Osteoblastic Transformation Research Articles

Aortic Stenosis Prevention: Is a New Cardiovascular Disease Paradigm Coming of Age?

Calcific aortic stenosis (CAS) is currently recognized as the third most frequent cardiovascular disorder in persons aged above 60 years, after atherosclerotic disease and hypertension, and together with its precursor aortic sclerosis it has been found in more than 30% of elderly individuals. CAS is an active multifactorial process characterized by a progressive fibro-calcific remodeling and thickening of the AV leaflets caused by hemodynamic flow factors, genetic factors, lipoprotein deposition, oxidation, chronic inflammation, immunomodulators, and finally osteoblastic transformation of cardiac. Herein a comprehensive state-of-the-art paper is presented regarding the underlying pathophysiological mechanisms of CAS and the potential preventive strategies as an alternative to surgical and interventional treatment.

Noncoding RNA, friend or foe for nephrolithiasis?

Nephrolithiasis is one of the most common diseases in urology, characterized by notable incidence and recurrence rates, leading to significant morbidity and financial burden. Despite its prevalence, the precise mechanisms underlying stone formation remain incompletely understood, thus hindering significant advancements in kidney stone management over the past three decades. Investigating the pivotal biological molecules that govern stone formation has consistently been a challenging and high-priority task. A significant portion of mammalian genomes are transcribed into noncoding RNAs (ncRNAs), which have the ability to modulate gene expression and disease progression. They are thus emerging as a novel target class for diagnostics and pharmaceutical exploration. In recent years, the role of ncRNAs in stone formation has attracted burgeoning attention. They have been found to influence stone formation by regulating ion transportation, oxidative stress injury, inflammation, osteoblastic transformation, autophagy, and pyroptosis. These findings contributes new perspectives on the pathogenesis of nephrolithiasis. To enhance our understanding of the diagnostic and therapeutic potential of nephrolithiasis-associated ncRNAs, we summarized the expression profiles, biological functions, and clinical significance of these ncRNAs in the current review.

Abstract P3070: Aortic Valve Calcification Is Accelerated By DNMT3A And TET2 Gene Driver Mutations Associated With Clonal Hematopoiesis

Background: Clonal hematopoiesis (CH) due to DNMT3A or TET2-driver mutations associates with heightened inflammation and a worse prognosis among patients with severe calcific aortic valve stenosis (AS). However, it is unknown what role CH plays in the pathogenesis of AS. AS is the most common age-related heart valve disease, with no medical therapy to halt progression. Methods and Results: Single-cell RNA-sequencing of immune cells of CAVD patients (n=13) with and without the most prevalent CH-mutations (DNMT3A and TET2), having a mean CH-driver gene variant allele frequency was 6.5% for DNMT3A and 17.9% for TET2. Monocytes of patients harboring DNMT3A and TET2 mutations shared 836 upregulated genes, which were associated with glycolytic, pro-inflammatory activation (CXCL10, IL38) along with paracrine pro-calcific factors (Oncostatin M (OSM), S100A9), signifying that CH might be causally involved in AS. Indeed, silencing of TET2 or DNMT3A in macrophages promotes M1-like polarization and increases release of pro-calcific mediators, particularly S100A9 and OSM. To assess whether CH-gene silenced macrophages may stimulate mesenchymal cells to secrete calcium, secreted factors from TET2 or DNMT3A-silenced macrophages were applied mesenchymal cells. Indeed, CH-gene silenced supernatants induced osteoblastic differentiation of mesenchymal cells, evidenced by increased Alizarin red calcium staining and elevated levels of ALP and RUNX2. This CH-mediated enhancement in osteoblastic transformation could be ablated by silencing of OSM. Finally, valves from atheroprone Ldlr-/- mice receiving Tet2-/- bone marrow transplants demonstrated increased total calcified area (p<0.05, 1.49-fold) along with increased numbers of calcification deposits (p<0.05, 2.33-fold) as shown by von Kossa staining, along with enhanced OSM and S100A9 levels. Conclusion: This is the first study to show that somatic CH-driver mutations in monocytes may accelerate AS. Moreover, calcification was ablated via silencing of OSM in CH-gene silenced macrophages. These data suggest Oncostatin M may be a therapeutically promising target in halting the development of AS in patients harboring CH mutations.

