High Vascular Calcification Research Articles

Prevotella copri promotes vascular calcification via lipopolysaccharide through activation of NF-κB signaling pathway

ABSTRACT Emerging evidence indicates that alteration of gut microbiota plays an important role in chronic kidney disease (CKD)-related vascular calcification (VC). We aimed to investigate the specific gut microbiota and the underlying mechanism involved in CKD-VC. We identified an increased abundance of Prevotella copri (P. copri) in the feces of CKD rats (induced by using 5/6 nephrectomy followed by a high calcium and phosphate diet) with aortic calcification via amplicon sequencing of 16S rRNA genes. In patients with CKD, we further confirmed a positive correlation between abundance of P. copri and aortic calcification scores. Moreover, oral administration of live P. copri aggravated CKD-related VC and osteogenic differentiation of vascular smooth muscle cells in vivo, accompanied by intestinal destruction, enhanced expression of Toll-like receptor-4 (TLR4), and elevated lipopolysaccharide (LPS) levels. In vitro and ex vivo experiments consistently demonstrated that P. copri-derived LPS (Pc-LPS) accelerated high phosphate-induced VC and VSMC osteogenic differentiation. Mechanistically, Pc-LPS bound to TLR4, then activated the nuclear factor κB (NF-κB) and nucleotide-binding domain, leucine-rich–containing family, pyrin domain–containing-3 (NLRP3) inflammasome signals during VC. Inhibition of NF-κB reduced NLRP3 inflammasome and attenuated Pc-LPS-induced VSMC calcification. Our study clarifies a novel role of P. copri in CKD-related VC, by the mechanisms involving increased inflammation-regulating metabolites including Pc-LPS, and activation of the NF-κB/NLRP3 signaling pathway. These findings highlight P. copri and its-derived LPS as potential therapeutic targets for VC in CKD.

Hypoxia-Inducible Factor-1α Regulates High Phosphate-Induced Vascular Calcification via Type III Sodium-Dependent Phosphate Cotransporter 1.

Vascular calcification (VC) has a high incidence in patients with chronic kidney disease, which is a worldwide public health problem and presents a heavy burden to society. Hypoxia-inducible factor (HIF)-1α, the active subunit of HIF-1, has been reported to play a vital role in high phosphate-induced VC. However, the underlying mechanism is still undetermined, and effective treatment is unavailable. In the present study, human aortic smooth muscle cells (HASMCs) were cultured under normal or high phosphate media conditions. HIF-1α small interfering RNA and overexpression plasmids were employed to regulate HIF-1α expression. Phosphonoformic acid was employed to restrain the function of type III sodium-dependent phosphate cotransporter 1 (Pit-1). The expression levels of HIF-1α, Pit-1, runt-related transcription factor 2 (Runx2), and smooth muscle 22 alpha (SM22α) were evaluated, and the calcium contents were also examined. Cell growth was assessed using an MTT assay. High phosphate stimulation caused an upregulation in HIF-1α and Pit-1 expression levels and induced calcium depositions in HASMCs. Upregulation of Runx2 expression accompanied by downregulation of SM22α expression was observed in the high phosphate group. Following the suppression of HIF-1α expression, there was a concomitant attenuation in Pit-1 expression, calcium deposition, the alteration of phenotypic transition marker genes, and vice versa. The most serious calcium deposition was noted in HASMCs cultured under high phosphate conditions which were pretreated with a HIF-1α overexpression plasmid. However, when the biological functions of Pit-1 were restrained, the putative serious calcium deposition was not formed even in HASMCs transfected with a HIF-1α overexpression plasmid. The findings confirmed that HIF-1α regulated Pit-1 expression and exerted its pro-calcifying effect through Pit-1, which identified HIF-1α and Pit-1 as therapeutic targets for high phosphate-induced VC.

