Vascular Calcification In Rats Research Articles

A machine learning-driven SERS platform for precise detection and analysis of vascular calcification.

Vascular calcification (VC) significantly increases the incidence and mortality rates of cardiovascular diseases, severely threatening public health as a global issue. Currently, there are no effective methods to prevent and treat vascular calcification. This study proposes a machine learning-assisted surface-enhanced Raman scattering (SERS) technique for label-free, highly sensitive analysis of VC rat serum. We prepared gold nanobipyramid (GNBP) substrates using seed-mediated and liquid-liquid interface self-assembly methods and measured the SERS spectra of the serum. The collected spectral data were processed using a Principal Component Analysis (PCA)-Linear Discriminant Analysis (LDA) model to achieve effective sample differentiation. In this analysis model, GNBP substrates enabled rapid, sensitive, and label-free serum spectral detection, achieving classification accuracy, sensitivity, and specificity of 96.0%, and an AUC value of 0.98, significantly outperforming currently used machine learning methods. By analyzing the PCA loading plots, key spectral features that distinguished VC were successfully captured. This study demonstrates that combining SERS technology with machine learning provides a new method and foundation for real-time diagnosis and identification of VC, showcasing the significant advantages of GNBP substrates in high-sensitivity and high-specificity detection, potentially improving the early diagnosis and treatment of VC significantly.

Effects of soluble Klotho and Wnt/β-catenin signaling pathway in vascular calcification in chronic kidney disease model rats and the intervention of Shenyuan granules

Objective This study aimed to investigate the effect of the soluble Klotho (sKlotho)/Wnt/β-catenin signaling pathway on vascular calcification in rat models of chronic kidney disease (CKD) and the intervention effect of Shenyuan granules. Methods Rats with 5/6 nephrectomy and high phosphorus feeding were used to establish the vascular calcification model. The rats were given gradient doses of Shenyuan granules aqueous solution and calcitriol solution by gavage for 8 weeks, which were divided into experimental group and positive control group. Results The 5/6 nephrectomy combined with high phosphorus feeding induced thoracic aortic calcification in rats. Shenyuan granules intervention increased the serum sKlotho level, inhibited the mRNA and protein expression of Wnt1, β-catenin, and Runx2 in the thoracic aorta, and alleviated thoracic aortic media calcification in rats. Conclusion Shenyuan granules may partially regulate the Wnt/β-catenin signaling pathway via serum sKl to interfere with the expression of Runx2, thereby improving vascular calcification in CKD.

Absolute iron deficiency, coronary artery calcification and cardiovascular mortality in maintenance haemodialysis patients.

The effects of iron on vascular calcification in rats and vascular smooth muscle cells were recently reported, but clinical studies on iron and vascular calcification are scant. We studied the associations of absolute iron deficiency, coronary artery calcification and mortality in patients with maintenance haemodialysis (MHD). Transferrin saturation (TSAT), ferritin, mean corpuscular haemoglobin (MCH) and Agatston coronary artery calcium score (CACS) were studied at baseline in MHD patients and followed up for 3 years. Cox proportional hazard analyses for mortality and linear regression analyses for CACS were performed. In 306 patients, the median age was 67 (56-81) years, dialysis duration was 76 (38-142) months, and diabetes prevalence was 42.5%. Fifty-two patients had died by 3 years. Patients with absolute iron deficiency (TSAT <20% and ferritin <100 ng/mL) (n = 102) showed significantly higher CACS (p = .0266) and C-reactive protein (p = .0011), but a lower frequency of iron formulation administration compared with patients without absolute iron deficiency at baseline (n = 204). Absolute iron deficiency was a significant predictor for 3-year cardiovascular (CV) mortality (hazard ratio: 2.08; p = .0466), but not for 3-year all-cause mortality. CACS was significant predictor for both 3-year CV and all-cause mortality (p <.05). Absolute iron deficiency and MCH were significant determinants of CACS (p < .05). MHD patients with absolute iron deficiency showed significantly higher CACS than others, and absolute iron deficiency was a significant risk factor for coronary artery calcification and 3-year CV mortality in MHD patients, but was not a significant predictor for 3-year all-cause mortality.