Mitochondria and vascular calcification in chronic kidney disease: Lessons learned from the past to improve future therapy.

Chronic kidney disease-mineral and bone disorders (CKD-MBD) is a common complication of CKD Stages 3-5. Hyperphosphatemia is one of the major metabolic components of CKD-MBD, frequently resulting in vascular calcification (VC) in advanced-stage patients. Also, a long duration of renal replacement therapy can cause the worsening of VC, leading to increased cardiovascular morbidity and mortality. Vascular smooth muscle cells play an important role in the development of VC through osteochondrogenic transformation and the apoptotic process. It has been shown that mitochondrial dysfunction is involved with CKD progression, and excessive oxidative stress can aggravate osteoblastic transformation and VC. Currently, novel interventions targeting mitochondrial function and dynamics, in addition to mitochondrial antioxidants, have been studied with the aim of attenuating VC. This review aims to comprehensively summarize and discuss the experimental and clinical reports concerning mitochondrial studies, along with the purpose of interventions that can improve the outcomes of VC among CKD patients.

32736 Characterizing calciphylaxis-like lesions in mice predisposed to vascular calcification

Calciphylaxis is a rare yet fatal form of vascular calcification often associated with end-stage renal disease (uremic calciphylaxis) and nonuremic causes. Calciphylaxis is associated with medial arteriolar vascular calcification and results in ischemic subcutaneous necrosis. We hypothesized that tissue-nonspecific alkaline phosphatase (TNAP), which is known to cause osteoblastic transformation of vascular cells, can contribute to the calcification of small arterioles in the skin. Our study aimed to characterize a mouse model of nonuremic calciphylaxis secondary to alkaline phosphatase overexpression in the vasculature. Eleven wild-type (WT) and 11 TNAP mice were evaluated at 23-week-old age on a regular diet. Alizarin and hematoxylin and eosin (H&E) staining of trunk skin cryosections revealed calcium deposits in TNAP mice. As visualized by vascular contrast micro-CT, calcifications appeared primarily vascular in nature and localized to the dermis and subcutaneous adipose tissue. Noncontrast micro-CT (n = 4 per group) revealed that calcification was present in all TNAP mice compared with its complete absence in WT mice. The presence of vascular calcification in adipose-rich trunks of the TNAP mice is consistent with the pattern of lesion formation in human calciphylaxis patients. Our mice tissues are most reflective of the earliest histologic manifestations, which lack ulcerations or necrotic lesions, but may be characterized by gross skin changes with intact epithelium. Our findings suggest that increased alkaline phosphatase activity in the vasculature can contribute to the pathophysiology of nonuremic calciphylaxis.

The Effect of Activated FXIII, a Transglutaminase, on Vascular Smooth Muscle Cells.

Plasma factor XIII (pFXIII) is a heterotetramer of FXIII-A and FXIII-B subunits. The cellular form (cFXIII), a dimer of FXIII-A, is present in a number of cell types. Activated FXIII (FXIIIa), a transglutaminase, plays an important role in clot stabilization, wound healing, angiogenesis and maintenance of pregnancy. It has a direct effect on vascular endothelial cells and fibroblasts, which have been implicated in the development of atherosclerotic plaques. Our aim was to explore the effect of FXIIIa on human aortic smooth muscle cells (HAoSMCs), another major cell type in the atherosclerotic plaque. Osteoblastic transformation induced by Pi and Ca2+ failed to elicit the expression of cFXIII in HAoSMCs. EZ4U, CCK-8 and CytoSelect Wound Healing assays were used to investigate cell proliferation and migration. The Sircol Collagen Assay Kit was used to monitor collagen secretion. Thrombospondin-1 (TSP-1) levels were measured by ELISA. Cell-associated TSP-1 was detected by the immunofluorescence technique. The TSP-1 mRNA level was estimated by RT-qPCR. Activated recombinant cFXIII (rFXIIIa) increased cell proliferation and collagen secretion. In parallel, a 67% decrease in TSP-1 concentration in the medium and a 2.5-fold increase in cells were observed. TSP-1 mRNA did not change significantly. These effects of FXIIIa might contribute to the pathogenesis of atherosclerotic plaques.