Mediating effect of vascular calcification in galectin-3-related mortality in hemodialysis patients

Galectin-3 levels have been studied as a potential biomarker for predicting cardiovascular (CV) risk and mortality in hemodialysis (HD) patients. Recently, a close relationship between galectin-3 and vascular calcification (VC) has been reported. Here, we investigated the role of VC as a mediating factor in the association between galectin-3 and mortality. Serum galectin-3 and baseline aortic arch calcification (AoAC) score were measured in 477 incident HD patients. Mortality data were obtained at a median follow-up of 40 months. Causal mediation analysis was performed to examine the effect of vascular risk factors on galectin-3-related mortality. The prevalence of AoAC in HD patients was 57% (n = 272), and elevated galectin-3 levels were associated with a significantly increased risk of AoAC. When the galectin-3 level was divided by the median level of 37 ng/mL, a higher galectin group increased the risk of all-cause mortality by 1.71-fold (95% CI 1.02–2.92, p = 0.048), even after adjustment for multiple CV risk factors. Mediation analysis showed that both the direct effect of the galectin-3 on mortality (β = 0.0368, bootstrapped 95% CI [0.0113–0.0622]) and the indirect effects were significant. AoAC score and high-sensitivity CRP levels significantly mediated the association between galectin-3 and mortality (total indirect effects: β = 0.0188, bootstrapped 95% CI [0.0066–0.0352]). This study suggests that the association between high galectin-3 and mortality may be partially mediated by higher VC and inflammatory state in HD patients.

GALNT3 protects against phosphate-induced calcification in vascular smooth muscle cells by enhancing active FGF23 and inhibiting the wnt/β-catenin signaling pathway

Vascular calcification (VC) acts as a notable risk factor in the cardiovascular system. Disorder of phosphorus (Pi) metabolism promotes VC. Recent findings show that polypeptide N-acetylgalactosaminyltransferase 3(GALNT3) is Pi responsive and with potent effects on Pi homeostasis. However, whether GALNT3 is involved in high Pi-induced VC remains unclear. The present study investigated the potential role of GALNT3 as a novel regulator of VC. In vitro, human aortic smooth muscle cells (HASMCs) calcification was induced by inorganic Pi, while in vivo, C57BL/6 J mice were used to determine the effects of GALNT3 on Vitamin D3-induced medial arterial calcification. Alizarin red staining, Von Kossa staining, calcium and alkaline phosphatase (ALP) activity were performed to test VC. We showed that expression of GALNT3 was increased in the calcified HASMCs and aortas of the calcified mice.In vitro, overexpression of GALNT3 increased the levels of active full-length FGF23, accompanied by suppression of the osteoblast-related factors (Runx2 and BMP2), and further inhibited the formation of calcified nodules. Moreover, the protein levels of Wnt3a and active β-catenin were determined and it was found that GALNT3 significantly inhibited their expression. LiCl, a Wnt/β-catenin signaling activator, was observed to reverse the protective effect of GALNT3 overexpression. The opposite results were observed in the GALNT3 knockdown cells. In vivo, overexpression of GALNT3 by adeno-associated virus decreased the serum Pi and slowed the formation of aortic calcification in the calcified mice. In conclusion, our results indicate that GALNT3 counteracts high Pi-induced osteoblastic differentiation of VSMCs and protects against the initiation and progression of VC by inhibiting the Wnt/β-catenin signaling pathway.

The combined clinical impact of red blood cell distribution width and vascular calcification on cardiovascular events and mortality in patients with endstage kidney disease

BackgroundLittle is known about how the interaction between red blood cell distribution width (RDW) and vascular calcification (VC) affects cardiovascular (CV) events and mortality in end-stage kidney disease (ESKD) patients. This study investigated the combined prognostic effect of RDW and VC in ESKD patients starting dialysis.MethodsA retrospective single-center study of 582 ESKD patients was conducted. VC was assessed by calculating the aortic calcification index (ACI) using computed tomography. Patients were divided into low ACI-low RDW, low ACI-high RDW, high ACI-low RDW, and high ACI-high RDW groups based on median ACI (17.12) and RDW (14.3) values. The association between RDW and VC and the composite endpoint of CV events and death was analyzed.ResultsDuring a median follow-up of 3.1 years (range, 1.5–5.5 years), 165 CV events (28.4%) and 124 deaths (21.4%) occurred. Cox regression showed that the low ACI-high RDW (adjusted hazard ratio [HR], 1.66; 95% confidence interval [CI], 1.04–2.66; p = 0.03) and high ACI-low RDW (adjusted HR, 1.95; 95% CI, 1.21–3.14; p = 0.006) groups had a greater risk of CV events and death than the low ACI-low RDW group. The high ACI-high RDW group had the greatest risk (adjusted HR, 2.23; 95% CI, 1.42–3.52; p = 0.001). The effect of the interaction between ACI and RDW on CV events and mortality was statistically significant (p = 0.005).ConclusionHigh RDW and VC interact to increase the risk of CV events and death in ESKD patients.