D-box-binding protein alleviates vascular calcification in rats with chronic kidney disease by activating microRNA-195-5p and downregulating cyclin D1.

Vascular calcification (VC) is a critical complication in chronic kidney disease (CKD), where transcription factors (TFs) and microRNAs (miRs) could potentially play a pivotal role in its pathogenesis and progression. To explore the potential molecular mechanism by which the TF D-box-binding protein (DBP) regulates the miR-195-5p/cyclin D1 (CCND1) axis and its impact on aortic VC in CKD rats, we established a rat model of CKD with VC through a 5/6 nephrectomy procedure. This model was treated with lentivirus overexpressing DBP or CCND1 to analyze their roles in aortic VC. Additionally, an in vitro cell model of VC was induced by high phosphorus. This model underwent transfection with lentivirus overexpressing DBP or miR-195-5p mimic/inhibitor to confirm their regulatory roles in aortic VC in vitro. We assessed the interactions between DBP and miR-195-5p, as well as between miR-195-5p and CCND1. Our results indicated that the expression of DBP and miR-195-5p was reduced, while CCND1 levels were elevated in both the rat and cell models. Overexpression of miR-195-5p inhibited VC in vascular smooth muscle cells (VSMCs). Bioinformatics prediction and dual luciferase assays confirmed that DBP could act as a TF to enhance miR-195-5p expression, with Ccnd1 identified as a downstream target gene of miR-195-5p. Overexpression of DBP inhibited aortic calcification in CKD rats, whereas overexpression of CCND1 produced the opposite effect. In conclusion, the TF DBP can inhibit CCND1 expression through transcriptional activation of miR-195-5p, thereby preventing VC in rats with CKD.

ENPP1 ameliorates vascular calcification via inhibiting the osteogenic transformation of VSMCs and generating PPi.

This study aims to investigate the impact of ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) on vascular calcification in rats. The rationale behind studying ENPP1's role in vascular calcification lies in its potential to modulate calcification processes. Understanding this relationship can offer insights into novel therapeutic avenues for addressing vascular calcification-related disorders. In this experiment, vascular smooth muscle cell (VSMC) calcification was induced using β-glycerophosphoric acid. Subsequently, recombinant AAV9-carrying ENPP1 was introduced into VSMCs to achieve both in vitro and in vivo overexpression of ENPP1. The findings indicate that ENPP1 overexpression significantly reduces calcium and phosphorus content in the aorta (P < 0.05). Alizarin red and von Kossa staining reveal notable reductions in calcium salt deposits in VSMCs and aorta, respectively. Notably, the expression levels of BMP-2, PINP, OC, and BALP were substantially decreased in VSMCs (P < 0.05), underscoring ENPP1's role in impeding osteoblast-like transdifferentiation of VSMCs. Additionally, ENPP1 overexpression led to a significant increase in pyrophosphate (PPi) levels compared to control rats (P < 0.05). In conclusion, this study suggests that ENPP1 contributes to alleviating vascular calcification by elevating PPi levels and inhibiting the phenotypic transformation of VSMCs. These findings shed light on the potential therapeutic role of ENPP1 in mitigating vascular calcification-related complications.

Antihypertensive treatment with hydrochlorothiazide-hydralazine combination aggravates medial vascular calcification in CKD rats with mineral bone disorder.