Abstract 199: Conditional Inactivation Of Lysyl Oxidase In Smooth Muscle Cells Reduces Atherosclerosis Burden And Plaque Calcification In Hyperlipidemic Mice Through Suppression Of The β-catenin/tcf4 Signaling Axis.

Introduction: In atherosclerosis development and complications, the importance of lysyl oxidase (LOX), a copper-dependent amino-oxidase that crosslinks collagen and elastin and controls gene expression, is not well established. Hypothesis: Inactivation of LOX in smooth muscle cells (SMC) decreases atherosclerosis burden and plaque calcification in hyperlipidemic mice. Methods: qRT-PCR, Western blot, and scRNAseq demonstrated the loss of Lox gene expression in SMC of ApoE conditional mice (Lox f/f Myh11-CreERT2 ApoE-/-) but not in littermate control animals after tamoxifen injections. Surprisingly, vascular loss of LOX did not increase the risk of aneurysms nor affected the aorta's stiffness. However, it reduced atherosclerosis burden with respect to control mice (13±2 versus 23±1%, p < 0.01) after 16 weeks of high fat diet (HFD). There was also a significant reduction in calcium deposition (5±0.4 versus11.8±3, p < 0.05) within the plaque of LOX knockout mice versus control. Aortic whole genome transcriptomic analysis revealed a positive correlation between Lox gene expression and Frizzled-related protein 3 ( Frzb ), a biphasic modulator of Wnt signaling that facilitates the association of β-catenin with TCF4 in the nucleus. scRNAseq analysis confirmed the downregulation of Bmp2 and Myc , both β-catenin/TCF4 regulated genes, in SMC following gene deletion of Lox and 8 weeks of HFD. LOX knockout SMCs also had lower expression of Frzb and were more resistant to osteoblastic transformation in vitro. Conclusions: Vascular inactivation of LOX protects the vasculature from the proatherogenic effects of FRZB in SMCs.

Irisin alleviates vascular calcification by inhibiting VSMC osteoblastic transformation and mitochondria dysfunction via AMPK/Drp1 signaling pathway in chronic kidney disease

Background and aimsVascular calcification (VC) is an intricate active process, significantly controlled by vascular smooth muscle cells (VSMCs). Mitochondrial dysfunction plays a pivotal role in VC and VSMCs osteoblastic transformation. We previously reported that decreased levels of Irisin were independently associated with VC in hemodialysis patients. The present study aimed to investigate the role of Irisin in VC, especially in VSMCs osteoblastic transformation and mitochondrial function. MethodsIn vitro, VSMCs calcification was induced by β‐glycerophosphate, while in vivo VC was triggered by adenine and high phosphorus diet. Alizarin red, Von Kossa staining, and calcium and Alp activity were performed to test VC. Western blot and immunohistochemical staining were employed to analyze the expression of proteins associated with VSMCs osteoblastic transformation and AMPK signaling. Mitochondrial membrane potential (MMP) and structures were observed by immunofluorescence staining. ResultsIrisin alleviated VSMCs calcification induced by β‐glycerophosphate. Mechanistically, Irisin activated AMPK and downregulated the expression of Drp1, further alleviating mitochondria fission and VSMCs osteoblastic transformation. In vivo, Irisin decreased serum creatinine, urea and phosphorous levels in chronic kidney disease (CKD) mice. Importantly, Irisin treatment postponed CKD-associated VC with the upregulation of α-Sma and p-AMPK expression, and the downregulation of Runx2 and Drp1 expression. ConclusionsOur results firstly reveal that Irisin inhibits CKD-associated VC. Irisin suppresses VSMCs osteoblastic transformation and mitochondria dysfunction via AMPK/Drp1 signaling.

Osteomodulin attenuates smooth muscle cell osteogenic transition in vascular calcification.