Low Muscle Mass in Patients Receiving Hemodialysis: Correlations with Vascular Calcification and Vascular Access Failure.

Background: Sarcopenia involves an age-related decline in skeletal muscle mass with functional disability or low muscle strength. Vascular calcification (VC) occurs commonly in patients with chronic kidney disease, in whom it is associated with cardiovascular disease. We aimed to investigate the correlations of low muscle mass with the quantified vascular calcification score (VCS) of the arm of vascular access, as well as whether low muscle mass is associated with the incidence of vascular access failure. Methods: The VCS was measured on non-contrast, arm computed tomography using the Agatston method. The lower muscle mass (LMM) group comprised subjects whose skeletal muscle mass of the lower extremities, as measured using bioelectrical impedance, was lower than the median. Higher VC was defined as a score of 500 or above, corresponding to the highest 40% of VCS. The relationship between LMM and VC was explored using univariate and multivariate logistic regression analyses. Results: Seventy-five patients were included, of whom forty-two (56.0%) were men. The median age was 64 years (interquartile range 58–72 years). Of the 75 patients, 73 satisfied the diagnostic criteria for sarcopenia. The median hemodialysis vintage was 49.4 months (range 32.1–99.2 months). No significant differences were found between the non-LMM and LMM groups in sex, end-stage renal disease etiology, and type of vascular access, although the LMM group showed significantly older age and hemodialysis vintage. LMM presented a significant association with VC (hazard ratio (HR) 3.562; 95% CI, 1.341–9.463; p = 0.011). Upon adjustment for hemodialysis vintage, diabetes, and systolic blood pressure, LMM demonstrated an independent association with VC (HR, 10.415; 95% CI, 2.357–46.024; p = 0.002). The risk of vascular access failure was higher in the LMM group (HR, 3.652; 95%, CI 1.135–11.749; p = 0.03). VC was a full mediator in the relationship of LMM with recurrent vascular access failure. Conclusions: We quantified LMM via bioimpedance analysis and found a heretofore-unreported association between LMM and vascular access failure. LMM increases the risk of VC and has the potential to predict vascular access failure.

Vascular Calcification as a Novel Risk Factor for Kidney Function Deterioration in the Nonelderly.

BackgroundThe relationship between vascular calcification and chronic kidney disease is well known. However, whether vascular calcification affects renal function deterioration remains unclear. We investigated whether kidney function deteriorated more rapidly in individuals with higher vascular calcification indicated by the coronary artery calcium score (CACS).Methods and ResultsIndividuals with a normal estimated glomerular filtration rate (>60 mL/min per 1.73 m2) who underwent cardiac computed tomography in our institution (a tertiary teaching hospital in Cheonan, Korea) from January 2010 to July 2012 were retrospectively reviewed. All participants were aged 20 to 65 years. Among 739 patients, 447, 175, and 117 had CACSs of 0, 1 to 99, and ≥100 units, respectively. The participants were followed for 7.8 (interquartile range, 5.5–8.8) years. The adjusted annual estimated glomerular filtration rates declined more rapidly in patients in the CACS ≥100 group compared with those in the CACS 0 group (adjusted‐β, −0.40; 95% CI, −0.80 to −0.03) when estimated using a linear mixed model. The adjusted hazard ratio in the CACS ≥100 group for Kidney Disease: Improving Global Outcomes criteria (a drop in estimated glomerular filtration rate category accompanied by a 25% or greater drop in estimated glomerular filtration rate) was 2.52 (1.13–5.61). After propensity score matching, more prevalent renal outcomes (13.2%) were observed in patients with a CACS of ≥100 compared with those with a CACS of 0 (1.9%), with statistical significance (P=0.004).ConclusionsOur results showed that renal function declined more rapidly in patients with higher CACSs, suggesting that vascular calcification might be associated with chronic kidney disease progression.