Arterial stiffness and medial vascular calcification, leading to isolated systolic blood pressure (BP), are major cardiovascular risk factors in patients with chronic kidney disease (CKD) and mineral bone disorders (MBD). The impact of BP on MBD-induced medial vascular calcification in CKD remains uncertain. We investigated whether BP reduction improves arterial stiffness and medial vascular calcification in a rat model of CKD-MBD. CKD was induced in Wistar rats by subtotal nephrectomy. Then, MBD was generated by a Ca/P-rich diet with calcitriol supplementation to induce medial vascular calcification. Two antihypertensive treatments were evaluated: (1) the angiotensin AT1 receptor antagonist losartan, and (2) the combination of the thiazide diuretic hydrochlorothiazide and the direct vasodilator hydralazine (HCTZ/HY). After 5 weeks, mean BP (MBP), pulse pressure (PP), and pulse wave velocity (PWV) were determined. Vascular calcification was assessed in the thoracic aorta. While MBP was similar in CKD-MBD and control CKD rats, PP and PWV were increased in CKD-MBD rats. The heightened arterial stiffness in CKD-MBD rats was associated with diffused medial calcification along the thoracic aorta. Although both losartan and HCTZ/HY reduced MBP in CKD-MBD rats, losartan did not affect PP and PWV nor medial vascular calcification, whereas HCTZ/HY, unexpectedly, further increased arterial stiffness and medial vascular calcification. In the rat model of CKD-MBD, antihypertensive treatment with losartan did not affect arterial stiffness or medial vascular calcification. However, HCTZ/HY treatment aggravated arterial stiffness and vascular calcification despite a similar reduction of MBP, suggesting a blood pressure-independent mechanism for vascular calcification.

Impaired renal ischemia reperfusion recovery after bilateral renal artery ligation in rats treated with adenine: role of renal mitochondria.

Vascular calcification (VC) and ischemia reperfusion (IR) injury is characterised to have mitochondrial dysfunction. However, the impact of dysfunctional mitochondria associated with vascular calcified rat kidney challenged to IR is not explored and is addressed in the present study. Male Wistar rats were treated with adenine for 20 days to induce chronic kidney dysfunction and VC. After 63 days, renal IR protocol was performed with subsequent recovery for 24h and 7 days. Various mitochondrial parameters and biochemical assays were performed to assess kidney function, IR injury and its recovery. Adenine-induced rats with VC, decreased creatinine clearance (CrCl), and severe tissue injury demonstrated an increase in renal tissue damage and decreased CrCl after 24h of IR (CrCl in ml: IR-0.220.02, VC-IR-0.050.01). Incidentally, the 24h IR pathology in kidney was similar in both VC-IR and normal rat IR. But, the magnitude of dysfunction was higher with VC-IR due to pre-existing basal tissue alterations. We found severed deterioration in mitochondrial quantity and quality supported by low bioenergetic function in both VC basal tissue and IR challenged sample. However, post 7 days of IR, unlike normal rat IR, VC rat IR did not improve CrCl and corresponding mitochondrial damage in terms of quantity and its function were observed. Based on the above findings, we conclude that IR in VC rat adversely affect the post-surgical recovery, mainly due to the ineffective renal mitochondrial functional restoration from the surgery.

Evaluation of prophylactic efficacy of sodium thiosulfate in combating I/R injury in rat brain: exploring its efficiency further in vascular calcified brain slice model.

Cerebral ischemia reperfusion injury (CIR) is one of the clinical manifestations encountered during the management of stroke. High prevalence of intracranial arterial calcification is reported in stroke patients. However, the impact of vascular calcification (VC) in the outcome of CIR and the efficacy of mechanical preconditioning (IPC) and pharmacological conditioning with sodium thiosulphate (STS) in ameliorating IR remains unclear. Two experimental models namely carotid artery occlusion (n=36) and brain slice models (n=18) were used to evaluate the efficacy of STS in male Wistar rats. IR was inflicted in rat by occluding carotid artery for 30min followed by 24-h reperfusion after STS (100mg/kg) administration. Brain slice model was used to reconfirm the results to account blood brain barrier permeability. Further, brain slice tissue was utilised to evaluate the efficacy of STS in VC rat brain by measuring the histological alterations and biochemical parameters. Pre-treatment of STS prior to CIR in intact animal significantly reduced the IR-associated histopathological alterations in brain, declined oxidative stress and improved the mitochondrial function found to be similar to IPC. Brain slice model data also confirmed the neuroprotective effect of STS similar to IPC in IR challenged tissue slice. Higher tissue injury was noted in VC brain IR tissue than normal IR tissue. Therapeutic efficacy of STS was evident in VC rat brain tissues and normal tissues subjected to IR. On the other hand, IPC-mediated protection was noted only in IR normal and adenine-induced VC brain tissues not in high-fat diet (HFD) induced VC brain tissues. Based on the results, we concluded that similar to IPC, STS was effective in attenuating IR injury in CIR rat brain. Vascular calcification adversely affected the recovery protocol of brain tissues from ischemic insult. STS was found to be an effective agent in ameliorating the IR injury in both adenine and HFD induced vascular calcified rat brain, but IPC-mediated neuroprotection was absent in HFD-induced VC brain tissues.