RationaleVascular calcification is a prominent feature of late‐stage diabetes, renal and cardiovascular disease (CVD), and has been linked to adverse events. Recent studies in patients reported that plasma levels of osteomodulin (OMD), a proteoglycan involved in bone mineralisation, associate with diabetes and CVD. We hypothesised that OMD could be implicated in these diseases via vascular calcification as a common underlying factor and aimed to investigate its role in this context.Methods and resultsIn patients with chronic kidney disease, plasma OMD levels correlated with markers of inflammation and bone turnover, with the protein present in calcified arterial media. Plasma OMD also associated with cardiac calcification and the protein was detected in calcified valve leaflets by immunohistochemistry. In patients with carotid atherosclerosis, circulating OMD was increased in association with plaque calcification as assessed by computed tomography. Transcriptomic and proteomic data showed that OMD was upregulated in atherosclerotic compared to control arteries, particularly in calcified plaques, where OMD expression correlated positively with markers of smooth muscle cells (SMCs), osteoblasts and glycoproteins. Immunostaining confirmed that OMD was abundantly present in calcified plaques, localised to extracellular matrix and regions rich in α‐SMA+ cells. In vivo, OMD was enriched in SMCs around calcified nodules in aortic media of nephrectomised rats and in plaques from ApoE −/− mice on warfarin. In vitro experiments revealed that OMD mRNA was upregulated in SMCs stimulated with IFNγ, BMP2, TGFβ1, phosphate and β‐glycerophosphate, and by administration of recombinant human OMD protein (rhOMD). Mechanistically, addition of rhOMD repressed the calcification process of SMCs treated with phosphate by maintaining their contractile phenotype along with enriched matrix organisation, thereby attenuating SMC osteoblastic transformation. Mechanistically, the role of OMD is exerted likely through its link with SMAD3 and TGFB1 signalling, and interplay with BMP2 in vascular tissues.ConclusionWe report a consistent association of both circulating and tissue OMD levels with cardiovascular calcification, highlighting the potential of OMD as a clinical biomarker. OMD was localised in medial and intimal α‐SMA+ regions of calcified cardiovascular tissues, induced by pro‐inflammatory and pro‐osteogenic stimuli, while the presence of OMD in extracellular environment attenuated SMC calcification.

The Activation of ROS/NF-κB/MMP-9 Pathway Promotes Calcium-Induced Kidney Crystal Deposition.

Idiopathic hypercalciuria is an important risk factor for the formation of calcium-containing kidney stones. Matrix metalloproteinase-9 (MMP-9) is closely related to cell and tissue remodeling and is involved in ectopic tissue calcification. However, little is known about its role in kidney stone formation. In this study, we found that the expression of MMP-9 and that of osteoblastic-related proteins was increased in normal rat kidney epithelial-like (NRK-52E) cells following treatment with a high concentration of calcium, while the knockout or overexpression of MMP-9 could, respectively, significantly inhibit or upregulate the expression of osteoblastic-related proteins and calcium crystal deposition. In addition, apoptosis and calcium crystal deposition were significantly reduced in Sprague–Dawley rats with 1,25(OH)2D3-induced hypercalciuria following MMP-9 inhibitor I treatment. Furthermore, inhibiting reactive oxygen species (ROS) production or the nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) pathway significantly reduced calcium-induced MMP-9 expression and calcium crystal deposition. In summary, our results suggested that a high calcium concentration promotes epithelial–osteoblastic transformation and calcium crystal deposition in renal tubule cells by regulating the ROS/NF-κB/MMP-9 axis and identified a novel role for MMP-9 in regulating calcium-induced calcium crystal deposition in renal tubules.

The Effects of Adipose Derived Stromal Vascular Fraction and Platelet-Rich Plasma on Bone Healing of a Rat Model With Chronic Kidney Disease.