Iron Sucrose: A Double-Edged Sword in High Phosphate Media-Induced Vascular Calcification

The high incidence of vascular calcification(VC) in patients with chronic kidney disease (CKD) has become an important clinical subject. Hyperphosphatemia is a primary cause of CKD-related VC. Intravenous iron sucrose (IS) is commonly used to treat anemia in CKD patients, and is effective and well tolerated worldwide. However, the interaction between iron and VC remains controversial, and the underlying mechanisms are yet to be clarified. In the present study, ex vivo normal rat aortic rings were cultured with various concentrations of phosphate and IS, and the levels of calcium and iron depositions, oxidative injury, as well as phenotypic marker genes were detected. To the best of our knowledge, the present study is the first to report that IS is a double-edged sword in high phosphate media-induced VC which not only alleviates VC in a dose-dependent manner but also leads to iron overload in vasculature when in high concentration. IS is a promising agent for VC prevention in patients with hyperphosphatemia and iron deficiency. Meanwhile, the appropriate blood concentration of IS in patients with hyperphosphatemia needs to be explored clinically.

OGT-Mediated KEAP1 Glycosylation Accelerates NRF2 Degradation Leading to High Phosphate-Induced Vascular Calcification in Chronic Kidney Disease.

Unraveling the complex regulatory pathways that mediate the effects of phosphate on vascular smooth muscle cells (VSMCs) may provide novel targets and therapies to limit the destructive effects of vascular calcification (VC) in patients with chronic kidney disease (CKD). Our previous studies have highlighted several signaling networks associated with VSMC autophagy, but the underlying mechanisms remain poorly understood. Thereafter, the current study was performed to characterize the functional relevance of O-linked N-acetylglucosamine (GlcNAc) transferase (OGT) in high phosphate-induced VC in CKD settings. We generated VC models in 5/6 nephrectomized rats in vivo and VSMC calcification models in vitro. Artificial modulation of OGT (knockdown and overexpression) was performed to explore the role of OGT in VSMC autophagy and VC in thoracic aorta, and in vivo experiments were used to substantiate in vitro findings. Mechanistically, co-immunoprecipitation (Co-IP) assay was performed to examine interaction between OGT and kelch like ECH associated protein 1 (KEAP1), and in vivo ubiquitination assay was performed to examine ubiquitination extent of nuclear factor erythroid 2-related factor 2 (NRF2). OGT was highly expressed in high phosphate-induced 5/6 nephrectomized rats and VSMCs. OGT silencing was shown to suppress high phosphate-induced calcification of VSMCs. OGT enhances KEAP1 glycosylation and thereby results in degradation and ubiquitination of NRF2, concurrently inhibiting VSMC autophagy to promote VSMC calcification in 5/6 nephrectomized rats. OGT inhibits VSMC autophagy through the KEAP1/NRF2 axis and thus accelerates high phosphate-induced VC in CKD.

OGT knockdown counteracts high phosphate-induced vascular calcification in chronic kidney disease through autophagy activation by downregulating YAP

AimsPathological vascular calcification (VC), a major risk factor for cardiovascular mortality, is a highly prevalent finding in patients with chronic kidney disease (CKD). We previously analyzed several pathways protecting against high phosphate-induced VC through induction of autophagy. Here, we explored how O-GlcNAc transferase (OGT) affected high phosphate-induced VC of CKD though mediation of autophagy. Main methodsIn the rats with CKD induced by 5/6 nephrectomy, the VC process was accelerated by a high phosphate diet. The calcification of vascular smooth muscle cells (VSMCs) was induced by high phosphate treatment. We then experimentally tested the effect of OGT on high phosphate-induced VC by conducting loss-of-function experiments. Co-immunoprecipitation and GST pull-down assays were performed to evaluate interaction between OGT and Yes-associated protein (YAP). In mechanistic studies of this pathway, we measured autophagy protein expression and autophagosome formation, as well as calcium deposition and calcium content in VSMCs and in vivo in response to altered expression of OGT and/or YAP. Key findingsOGT was up-regulated in high phosphate-induced VC models in vitro and in vivo. High phosphate-induced calcification in the rat aorta and VSMCs were suppressed by OGT silencing. OGT promoted the glycosylation of YAP to enhance its stability. Importantly, over-expressing YAP reduced autophagy and OGT expedited high phosphate-induced VC by inhibiting autophagy through upregulation of YAP. SignificanceOGT silencing downregulated YAP to induce autophagy activation, thus suppressing high phosphate-induced VC, which highlighted a promising preventive target against high phosphate-induced VC in CKD.

