Phosphonoformic acid reduces hyperphosphatemia-induced vascular calcification via Pit-1.

OBJECTIVE: This study aimed to examine the mechanism of hyperphosphatemia-induced vascular calcification (HPVC). METHODS: Primary human aortic smooth muscle cells and rat aortic rings were cultured in Dulbecco's modified Eagle's medium supplemented with 0.9 mM or 2.5 mM phosphorus concentrations. Type III sodium-dependent phosphate cotransporter-1 (Pit-1) small interfering RNA and phosphonoformic acid (PFA), a Pit-1 inhibitor, were used to investigate the effects and mechanisms of Pit-1 on HPVC. Calcium content shown by Alizarin red staining, expression levels of Pit-1, and characteristic molecules for phenotypic transition of vascular smooth muscle cells were examined. RESULTS: Hyperphosphatemia induced the upregulation of Pit-1 expression, facilitated phenotypic transition of vascular smooth muscle cells, and led to HPVC in cellular and organ models. Treatment with Pit-1 small interfering RNA or PFA significantly inhibited Pit-1 expression, suppressed phenotypic transition, and attenuated HPVC. CONCLUSIONS: Our findings suggest that Pit-1 plays a pivotal role in the development of HPVC. The use of PFA as a Pit-1 inhibitor has the potential for therapeutic intervention in patients with HPVC. However, further rigorous clinical investigations are required to ensure the safety and efficacy of PFA before it can be considered for widespread implementation in clinical practice.

In vitro Evaluation of the Calcification Inhibitory Properties of Policosanol, Genistein, and Vitamin D (Reduplaxin®) either Alone or in Combination.

INTRODUCTION: The process of vascular calcification has severe clinical consequences in a number of diseases, including diabetes, atherosclerosis, and end-stage renal disease. In the present study, we investigated the effect of policosanol (Poli), genistein (Gen), and vitamin D (VitD) separately and in association to evaluate the possible synergistic action on inorganic phosphate (Pi)-induced calcification of vascular smooth muscle cells (VSMCs). METHODS: Primary human VSMCs were cultured with either growth medium or growth medium supplemented with calcium and phosphorus (calcification medium) in combination with Poli, Gen, and VitD. Alizarin Red staining, mineralization, and the protein expression of RUNX2 and superoxide dismutase-2 (SOD2) were investigated. RESULTS: All three substances tested were effective at reducing osteogenic differentiation of VSMCs in a dose-dependent manner. Poli+Gen, Poli+VitD, Gen+VitD treatment induced a greater inhibition of calcification and RUNX2 expression compared to single compounds treatments. Moreover, the association of Poli+Gen+VitD (Reduplaxin®) was more effective at inhibiting VSMCs mineralization and preventing the increase in RUNX2 expression induced by calcification medium but not modified SOD2 expression. CONCLUSIONS: The association of Pol, Gen, and VitD (Reduplaxin®) has an additive inhibitory effect on the calcification process of VSMCs induced in vitro by a pro-calcifying medium.

Efficacy and mechanism of Shenqi Compound in inhibiting diabetic vascular calcification.