Chronic kidney disease (CKD) impairs osteoblast/osteoclast balance and damages bone structure with diminished mineralization and results in bone restoration disorders. In this study, we investigate the effects of adipose-derived stromal vascular fraction and platelet-rich plasma (PRP) on bone healing model in rats with CKD. Sprague-Dawley rats were separated into 4 groups. All groups except group I (healthy control) had CKD surgery using 5/6 nephrectomy model. All groups had intramedullary pin fixation after receiving bone fracture using drilling tools. Group II rats were used as control group for CKD. Group III rats received PRP treatment on fracture site. Group IV rats received PRP and stromal vascular fraction treatment on fracture site.Weight loss and blood samples were followed at the time of kidney surgery, third, sixth, and 12th weeks. Bone healing and callus formations were compared, biomechanically, radiologically, histopathologically, and immunohistochemically. Osteoblastic transformation of stem cells was assessed with DiI staining. Negative effects of CKD on bone healing were reduced by increasing mechanical, histological, radiological, and biochemical properties of the bone with stromal vascular fraction and PRP treatments. Although thickness of callus tissue delayed bone healing process, it also enhanced biomechanical features and bone tissue organization. Platelet-rich plasma and adipose-derived stromal vascular fraction treatments were effective for bone healing in animal model, which can be promising for clinical trials.

Hepatoma-derived growth factor enhances osteoblastic transformation of rat aortic vascular smooth muscle cells in vitro

AimsVascular smooth muscle cells (VSMCs) are important regulators of vascular functions and their conversion to osteoblasts is a key to development of vascular calcification. This study aimed to characterize in vitro effect of hepatoma-derived growth factor (HDGF) on phenotypic conversion of cultured aortic VSMCs into osteoblast-like cells. Materials and methodsCell proliferation and migration assays were used to examine cell behaviors. Western blotting, alkaline phosphatase activity and calcium staining were used to evaluate osteoblastic marker expression and function, respectively. Key findingsRecombinant HDGF treatment enhanced VSMC growth and motility. Treatment of osteogenic medium (OM) increased expression of not only HDGF but also osteoblastic markers, including Runx2 and osteopontin (OPN), while VSMC marker α-smooth muscle actin (α-SMA) declined. Coincidentally, HDGF and OM treatment alone stimulated signaling activities in both PI3K/Akt and MAPK pathways. Conversely, inhibition of Akt and p38 significantly blocked the OM-upregulated HDGF, Runx2, and OPN expression and NF-κB phosphorylation, but did not reversed the α-SMA downregulation, implicating the involvement of Akt and p38 activities in the osteoblastic transformation of VSMCs. Small interfering RNA-mediated HDGF gene silencing effectively prevented the Runx2 and OPN upregulation, alkaline phosphatase activation, and calcium deposition, but did not affect the α-SMA levels in the transformed cells, supporting the involvement of HDGF in regulation of Runx2 and OPN expression. SignificanceIn conclusion, in synergism with other osteogenic factor, HDGF may promote the progression of osteobastic transformation of VSMCs via Akt and p38 signaling pathways and contribute to vascular calcification in arteriosclerosis. Chemical compounds studied in this studyHDGF (PubChem CID:); LY294002 (PubChem CID: 3973); PD98059 (PubChem CID: 4713); SB203580 (PubChem CID: 176155); SB431542 (PubChem CID: 4521392); SP600125 (PubChem CID: 8515); Wortmannin (PubChem CID: 312145).

Evaluation of Osteoblastic Gene Expression in Human Mesenchymal Stem Cells

BackgroundOsteoblastic differentiation is a primary pathway to stimulate bone remodeling within regenerative medicine. Human mesenchymal stem cells (hMSCs) are an attractive model for bone regeneration due to their ability to differentiate into osteoblasts, thus promoting an osteogenic environment. With the development of bone biomaterials, it is important to establish a solid baseline of the genes involved in osteoblast differentiation. Once established, the genes regulating osteoblastic differentiation can be compared to any biomaterial product that may potentially speed up the process of bone regeneration. Therefore, the purpose of this study is to examine gene expression as hMSCs undergo osteoblastic differentiation.MethodshMSCs were extracted from human adipose tissue and cultured in DMEM‐F12 medium, 10% FBS. For experimental conditions, cells were cultured in either a growth medium (GM) or an osteogenic induced medium (OIM), supplemented with β‐glycerophosphate, ascorbic‐2‐phosphate, and dexamethasone. Cells were stained with Alizarin Red S staining to confirm calcium deposits of cells cultured in GM or OIM. hMSCs cultured in OIM were extracted for total RNA at days 3, 7, and 21, followed by cDNA synthesis for RT2 Profiler PCR Human Osteogenesis Array (Qiagen, Hilden, Germany). Gene expressions were analyzed using Qiagen Gene Globe software to determine the relative fold change.ResultshMSCs appear to express alkaline phosphatase (ALPL) and bone gammacarboxyglutamate protein (BGLAP) early throughout OIM culture. In contrast, Sp7 expression increases over time, while Sox9 peaks at Day 7 in comparison to the control at Day 3.ConclusionOur data collectively show a change in gene expressions associated with osteoblastic differentiation such as ALPL, BGLAP, Sp7, and SOX9. Sp7, a master osteoblastic transcription factor, increases over time suggesting hMSCs favoring into osteoblastic transformation. Interestingly, Sox9 is another transcription regulator known to commit hMSCs into osteo‐chondrogenic progenitors, and our data show its expression to increase before diminishing near control levels. However, further studies are needed to confirm the genetic pattern of hMSCS undergoing osteoblastic differentiation as well as the upstream mechanisms controlling their activation. This information will be useful in the application of bone biomaterials as a therapeutic treatment for bone regeneration.