P1234SNF472 IMPROVES LIMB BLOOD PERFUSION AND WALKING ABILITY IN A PERIPHERAL ARTERY DISEASE VASCULAR CALCIFICATION RAT MODEL

Abstract Background and Aims Peripheral Artery disease (PAD) is a common vascular disease associated with functional impairment and increased risk of cardiovascular events in End Stage Kidney Disease (ESKD) patients undergoing dialysis. Poor limb salvage outcomes and high post-amputation mortality in hemodialysis (HD) patients highlight the need for earlier medical therapies. Cilostazol and pentoxifylline are approved for PAD. Their use in HD patients stays limited and cilostazol use requires caution in this population. Clinical studies demonstrate associations between arterial calcification and adverse outcomes in PAD patients. SNF472, a selective calcification inhibitor that interferes in the formation and growth of hydroxyapatite, is in Phase 3 for calciphylaxis treatment. This study aims to evaluate the effects of SNF472 on limb functional recovery and blood perfusion in a Vitamin D3 (VitD)-induced arterial calcification rat model. Method Arterial calcification was induced in 32 Sprague Dawley rats by 3 consecutive daily s.c. doses of 120 kIU/kg VitD. Rats were divided into four groups and treated during 12 days by: placebo s.c, placebo p.o, SNF472 (20 mg/kg/day, s.c.) or cilostazol (20 mg/kg/day, p.o.). An additional group of 8 rats without VitD received vehicle only (sham). Efficacy was evaluated at day 12 and 17 (5 days after treatment stop). Posterior limb blood perfusion was measured using Laser Doppler Imaging and limb walking ability was evaluated by measuring Maximum Walking Distance (MWD) and Maximum Walking Time (MWT) using a treadmill. Rats were sacrificed at day 26 (14 days after treatment stop), and aortas were collected for calcium analysis. Results VitD-induced arterial calcification was associated with decreased blood perfusion and impairment of limb walking ability (MWT and MWD) compared to sham. SNF472 reduced aorta calcification by 41% compared to placebo. No effects of cilostazol on vascular calcification were observed. The inhibition of calcification in SNF472-treated animals was associated with significant higher limb blood perfusion compared to placebo or Cilostazol (1.28 and 1.37-fold higher, respectively at day 12: p< 0.001) and it was translated into a significant improvement in walking ability compared to placebo (515±114 meters vs 334±187 meters, respectively: p<0.05). Conclusion SNF472 shows improvements in vascular calcification, blood perfusion and a functional parameter like walking distance in a PAD vascular calcification rat model. These results suggest that SNF472 may represent a new therapeutic approach for the treatment of PAD associated with high vascular calcification such as in renal disease.

P1433VALIDATION OF A SERUM CALCIFICATION PROPENSITY TEST FOR THE PREDICTION OF ALL-CAUSE MORTALITY AMONG DIALYSIS PATIENTS

Abstract Background and Aims Vascular calcification as cause of vascular stiffness is a major contributor to the high cardiac burden among stage 5 chronic kidney disease dialysis (CKD5HD) patients. Early identification of patients with high calcification propensity is crucial for proper risk stratification and management of these patients. Recently, a novel in vitro test (T50-test) was developed which determines calcification propensity of human serum and predicts mortality among ND-CKD (non-dialysis chronic kidney disease) patients, kidney transplant recipients and CKD5HD patients suffering from secondary hyperparathyroidism. We now evaluated whether these results can be confirmed in an unselected cohort of CKD5HD patients and can be used in the future for improving care of these patients. Method This prospective clinical study included 776 incident and prevalent hemodialysis patients from 8 Fresenius Medical Care NephroCare centers in Cataluña (Spain). T50 was determined at Calciscon AG, all other clinical data were retrieved from the European Clinical Database (EuCliD®). After their baseline T50 measurement, patients were followed for two years for the occurrence of the primary endpoint all-cause mortality. The association between T50 and all-cause mortality was examined by using proportional subdistribution hazards regression modelling accounting for kidney transplantation as competing event. In sensitivity analyses we adjusted for age, sex, vascular access (fistula/graft, catheter), treatment modality (HD, HDF), Charlson comorbidity index and dialysis vintage. Results Mean age of the study population was 72.2 years and 63.8% were male. 42.5% had diabetes and 55.0% past history of cardiovascular disease. Mean T50 was 283.4 min among the total cohort. During follow-up, 185 (23.8%) patients died. Patients who reached the endpoint had a significantly lower T50 at baseline as compared to those who survived during follow-up (269.6 vs 287.7 min, p = 0.001). A cross-validated model (mean c statistic: 0.5767) identified T50 as a linear predictor of all-cause-mortality (subdistribution hazard ratio (per min): 0.9957, 95% CI [0.9933;0.9981]). T50 remained a significant predictor of all-cause mortality after adjusting for relevant confounding in sensitivity analyses. Conclusion In this prospective study, we confirmed that T50 is an independent predictor of all-cause mortality among a large unselected cohort of hemodialysis patients. T50 may be used to identify patients with high vascular calcification propensity and mortality risks and may help physicians to implement a personalized and more precise renal replacement therapy option.