BACKGROUND: Shenqi Compound (SQC) has been used in clinic for several decades in the prevention and treatment of diabetes and its complications. But this is merely a heritage of experience. The primary aim of this study is to scientifically validate the therapeutic effects of SQC on diabetic vascular calcification (DVC) in an animal model and, simultaneously, uncover its potential underlying mechanisms. METHOD: Spontaneous diabetic rat- Goto Kakizaki (GK) rats were selected for rat modeling. We meticulously designed three distinct groups: a control group, a model group, and an SQC treatment group to rigorously evaluate the influence of SQC. Utilizing a comprehensive approach that encompassed methods such as pathological staining, western blot analysis, qRT-PCR, and RNA sequencing, we thoroughly investigated the therapeutic advantages and the underlying mechanistic pathways associated with SQC in the treatment of DVC. RESULT: The findings from this investigation have unveiled the extraordinary efficacy of SQC treatment in significantly mitigating DVC. The underlying mechanisms driving this effect encompass multifaceted facets, including the restoration of aberrant glucose and lipid metabolism, the prevention of phenotypic transformation of vascular smooth muscle cells (VSMCs) into osteogenic-like states, the subsequent inhibition of cell apoptosis, the modulation of inflammation responses, the remodeling of the extracellular matrix (ECM), and the activation of the Hippo-YAP signaling pathway. Collectively, these mechanisms lead to the dissolution of deposited calcium salts, ultimately achieving the desired inhibition of DVC. CONCLUSION: Our study has provided compelling and robust evidence of the remarkable efficacy of SQC treatment in significantly reducing DVC. This reduction is attributed to a multifaceted interplay of mechanisms, each playing a crucial role in the observed therapeutic effects. Notably, our findings illuminate prospective directions for further research and potential clinical applications in the field of cardiovascular health.

Effect of iron administration on the aortic iron content and vascular calcification in phosphorus-loaded chronic kidney disease rats.

BACKGROUND: Cardiovascular disease (CVD) is a major cause of morbidity and mortality in patients with chronic kidney disease (CKD) and could be related to oxidative stress. Vascular calcification (VC) has been established as a critical risk factor for accelerated CVD. In CKD, phosphorus (Pi), iron (Fe) and Nrf2 are modulators of VC and important agonists and antagonists of oxidative stress. The aim of this study was to determine whether Fe administration, which is commonly used to treat renal anemia, affects aortic Fe overload and VC, and whether Nrf2 and its related genes, ferritin H and HIF-1α, are involved in the development of VC. METHODS: A CKD model was created in rats by administering adenine and simultaneously feeding a high-Pi diet. In addition to control and CKD rats without Fe administration (No-Fe group), Fe was administered orally (PO-Fe group) or intraperitoneally (IP-Fe group) to CKD animals to clarify the effects of Fe administration on the aortic Fe and calcium (Ca) contents and the involvement of Nrf2 and its induced antioxidative proteins, ferritin H and HIF-1α, in VC. RESULTS: The aortic Fe content increased significantly in the IP-Fe group, which was closely correlated with liver HAMP (hepcidin) expression in all animals. Fe administration had no significant effect on the aortic Ca and Pi contents regardless of the route of Fe administration. The aortic mRNA level of Nrf2 was significantly increased in the IP-Fe group and correlated with serum Pi levels and aortic Fe contents, which could respond to oxidative stress. Notably, the mRNA level of Nrf2 was also significantly correlated with the mRNA levels of ferritin H and HIF-1α. Since we could not measure Nrf2 protein levels in this study, we confirmed the upregulation of HMOX1 and NQO1 mRNA expression in parallel with Nrf2 mRNA. CONCLUSION: Parenteral Fe administration increased aortic Fe in parallel with the liver HAMP mRNA level but did not affect VC. Aortic Nrf2 mRNA levels correlated significantly with aortic Fe and serum Pi levels and with aortic mRNA levels of ferritin H and HIF-1α as well as HMOX1 and NQO1.

Interventions to Attenuate Cardiovascular Calcification Progression: A Systematic Review of Randomized Clinical Trials.