PS-BPB01-8: IMPACT OF DIFFERENT DIURETIC TYPES ON ARTERIAL STIFFNESS AND CALCIFICATION IN A RAT MODEL OF CHRONIC KIDNEY DISEASE

Introduction: We reported that antihypertensive therapy with a hydrochlorothiazide (HCTZ)-based regimen aggravates arterial stiffness and vascular calcification in a rat model of chronic kidney disease (CKD) with mineral and bone disorder (MBD). In this study, we investigated whether the aggravation of arterial stiffness and vascular calcification in CKD-MBD rats is induced by HCTZ and whether these abnormalities could also occur with other types of diuretic currently used in clinic. Methods: In rats with renal mass ablation-induced CKD, MBD was generated by a Ca/P-rich diet and calcitriol supplementation. The animals were divided into 4 groups; 1) CKD-MBD control; 2) CKD-MBD + HCTZ (thiazide diuretic, 5 mg/kg/d); 3) CKD-MBD + Chlorthalidone (thiazide-like diuretic, 5 mg/kg/d), and; 4) CKD-MBD + Furosemide (loop diuretics, 10 mg/kg/d). At week 6, hemodynamic parameters including systolic and mean blood pressure (SBP and MBP), pulse pressure (PP) and pulse wave velocity (PWV) were determined. The thoracic aorta was harvested to assess vascular calcification. Results: SBP was reduced by all types of diuretic in CKD-MBD rats. Contrary to chlortalidone nor furosemide, HCTZ treatment led to a reduction of MBP, but an increase in PP and PWV in CKD-MBD rats (p &lt; 0.05). As expected from these hemodynamic changes, medial calcification in the thoracic aorta was significantly greater in CKD-MBD rats treated with HCTZ as compared to all the other groups of rats (p &lt; 0.05). Conclusion: In rats with CKD-MBD, antihypertensive therapy with the thiazide diuretic HCTZ, but not other types of diuretic, exacerbated arterial stiffness and vascular calcification despite a reduction in SBP. The deleterious effect of HCTZ in CKD-MBD rats may have major clinical impact as this diuretic is widely use in patients with CKD that often develop MBD.

Pharmacological inhibition of eIF2alpha phosphorylation by integrated stress response inhibitor (ISRIB) ameliorates vascular calcification in rats.

Vascular calcification (VC) is an independent risk factor for cardiovascular events and all-cause mortality with the absence of current treatment. This study aimed to investigate whether eIF2alpha phosphorylation inhibition could ameliorate VC. VC in rats was induced by administration of vitamin D3 (3×10(5) IU/kg, intramuscularly) plus nicotine (25 mg/kg, intragastrically). ISRIB (0.25 mg/kg·week), an inhibitor of eIF2alpha phosphorylation, ameliorated the elevation of calcium deposition and ALP activity in calcified rat aortas, accompanied by amelioration of increased SBP, PP, and PWV. The decreased protein levels of calponin and SM22alpha, and the increased levels of RUNX2 and BMP2 in calcified aorta were all rescued by ISRIB, while the increased levels of the GRP78, GRP94, and C/EBP homologous proteins in rats with VC were also attenuated. Moreover, ISRIB could prevent the elevation of eIF2alpha phosphorylation and ATF4, and partially inhibit PERK phosphorylation in the calcified aorta. These results suggested that an eIF2alpha phosphorylation inhibitor could ameliorate VC pathogenesis by blocking eIF2alpha/ATF4 signaling, which may provide a new target for VC prevention and treatment.

Potential Actions of Baicalein for Preventing Vascular Calcification of Smooth Muscle Cells In Vitro and In Vivo.