CC chemokine receptor 2 functions in osteoblastic transformation of valvular interstitial cells

AimsCalcific aortic valve disease (CAVD) emerges as a challenging clinical issue, which is associated with high cardiovascular mortality. It has been demonstrated that osteoblastic transformation of AVICs is a key mechanism of CAVD and C-C motif chemokine receptors (CCRs) may favor this process. Thus, we aimed to investigate whether CCRs were involved in osteoblastic transformation of AVICs during the development CAVD. Main methodsWe first analyzed microarray data (GSE51472 and GSE12644) to identify differentially expressed genes between CAVD aortic valve tissue and normal samples, followed by verification of immunohistochemistry, qPCR and western blotting. Primary aortic valvular interstitial cells (AVICs) were incubated with specific inhibitors and/or siRNA of CCR2 and CCL2 under pro-calcifying medium. The levels of CCL2 in the medium were measured by ELISA. In addition, we used recombinant CCL2 to activate CCR2 in calcifying AVICs. Alizarin red S staining and calcium deposition were used to evaluate the degree of calcification. Western blotting was used to determine osteoblastic transformation of AVIC and total Akt and phosphorylated Akt expression. Key findingCCR2 levels were remarkably up-regulated in calcified aortic valve and calcifying AVICs. Silencing CCR2 inhibited the osteoblastic transformation and calcification of AVICs. In addition, recombinant CCL2 activated CCR2 and accelerated AVICs calcification through PI3K/Akt pathway. SignificanceWe characterize abnormal activation of CCL2/CCR2 axis as a promoter of AVICs osteoblastic transformation and calcification. Our findings implicate the CCL2/CCR2-PI3K/Akt pathway as a potential target for treatment of CAVD.

Uremic Patients with Increased Vascular Calcification Score Have Serum with High Calcific Potential: Role of Vascular Smooth Muscle Cell Osteoblastic Differentiation and Apoptosis