Apelin-13 attenuates high glucose-induced calcification of MOVAS cells by regulating MAPKs and PI3K/AKT pathways and ROS-mediated signals

Vascular calcification (VC) is an inducement of many cardiovascular diseases. Clinic evidences have confirmed that diabetes was the independent risk factor for VC, and the mechanism has not been well explored. Apelin as a ligand molecule is widely found in the cardiovascular system and showed potential in inhibiting VC, but the inhibitory effect and mechanism of apelin-13 against high glucose-induced VC have not been investigated yet. Herein, apelin-13 was employed to inhibit high glucose-induced VC in mouse aortic vascular smooth muscle cells (MOVAS), and the underlying mechanism was explored. The results showed that apelin-13 significantly inhibited high glucose-induced cells proliferation, migration and invasion of MOVAS cells. Apelin-13 also effectively attenuated high glucose-induced calcification by inhibiting alkaline phosphatase (ALP) activity and expression. Further investigation revealed that apelin-13 dramatically suppressed high glucose-induced DNA damage through inhibiting reactive oxide species (ROS) generation. Moreover, apelin-13 also effectively improved high glucose-induced dysfunction of MAPKs and PI3K/AKT. Inhibition of ERK by inhibitor (U0126) significantly blocked high glucose-induced calcification, which further confirmed the significance of MAPKs. Taken together, these results suggested that apelin-13 had the potential to attenuate high glucose-induced calcification of MOVAS cells by inhibiting ROS-mediated DNA damage and regulating MAPKs and PI3K/AKT pathways. Our findings validated the strategy of using apelin-13 maybe a novel way in treating high glucose-mediated VC.

Matrix Gla protein is an independent predictor of both intimal and medial vascular calcification in chronic kidney disease

Matrix Gla protein (MGP) is a potent inhibitor of vascular calcification (VC) and requires carboxylation by vitamin K to exert calcification inhibition. Chronic kidney disease (CKD) patients undergo early vascular aging often involving extensive VC. The present cross-sectional study investigated the association between circulating dp-ucMGP levels, MGP expression in vascular tissue and MGP polymorphisms. In 141 CKD stage 5 patients, CAC score was significantly increased in the highest tertile of dp-ucMGP (p = 0.002), and a high medial VC score was associated with elevated dp-ucMGP levels. MGP vascular expression was associated with increased circulating dp-ucMGP and CAC scores. MGP SNP analysis revealed that patients homozygous for the C allele of the rs1800801 variant had a higher CAC score (median 15 [range 0–1312]) compared to patients carrying a T allele (median 0 [range 0–966] AU). These results indicate that plasma levels of dp-ucMGP are an independent predictor of increased VC in CKD5 patients and correlate with both higher CAC scores and degree of medial calcification. Additionally, high vascular expression of MGP was associated with higher CAC scores and plasma dp-ucMGP levels. Taken together, our results support that MGP is involved in the pathogenesis of VC.

Vascular Calcification in Chronic Kidney Disease: The Role of Inflammation.

Cardiovascular complications are extremely frequent in patients with chronic kidney disease (CKD) and death from cardiac causes is the most common cause of death in this particular population. Cardiovascular disease is approximately 3 times more frequent in patients with CKD than in other known cardiovascular risk groups and cardiovascular mortality is approximately 10-fold more frequent in patients on dialysis compared to the age- and sex-matched segments of the nonrenal population. Among other structural and functional factors advanced calcification of atherosclerotic plaques as well as of the arterial and venous media has been described as potentially relevant for this high cardiovascular morbidity and mortality. One potential explanation for this exceedingly high vascular calcification in animal models as well as in patients with CKD increased systemic and most importantly local (micro)inflammation that has been shown to favor the development of calcifying particles by multiple ways. Of note, local vascular upregulation of proinflammatory and proosteogenic molecules is already present at early stages of CKD and may thus be operative for vascular calcification. In addition, increased expression of costimulatory molecules and mast cells has also been documented in patients with CKD pointing to a more inflammatory and potentially less stable phenotype of coronary atherosclerotic plaques in CKD.

