hormone ghrelin is secreted in the stomach, with unique N-octanoylation at serine 3, which is a requirement for its functionality. These functions include growth hormone release, appetite stimulation, gastrointestinal motility, glucose regulation, and cell proliferation. The enzyme responsible for ghrelin acylation was recently identified as ghrelin O-acyltransferase (GOAT). In this study, porcine GOAT was cloned and characterized. A full-length cDNA of GOAT of 2013 bp was obtained, which included a 70-bp 5' UTR, a 635-bp 3' UTR, and a 1308-bp open reading frame encoding a protein of 415 amino acids. The GOAT and ghrelin mRNAs are co-expressed in stomach, pancreas, and duodenum at high levels. GOAT was also detected in liver, lung, brain, testis, spleen, kidney, heart, muscle, lipid, and ovary. Our results provide an important basis for further research on GOAT function and the relationship between ghrelin and GOAT.

Repression of Slc24a5 can reduce pigmentation in chicken.

Slc24a5 is a putative cation transporter, which is a member of the potassium-dependent sodium-calcium ion exchanger family. Association of the Slc24a5 gene with pigmentation has been established in Zebrafish, mice and humans. Despite these findings, its function in chicken remains unknown. The intent of this study was to describe the association of Slc24a5 with respect to melanin deposition in the chicken using RNAi. The objective was to detect the variety of melanin deposition caused by the down-regulation of Slc24a5 in chicken retinal pigment epithelium (RPE) cells. Nine siRNAs that targeted against Slc24a5 mRNA were found to be effective in suppressing Slc24a5 gene expression in 293FT cells. The most effective target tested effectively inhibited Slc24a5 expression in mRNA and subsequently reduced protein levels in RPE cells. These results show that down-regulation of Slc24a5 results in a reduction of melanin content, as well as a decrease of melaeneous type ß and type χ melanosomes simultaneously. Taken together, this work suggests that Slc24a5 function is conserved in the chicken, and necessary for melanin synthesis.

Knockdown of the prion gene expression by RNA interference in bovine fibroblast cells.

PRNP is the gene encoding prion protein whose misfolded and ß-sheet-rich isoform is the infectious agent of transmissible spongiform encephalopathy (TSE). TSE, also called prion diseases, cause fatal neurodegenerative and transmissible disorders in human and animals. Among these diseases, bovine spongiform encephalopathy (BSE) has tremendous impact on economy and human health in the world. In the present study, we hypothesize suppression of the PRNP gene expression could raise resistance to BSE in cattle by using vector-based small interfering RNA (siRNA) expression systems. Therefore, the objective was to screen effective DNA-encoding short hairpin RNAs (shRNAs) which could knockdown the PRNP gene expression in bovine fibroblast cells. Human U6 promoter was employed to drive shRNA transcription from the DNA vector, and seven shRNAs, that designed to target coding region and 3' untranslated region of the PRNP gene, were selected. Four out of seven shRNAs tested were found to be effective in inhibiting the PRNP gene expression, and the most significant suppression level was as much as 62.9% evidenced by real-time RT-PCR. Furthermore, the protein abundance was obviously reduced compared to the control. Overall, the present study demonstrated that vector-based siRNA expression systems is an efficient approach to knockdown the PRNP gene expression in bovine fibroblast cells and thereby provide donor cells for somatic cell nuclear cloning to produce cattle that is resistant to prion related diseases.