Background/Aims: Uremic patients experience premature vascular ageing that causes cardiovascular morbidity. In this study, we investigated the relationship between uremic serum calcific potential induced by high phosphate (Pi) and vascular calcification score (VCS). Methods: Vascular smooth muscle cells (VSMCs) were cultured with 3.5 mM Na₃PO₄ (Pi) with 10% uremic serum and calcium deposition, markers of osteoblastic transformation, and apoptosis were evaluated. Results: Culture with uremic serum and high-Pi significantly induced calcification (0.21 ± 0.03 vs. 8.05 ± 0.6; ctr vs. Pi; OD/mg protein; p < 0.01). We next stratified patients with respect of the degree of VCS in 2 groups: absence of vascular calcification (VC) “no VC group” and presence of VC “VC group”. We found that there was a significant correlation between VCS and uremic serum calcific potential induced by high Pi in vitro (p < 0.01). Interestingly, uremic sera of the “VC group” were more effective than sera from the “no VC group”, in downregulating α-actin and SM22α, after treatment with high-Pi (41.3 ± 4.7 vs. 23.3 ± 2.9 and 25.6 ± 6.8 vs. 8.14 ± 2.3; VC vs. no VC group, α-actin and SM22α respectively; Δ intensity area; p < 0.01). Similarly, sera from “VC group” were more effective than sera from “no VC group” in adjuvanting the high-Pi effect of increasing osteoblastic markers, such as bone morphogenic protein 2 (BMP2), osteocalcin (OC), and runt-related transcription factor 2 (RUNX2; 39.1 ± 11.3 vs. 5.0 ± 2.6 BMP2; 12.2 ± 4.2 vs. 1.7 ± 0.3 OC; 2.9 ± 0.4 vs. 1.2 ± 0.2 RUNX2; VC vs. no VC group respectively; p < 0.05). We found a similar pattern with significantly higher apoptosis and necrosis induction by sera from the “VC group” compared to the “no VC group” (2.05 ± 0.33 vs. 1.29 ± 0.13 and 54.1 ± 19.5 vs. 27.4 ± 10.6; Pi; VC group vs. no VC group; enrichment factor of apoptotic or necrotic fragments, respectively; p < 0.05). Conclusions: We conclude that VCS of end-stage renal disease patients significantly correlates with serum-calcific potential induced by high Pi. In addition, uremic patients with higher VCS have sera with a higher potential to induce VSMC osteoblastic trans-differentiation, apoptosis, and necrosis.

Emerging Role of Vitamins D and K in Modulating Uremic Vascular Calcification: The Aspect of Passive Calcification.

Vascular calcification is a critical complication in patients with chronic kidney disease (CKD) because it is predictive of cardiovascular events and mortality. In addition to the traditional mechanisms associated with endothelial dysfunction and the osteoblastic transformation of vascular smooth muscle cells (VSMCs), the regulation of calcification inhibitors, such as calciprotein particles (CPPs) and matrix vesicles plays a vital role in uremic vascular calcification in CKD patients because of the high prevalence of vitamin K deficiency. Vitamin K governs the gamma-carboxylation of matrix Gla protein (MGP) for inhibiting vascular calcification, and the vitamin D binding protein receptor is related to vitamin K gene expression. For patients with chronic kidney disease, adequate use of vitamin D supplements may play a role in vascular calcification through modulation of the calciprotein particles and matrix vesicles (MVs).

Doxorubicin induces ZAKα overexpression with a subsequent enhancement of apoptosis and attenuation of survivability in human osteosarcoma cells

Human osteosarcoma (OS) is a malignant cancer of the bone. It exhibits a characteristic malignant osteoblastic transformation and produces a diseased osteoid. A previous study demonstrated that doxorubicin (DOX) chemotherapy decreases human OS cell proliferation and might enhance the relative RNA expression of ZAK. However, the impact of ZAKα overexpression on the OS cell proliferation that is inhibited by DOX and the molecular mechanism underlying this effect are not yet known. ZAK is a protein kinase of the MAPKKK family and functions to promote apoptosis. In our study, we found that ZAKα overexpression induced an apoptotic effect in human OS cells. Treatment of human OS cells with DOX enhanced ZAKα expression and decreased cancer cell viability while increasing apoptosis of human OS cells. In the meantime, suppression of ZAKα expression using shRNA and inhibitor D1771 both suppressed the DOX therapeutic effect. These findings reveal a novel molecular mechanism underlying the DOX effect on human OS cells. Taken together, our findings demonstrate that ZAKα enhances the apoptotic effect and decreases cell viability in DOX-treated human OS cells.