Vascular calcification (VC) is associated with cardiovascular disease. Baicalein, a natural flavonoid extract of Scutellaria baicalensis rhizome has several biological properties which may inhibit VC. We investigated whether baicalein suppresses Runt-related transcription factor 2 (Runx2) and bone morphogenetic protein 2 (BMP-2) and upregulates smooth muscle 22-alpha (SM22-α) and alpha-smooth muscle actin (α-SMA). In an in vitro experiment, primary rat aortic vascular smooth muscle cells (VSMCs) were pretreated with 0.1, 1, and 5 μM baicalein, followed by β-glycerophosphate (β-GP) to induce calcification. In an in vivo experiment, VC was generated by vitamin D3 plus nicotine (VDN) administration to male Sprague Dawley (SD) rats randomly assigned into a control group, a VC group, a VC group pretreated with baicalein, and a baicalein alone group. Each group comprised 10 rats. Left ventricular (LV) morphology, function and performance were assessed by echocardiography. Calcium content was measured by Alizarin red S staining and alkaline phosphatase (ALP) activity assays. Apoptotic VSMCs were detected by flow cytometry. Protein levels and superoxide changes were evaluated using Western blotting and immunofluorescence assays respectively. Plasma malondialdehyde (MDA) was assayed. Baicalein pretreatment significantly reduced calcium content in calcified VSMCs (p < 0.001) as well as in VC rat aortic smooth muscle (p < 0.001). Additionally, ALP activity was decreased in calcified VSMCs and VC rat aortic smooth muscle (p < 0.001). Apoptosis was significantly attenuated by 1 μM baicalein pretreatment in calcified VSMCs. Runx2 and BMP-2 expressions were downregulated by the baicalein in calcified VSMCs. Baicalein pretreatment increased typical VSMCs markers SM22-α and α-SMA in calcified VSMCs. Baicalein pretreatment was associated with adverse changes in LV morphometry. Markers of oxidative stress declined, and endogenous antioxidants increased in VC rats pretreated with baicalein. Baicalein mitigates VC through the inhibition of Runx2/BMP-2 signaling pathways, enhancement of vascular contractile phenotype and oxidative stress reduction. However, our study is of basic experimental design; more advanced investigations to identify other molecular regulators of VC and their mechanisms of action is required.

A long non-coding RNA H19/microRNA-138/TLR3 network is involved in high phosphorus-mediated vascular calcification and chronic kidney disease

ABSTRACT Vascular calcification, characterized by the accumulation of calcium-phosphate crystals in blood vessels, is a major cause of cardiovascular complications and chronic kidney disease (CKD)-related death. This work focuses on the molecules involved in high-phosphorus-mediated vascular calcification in CKD. A rat model of CKD was established by 5/6 nephrectomy, and the rats were given normal phosphorus diet (NPD) or high phosphorus diet (HPD). HPD decreased kidney function, increased the concentration of calcium ion and damaged vascular structure in the thoracic aorta of diseased rats. A high phosphorus condition enhanced calcium deposition in vascular smooth muscle cells (VSMCs). High phosphorus also increased the expression of RUNX2 whereas reduced the expression of α-SM actin in the aortic tissues and VSMCs. Long non-coding RNA (lncRNA) H19 was upregulated in the aortic tissues after HPD treatment. H19 bound to microRNA (miR)-138 to block its inhibitory effect on TLR3 mRNA and activated the NF-κB signaling pathway. Downregulation of H19 or TLR3 alleviated, whereas downregulation of miR-138 aggravated the calcification and vascular damage in model rats and VSMCs. In conclusion, this study demonstrates that the H19/miR-138/TLR3 axis is involved in high phosphorus-mediated vascular calcification in rats with CKD.

Dihydromyricetin ameliorates vascular calcification in chronic kidney disease by targeting AKT signaling.