Bone mineral density of extremities is associated with coronary calcification and biopsy-verified vascular calcification in living-donor renal transplant recipients.

Chronic kidney disease (CKD) mineral and bone disorders (CKD-MBD) may lead to low bone mineral density (BMD) and vascular calcification (VC), but links to the latter are unclear. Here we investigated associations between BMD, coronary artery calcium (CAC) scores, and histological signs of VC in end-stage renal disease (ESRD) patients undergoing living-donor kidney transplantation (LD-Rtx). In 66 ESRD patients (median age 45years, 68% males), BMD (by dual-energy X-ray absorptiometry, DXA), CAC score (by computed tomography, CT; n=54), and degree of VC score (graded by histological examination of epigastric artery specimens collected at LD-Rtx; n=55) were assessed at the time of LD-Rtx. Of the patients, 26% had osteopenia and 7% had osteoporosis. Of those undergoing artery biopsy, 16% had extensive VC, and of those undergoing CT 28% had high CAC score (>100 Agatston units). CAC scores correlated with BMD of legs and pelvis. BMDs of leg and pelvic sub-regions were significantly lower in patients with extensive VC. In multivariate regression analysis adjusted for age and gender, lower BMD of leg sub-region was associated with CAC score >100 AUs and extensive VC, and patients with extensive VC had significantly higher CAC score. Both high CAC and extensive VC were independently predicted by low BMD of legs. Low BMD has the potential to identify ESRD patients at risk of vascular calcification.

Iron citrate reduces high phosphate-induced vascular calcification by inhibiting apoptosis

Background and aimsHigh phosphate-induced vascular calcification (VC) and iron deficiency-induced anemia are two major contributors of cardiovascular morbidity and mortality in patients affected by chronic kidney disease (CKD). Since phosphate (Pi) control and iron replacement are common therapies in CKD, the aim of our study was to investigate the effect of iron on high Pi-induced VC in rat vascular smooth muscle cells (VSMCs). MethodsWe treated VSMCs with 5 mM Pi and iron citrate (Fe3+) to evaluate Ca deposition by Alizarin Red destaining, DNA fragmentation by ELISA, gene expression by RT-PCR and protein expression by Western blot. ResultsPretreatment with Fe3+ prevents high Pi-induced calcium (Ca) deposition concentration-dependently, with 90.1% inhibition at 50 μM (0.716 ± 0.04 vs. 0.071 ± 0.01, OD/mg protein; Pi vs. Pi + Fe3+, p < 0.01). We found that 50 μM Fe3+ completely prevents high Pi-induced apoptosis measured as DNA fragmentation (1.51 ± 0.08 vs. 1.03 ± 0.06, Pi vs. Pi + Fe3+; p < 0.01), through the prevention of the downregulation of the pro-survival pathway GAS6/AXL. Moreover, Fe3+ stimulates autophagy, a protective phenomenon in VC, as demonstrated by electron microscopy and by autophagy flux detected by LC3IIβ protein expression. Finally, high Pi-induced osteoblastic differentiation is partially affected by Fe3+, since BMP2 increase is prevented and OPN is enhanced, but RUNX2 increase and α-actin and SM22α decrease are not modified. Interestingly, the addition of Fe3+ at different time points after Pi challenge completely blocks the progression of Ca deposition. ConclusionsIn conclusion, iron citrate inhibits high Pi-induced Ca deposition by prevention of apoptosis, induction of autophagy, and partially affecting osteoblastic differentiation.

Endoplasmic Reticulum Stress-Mediated Apoptosis Contributing to High Glucose-Induced Vascular Smooth Muscle Cell Calcification

Vascular calcification (VC) is a common feature in patients with type 2 diabetes mellitus, a metabolic disorder that is characterized by hyperglycemia (high blood glucose) in the context of insulin resistance and a relative lack of insulin. Recently, a few studies have indicated that a high concentration of glucose amplifies the osteogenesis of vascular smooth muscle cells (VSMCs). Some previous reports state that endoplasmic reticulum (ER) stress-mediated apoptosis was activated in and contributed to VC. However, whether or not high glucose could induce ER stress-mediated apoptosis and then involve the pathogenesis of VC remains unclear. The purpose of the present study was to investigate whether high blood glucose-induced VC in diabetes mellitus is caused by the ER response and subsequent apoptosis. We examined the effects of high glucose on the ER stress response of VSMCs. High glucose treatment drastically increased the ER stress response in VSMCs. The high glucose-induced osteoblastic differentiation of VSMCs was significantly attenuated by pretreatment with 500 μ<smlcap>M</smlcap> of 4-PBA (an ER stress inhibitor) prior to the exposure to high glucose, as evidenced by decreases in the expression of Runx2 and activity of alkaline phosphatase, as well as calcium nodules. These results suggest that high glucose induces the ER stress response and apoptosis, leading to high glucose-elicited VC.