Apelin: A novel inhibitor of vascular calcification in chronic kidney disease

BackgroundVascular calcification (VC) is closely related to cardiovascular events in chronic kidney disease (CKD). Apelin has emerged as a potent regulator of cardiovascular function, but its role in VC during CKD remains unknown. We determined whether apelin plays a role in phosphate-induced mineralization of human aortic smooth muscle cells (HASMCs) and in adenine-induced CKD rats with aortic calcification. Methods and resultsIn vitro, apelin-13 was found to inhibit calcium deposition in HASMCs (Pi+ Apelin+ group vs Pi+ Apelin− group: 50.1 ± 6.21 ug/mg vs 146.67 ± 10.02 ug/mg protein, p = 0.012) and to suppress the induction of the osteoblastic transformation genes BMP-2, osteoprotegerin (OPG) and Cbfa1. This effect was mediated by interference of the sodium-dependent phosphate cotransporter (Pit-1) expression and phosphate uptake. In vivo, decreased plasma apelin levels (adenine+ apelin− vs vehicle: 0.37 ± 0.09 ng/ml vs 0.68 ± 0.16 ng/ml, p = 0.003) and downregulation of APJ in the aorta were found in adenine-induced CKD rats with hyperphosphatemia (adenine+ apelin− vs vehicle: 6.91 ± 0.23 mmoL/L vs 2.3 ± 0.07 mmoL/L, p = 0.001) and aortic calcification. Exogenous supplementation of apelin-13 normalized the level of the apelin/APJ system and significantly ameliorated aortic calcification, as well as the suppression of Runx2, OPG and Pit-1 expression. ConclusionsApelin ameliorates VC by suppressing osteoblastic differentiation of VSMCs through downregulation of Pit-1. These results suggest apelin may have potential therapeutic value for treatment of VC in CKD.

Pharmacological induction of ferritin prevents osteoblastic transformation of smooth muscle cells.

Vascular calcification is a frequent complication of atherosclerosis, diabetes and chronic kidney disease. In the latter group of patients, calcification is commonly seen in tunica media where smooth muscle cells (SMC) undergo osteoblastic transformation. Risk factors such as elevated phosphorus levels and vitamin D3 analogues have been identified. In the light of earlier observations by our group and others, we sought to inhibit SMC calcification via induction of ferritin. Human aortic SMC were cultured using β-glycerophosphate with activated vitamin D3 , or inorganic phosphate with calcium, and induction of alkaline phosphatase (ALP) and osteocalcin as well as accumulation of calcium were used to monitor osteoblastic transformation. In addition, to examine the role of vitamin D3 analogues, plasma samples from patients on haemodialysis who had received calcitriol or paricalcitol were tested for their tendency to induce calcification of SMC. Addition of exogenous ferritin mitigates the transformation of SMC into osteoblast-like cells. Importantly, pharmacological induction of heavy chain ferritin by 3H-1,2-Dithiole-3-thione was able to inhibit the SMC transition into osteoblast-like cells and calcification of extracellular matrix. Plasma samples collected from patients after the administration of activated vitamin D3 caused significantly increased ALP activity in SMC compared to the samples drawn prior to activated vitamin D3 and here, again induction of ferritin diminished the osteoblastic transformation. Our data suggests that pharmacological induction of ferritin prevents osteoblastic transformation of SMC. Hence, utilization of such agents that will cause enhanced ferritin synthesis may have important clinical applications in prevention of vascular calcification.

Bone-forming capacity of adult human nasal chondrocytes.

Nasal chondrocytes (NC) derive from the same multipotent embryological segment that gives rise to the majority of the maxillofacial bone and have been reported to differentiate into osteoblast-like cells in vitro. In this study, we assessed the capacity of adult human NC, appropriately primed towards hypertrophic or osteoblastic differentiation, to form bone tissue in vivo. Hypertrophic induction of NC-based micromass pellets formed mineralized cartilaginous tissues rich in type X collagen, but upon implantation into subcutaneous pockets of nude mice remained avascular and reverted to stable hyaline-cartilage. In the same ectopic environment, NC embedded into ceramic scaffolds and primed with osteogenic medium only sporadically formed intramembranous bone tissue. A clonal study could not demonstrate that the low bone formation efficiency was related to a possibly small proportion of cells competent to become fully functional osteoblasts. We next tested whether the cues present in an orthotopic environment could induce a more efficient direct osteoblastic transformation of NC. Using a nude rat calvarial defect model, we demonstrated that (i) NC directly participated in frank bone formation and (ii) the efficiency of survival and bone formation by NC was significantly higher than that of reference osteogenic cells, namely bone marrow-derived mesenchymal stromal cells. This study provides a proof-of-principle that NC have the plasticity to convert into bone cells and thereby represent an easily available cell source to be further investigated for craniofacial bone regeneration.