Vascular calcification is highly prevalent in chronic kidney disease (CKD), and is characterized by transdifferentiation from contractile vascular smooth muscle cells (VSMCs) into an osteogenic phenotype. However, no effective and therapeutic option to prevent vascular calcification is yet available. Dihydromyricetin (DMY), a bioactive flavonoid isolated from Ampelopsis grossedentata, has been found to inhibit VSMCs proliferation and the injury-induced neointimal formation. However, whether DMY has an effect on osteogenic differentiation of VSMCs and vascular calcification is still unclear. In the present study, we sought to investigate the effect of DMY on vascular calcification in CKD and the underlying mechanism. DMY treatment significantly attenuated calcium/phosphate-induced calcification of rat and human VSMCs in a dose-dependent manner, as shown by Alizarin Red S staining and calcium content assay, associated with down-regulation of osteogenic markers including type I collagen (COL I), Runt-related transcription factor 2 (RUNX2), bone morphogenetic protein 2 (BMP2) and osteocalcin (OCN). These results were further confirmed in aortic rings ex vivo. Moreover, DMY ameliorated vascular calcification in rats with CKD. Additionally, we found that AKT signaling was activated during vascular calcification, whereas significantly inhibited by DMY administration. DMY treatment significantly reversed AKT activator-induced vascular calcification. Furthermore, inhibition of AKT signaling efficiently attenuated calcification, which was similar to that after treatment with DMY alone, and DMY had a better inhibitory effect on calcification as compared with AKT inhibitor. The present study demonstrated that DMY has a potent inhibitory role in vascular calcification partially by inhibiting AKT activation, suggesting that DMY may act as a promising therapeutic candidate for patients suffering from vascular calcification.

Flavocoxid Ameliorates Aortic Calcification Induced by Hypervitaminosis D3 and Nicotine in Rats Via Targeting TNF-α, IL-1β, iNOS, and Osteogenic Runx2.

This research was designed to investigate the effects and mechanisms of flavocoxid (FCX) on vascular calcification (VC) in rats. Vitamin D3 and nicotine were administered to Wistar rats, which then received FCX (VC-FCX group) or its vehicle (VC group) for 4weeks. Control and FCX groups served as controls.Systolic (SBP) and diastolic (DBP) blood pressures, heart rate (HR), and left ventricular weight (LVW)/BW were measured. Serum concentrations of calcium, phosphate, creatinine, uric acid, and alkaline phosphatase were determined. Moreover, aortic calcium content and aortic expression of runt-related transcription factor (Runx2), osteopontin (OPN), Il-1β, α-smooth muscle actin (α-SMA), matrix metalloproteinase-9 (MMP-9), inducible nitric oxide synthase (iNOS), and tumor necrosis factor-α (TNF-α) were assessed. Oxidative status in aortic homogenates was investigated. Compared to untreated VC rats, FCX treatment prevented body weight loss, reduced aortic calcium deposition, restored normal values of SBP, DBP, and HR, and attenuated LV hypertrophy. FCX also improved renal function and ameliorated serum levels of phosphorus, calcium, and ALP in rats with VC. FCX abolished aortic lipid peroxidation in VC rats. Moreover, VC-FCX rats showed marked reductions in aortic levels of Il-1β and osteogenic marker (Runx2) and attenuated aortic expression of TNF-α, iNOS, and MMP-9 proteins compared to untreated VC rats. The expression of the smooth muscle lineage marker α-SMA was greatly enhanced in aortas from VC rats upon FCX treatment. These findings demonstrate FCX ability to attenuate VDN-induced aortic calcinosis in rats, suggesting its potential for preventing arteiocalcinosis in diabetic patients and those with chronic kidney disease.

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&amp;lt; 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&amp;lt;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.

Intermedin1-53 attenuates aging-associated vascular calcification in rats by upregulating sirtuin 1.