The effects of vitamin K supplementation and vitamin K antagonists on progression of vascular calcification: ongoing randomized controlled trials.

BackgroundThe extent and the progression of vascular calcification (VC) are independent predictors of cardiovascular risk in the haemodialysis population. Vitamin K is essential for the activation of matrix gla protein (MGP), a powerful inhibitor of tissue calcification. Functional vitamin K deficiency may contribute to the high VC burden in haemodialysis patients. In addition, haemodialysis patients are frequently treated with vitamin K antagonists, mainly to prevent stroke in atrial fibrillation, potentially compounding the cardiovascular risk in these already vulnerable patients. New oral anticoagulants (NOACs) are valuable alternatives to vitamin K antagonists in the general population, but their use in dialysis has been encumbered by substantial renal clearance. However, a recent pharmacokinetic study provided information on how to use rivaroxaban in haemodialysis patients.MethodsWe conduct a randomized, prospective, multicentre, open-label interventional clinical trial that will include 117 chronic haemodialysis patients with non-valvular atrial fibrillation, treated with or candidates for treatment with vitamin K antagonists. Patients will be randomized to a vitamin K antagonist titrated weekly to an international normalized ratio between 2 and 3, a daily dose of rivaroxaban of 10 mg, or a daily dose of rivaroxaban 10 mg with a thrice weekly supplement of 2000 µg vitamin K2. Cardiac computed tomography, pulse wave velocity (PWV) measurements and MGP sampling will be performed at baseline, 6 months, 12 months and 18 months. Primary endpoints include progression of coronary artery and thoracic aorta calcification and changes in PWV. Secondary endpoints are progression of aortic and mitral valve calcification, all-cause mortality, major adverse cardiovascular events, stroke and bleeding. The ClinicalTrials.gov database was searched to retrieve related trials.ResultsSeven trials, three of which are performed in the haemodialysis population, evaluate whether pharmacological doses of vitamin K1 or K2 retard progression of VC. Five studies compare the effect of warfarin and NOACs on progression of VC, the present study being the only conducted in the dialysis population.ConclusionVitamin K deficiency may be a modifiable cardiovascular risk factor in the haemodialysis population. Conversely, vitamin K antagonists may aggravate VC burden in haemodialysis patients. Several ongoing trials may provide an answer to these questions in the near future.

Vascular calcification on plain radiographs is related with the severity of lesions detected by coronary angiography in dialysis patients.

Coronary artery disease (CAD) is a primary cause of mortality and morbidity in dialysis patients. However, it is difficult to select the proper point for coronary angiographic procedure, because dialysis patients frequently do not display typical symptoms. Vascular calcification (VC) scores of artery or aorta on plain radiographs are associated with CAD events and may be predictive of CAD in dialysis patients. Therefore, we evaluated whether high or meaningful VC scores on plain radiographs are related with the severity of lesions detected by coronary angiography (CAG) in dialysis patients. We retrospectively enrolled dialysis patients who underwent CAG and checked several plain radiographs within one year before or after CAG. Significant VC is defined as high or meaningful VC scores, such as long abdominal aortic calcification and medial artery calcification on feet. Of all 55 patients, 41 patients (74.5%) exhibited significant VC on plain radiographs and 23 patients (41.8%) underwent stent insertion. Among the 23 patients, longer stents were used in 18 patients with significant VC (34.1 ± 19.5 mm vs. 16.6 ± 15.2 mm, P = 0.029). Patients with significant VC showed higher prevalence rate of severe coronary artery calcification (P = 0.007) and diffuse/tubular stenosis (P = 0.012), detected by CAG, than those without significant VC. Thus, high or meaningful VC scores on plain radiographs were associated with the degree of calcification or stenosis detected by CAG. In conclusion, VC scores on plain radiographs may be predictive of calcification or stenosis of coronary artery before CAG in dialysis patients.