Vascular calcification is a common phenomenon in older adults. Intermedin (IMD) is a cardiovascular bioactive peptide inhibiting vascular calcification. In this study, we aimed to investigate whether IMD1-53 attenuates aging-associated vascular calcification. Vascular calcification was induced by vitamin D3 plus nicotine (VDN) in young and old rats. The calcification in aortas was more severe in old rats treated with VDN than young control rats, and IMD expression was lower. Exogenous administration of IMD1-53 significantly inhibited the calcium deposition in aortas and the osteogenic transdifferentiation of vascular smooth muscle cells (VSMCs) in VDN-treated old rats. Moreover, levels of aging-related p16, p21 and β-galactosidase were all greatly decreased by IMD1-53. These results were further confirmed in rat and human VSMCs in vitro. In addition, IMD-deficient mouse VSMCs showed senescence features coinciding with osteogenic transition as compared with wild-type mouse VSMCs. Mechanistically, IMD1-53 significantly increased the expression of the anti-aging factor sirtuin 1 (sirt1); the inhibitory effects of IMD1-53 on calcification and senescence were blocked by sirt1 knockdown. Furthermore, preincubation with inhibitors of PI3K, AMPK or PKA efficiently blunted the upregulatory effect of IMD1-53 on sirt1. Consequently, IMD1-53 could attenuate aging-associated vascular calcification by upregulating sirt1 via activating PI3K/Akt, AMPK and cAMP/PKA signaling.

Estrogen inhibits vascular calcification in rats via hypoxia-induced factor-1α signaling.

ObjectiveCalcification serves as a surrogate for atherosclerosis-associated vascular diseases, and coronary artery calcification is mediated by multiple pathogenic factors. Estrogen is a known factor that protects the arterial wall against atherosclerosis, but its role in the coronary artery calcification development remains largely unclear. This study tested the hypothesis that estrogen inhibits coronary artery calcification via the hypoxia-induced factor-1α pathway.MethodsEight-week-old healthy female Sprague–Dawley rats were castrated, and vitamin D3 was administered orally to establish. Hypoxia-induced factor-1 inhibitor was administered to test its effect on vascular calcification and expression of bone morphogenetic protein 2 and runt-related transcription factor-2. Vascular smooth muscle cell calcification was induced with CaCl2 in rat aortic smooth muscle cells in the presence or absence of E2(17β-estradiol) and bone morphogenetic protein 2 siRNA intervention.ResultsThe estrogen levels in ovariectomized rats were significantly decreased, as determined by ELISA. Expression of hypoxia-induced factor-1α mRNA and protein was significantly increased in vascular cells with calcification as compared to those without calcification (p < 0.01). E2 treatment decreased the calcium concentration in vascular cell calcification and cell calcium nodules in vitro (p < 0.05). E2 also lowered the levels of hypoxia-induced factor-1α mRNA and protein (p < 0.01). Oral administration of the hypoxia-induced factor-1α inhibitor dimethyloxetane in castrated rats alleviated vascular calcification and expression of osteogenesis-related transcription factors, bone morphogenetic protein 2 and RUNX2 (p < 0.01). Finally, bone morphogenetic protein 2 siRNA treatment decreased the levels of p-Smad1/5/8 in A7r5 calcification cells (p < 0.01).ConclusionEstrogen deficiency enhances vascular calcification. Treatment with estrogen reduces the expression of hypoxia-induced factor-1α as well as vascular calcification in rats. The estrogen effects occur in a fashion dependent on hypoxia-induced factor-1α regulation of bone morphogenetic protein-2 and downstream Smad1/5/8.

Trimethylamine-N-Oxide Promotes Vascular Calcification Through Activation of NLRP3 (Nucleotide-Binding Domain, Leucine-Rich-Containing Family, Pyrin Domain-Containing-3) Inflammasome and NF-κB (Nuclear Factor κB) Signals.

Vascular calcification is highly prevalent in patients with chronic kidney disease. Increased plasma trimethylamine N-oxide (TMAO), a gut microbiota-dependent product, concentrations are found in patients undergoing hemodialysis. However, a clear mechanistic link between TMAO and vascular calcification is not yet established. In this study, we investigate whether TMAO participates in the progression of vascular calcification using in vitro, ex vivo, and in vivo models. Approach and Results: Alizarin red staining revealed that TMAO promoted calcium/phosphate-induced calcification of rat and human vascular smooth muscle cells in a dose-dependent manner, and this was confirmed by calcium content assay. Similarly, TMAO upregulated the expression of bone-related molecules including Runx2 (Runt-related transcription factor 2) and BMP2 (bone morphogenetic protein-2), suggesting that TMAO promoted osteogenic differentiation of vascular smooth muscle cells. In addition, ex vivo study also showed the positive regulatory effect of TMAO on vascular calcification. Furthermore, we found that TMAO accelerated vascular calcification in rats with chronic kidney disease, as indicated by Mico-computed tomography analysis, alizarin red staining and calcium content assay. By contrast, reducing TMAO levels by antibiotics attenuated vascular calcification in chronic kidney disease rats. Interestingly, TMAO activated NLRP3 (nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3) inflammasome and NF-κB (nuclear factor κB) signals during vascular calcification. Inhibition of NLRP3 inflammasome and NF-κB signals attenuated TMAO-induced vascular smooth muscle cell calcification. This study for the first time demonstrates that TMAO promotes vascular calcification through activation of NLRP3 inflammasome and NF-κB signals, suggesting the potential link between gut microbial metabolism and vascular calcification. Reducing the levels of TMAO could become a potential treatment strategy for vascular calcification in chronic kidney disease.

Expression characteristics of adiponectin and receptor activator of nuclear factor kappa B ligand in the alveolar bone of rats with periodontitis and its effect

ABSTRACT We sought to investigate the expression characteristics of adiponectin (APN) and receptor activator of nuclear factor kappa B ligand (RANKL) in the periodontal tissues of rats with periodontitis and the effect on vascular calcification. Thirty-six Sprague Dawley rats were randomly divided into a normal control group, periodontitis group, and vascular calcification group. Western blotting was employed to detect the expressions of APN and RANKL in the periodontal tissues of rats. Calcium content and alkaline phosphatase activity in the vascular tissue of rats were determined. Compared to the normal control group, the periodontitis and vascular calcification groups presented lower expressions of APN and RANKL. The expression of APN in the periodontal tissues of rats in the vascular calcification group was significantly lower than that in the periodontitis group. As for the calcium content and alkaline phosphatase activity in rats’ vascular tissues, the vascular calcification group displayed significantly higher rates than the other two groups, whereas the periodontitis group presented lower calcium content and alkaline phosphatase activity than the normal control group. APN and RANKL may be involved in the process of periodontitis and vascular calcification in rats, and some kinds of relationship may exist between periodontitis and vascular calcification.

Salusin-β Promotes Vascular Calcification via Nicotinamide Adenine Dinucleotide Phosphate/Reactive Oxygen Species-Mediated Klotho Downregulation.

Aims: Vascular calcification (VC) is a hallmark feature of cardiovascular disease and a significant risk factor for morbidity and mortality. Salusin-β exerts cardiovascular regulating effects in hypertension, atherosclerosis, and diabetes. The present study was designed to examine the roles of salusin-β in the progression of VC and its downstream signaling mechanisms. Results: Salusin-β expression in both the aortas of VC rats induced by vitamin D3 and nicotine and vascular smooth muscle cells (VSMCs) incubated with calcifying media was increased. Salusin-β knockdown remarkably reduced VC, whereas overexpression of salusin-β exacerbated VC both in vitro and in vivo. Overexpression of salusin-β promoted the VSMC osteochondrogenic transition, decreased Klotho protein levels, enhanced Ras-related C3 botulinum toxin substrate 1 (Rac1) activity and the translocation of p47phox to the membrane, increased the expression of nicotinamide adenine dinucleotide phosphate [NAD(P)H] oxidase subunits and the production of reactive oxygen species (ROS) with or without calcifying media; however, salusin-β deficiency played the opposite roles. The calcification and downregulated Klotho protein levels induced by salusin-β were restored by ROS scavenger N-acetyl-l-cysteine, diphenyleneiodonium chloride [an inhibitor of flavin-containing enzyme, including NAD(P)H oxidase], or gene knockdown of NAD(P)H oxidase (NOX)-2, p22phox, or p47phox but were not affected by NOX-1 and NOX-4 knockdown. Klotho knockdown attenuated the protective effect of salusin-β deficiency on VSMC calcification. By contrast, exogenous Klotho ameliorated the development of VC and ROS generation induced by salusin-β overexpression. Innovation: Salusin-β is a critical modulator in VC. Conclusion: Salusin-β regulates VC through activation of NAD(P)H/ROS-mediated Klotho downregulation, suggesting that salusin-β may be a novel target for treatment of VC.