BACKGROUND: Cardiovascular calcification, characterized by deposition of calcium phosphate in the arterial wall and heart valves, is associated with cardiovascular morbidity and mortality and is commonly seen in aging, diabetes, and chronic kidney disease. Whether evidence-based interventions could significantly attenuate cardiovascular calcification progression remains uncertain. METHODS AND RESULTS: We conducted a systematic review of randomized controlled trials involving interventions, compared with placebo, another comparator, or standard of care, to attenuate cardiovascular calcification. Included clinical trials involved participants without chronic kidney disease, and the outcome was cardiovascular calcification measured using radiological methods. Quality of evidence was determined by the Cochrane risk of bias and Grading of Recommendations, Assessment, Development, and Evaluations assessment. Forty-nine randomized controlled trials involving 9901 participants (median participants 104, median duration 12 months) were eligible for inclusion. Trials involving aged garlic extract (n=6 studies) consistently showed attenuation of cardiovascular calcification. Trials involving 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (n=14), other lipid-lowering agents (n=2), hormone replacement therapies (n=3), vitamin K (n=5), lifestyle measures (n=4), and omega-3 fatty acids (n=2) consistently showed no attenuation of cardiovascular calcification with these therapies. Trials involving antiresorptive (n=2), antihypertensive (n=2), antithrombotic (n=4), and hypoglycemic agents (n=3) showed mixed results. Singleton studies involving salsalate, folate with vitamin B6 and 12, and dalcetrapib showed no attenuation of cardiovascular calcification. Overall, Cochrane risk of bias was moderate, and the Grading of Recommendations, Assessment, Development, and Evaluations assessment for a majority of analyses was moderate certainty of evidence. CONCLUSIONS: Currently, there are insufficient or conflicting data for interventions evaluated in clinical trials for mitigation of cardiovascular calcification. Therapy involving aged garlic extract appears most promising, but evaluable studies were small and of short duration.

Sodium-glucose cotransporter 2 inhibitor canagliflozin alleviates vascular calcification through suppression of nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 inflammasome.

AIMS: Vascular calcification (VC) is prevalent in pathological processes such as diabetes, chronic kidney disease (CKD), and atherosclerosis, but effective therapies are still lacking by far. Canagliflozin (CANA), a sodium-glucose cotransporter 2 inhibitor, has been approved for the treatment of type 2 diabetes mellitus and exhibits beneficial effects against cardiovascular disease. However, the effect of CANA on VC remains unknown. In this study, we hypothesize that CANA protects against VC. METHODS AND RESULTS: Micro-computed tomography analysis and alizarin red staining revealed that CANA treatment prevented aortic calcification in CKD rats and in VitD3-overloaded mice. Moreover, CANA alleviated the calcification of rat and human arterial rings. Alizarin red staining revealed that calcification of rat and human vascular smooth muscle cells (VSMCs) was attenuated by CANA treatment and this phenomenon was confirmed by calcium content assay. In addition, CANA downregulated the expression of osteogenic differentiation markers Runx2 and BMP2. Of interest, qPCR and western blot analysis revealed that CANA downregulated the expression of the nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3), and the downstream signalling molecules Caspase-1 and IL-1ß in VSMCs as well. Both NLRP3 inhibitor MCC950 and knockdown of NLRP3 by siRNA independently resulted in decreased calcification of VSMCs. By contrast, activation of NLRP3 exacerbated VSMC calcification, and this effect was prevented by the addition of CANA. CONCLUSIONS: Our study for the first time demonstrates that CANA exerts a protective effect on VC at least partially via suppressing the NLRP3 signalling pathway. Therefore, supplementation of CANA as well as inhibition of NLRP3 inflammasome presents a potential therapy for VC.

Panax quinquefolius saponin inhibits vascular smooth muscle cell calcification via activation of nuclear factor-erythroid 2-related factor 2.

BACKGROUND: Panax quinquefolius saponin (PQS) is the main active component of Panax quinquefolius. Emerging evidence suggests that PQS exerts beneficial effects against cardiovascular diseases. However, the role and mechanism of PQS in vascular calcification are not unclear. The present study investigated the effects of PQS on the calcification of vascular smooth muscle cell (VSMCs). METHODS: The present study used calcification medium containing 3 mM inorganic phosphate (Pi) to induce rat VSMCs calcification. We investigated the effects of PQS on VSMCs calcification using alizarin red staining and alkaline phosphatase (ALP) activity assays. The intracellular reactive oxygen species (ROS) levels and the transcriptional activity of nuclear factor-erythroid 2-related factor 2 (Nrf2) were determined. The mRNA and protein expression levels of Nrf2, the antioxidant gene heme oxygenase-1 (HO-1), osteogenic markers, including runt-related transcription factor 2 (Runx2) and bone morphogenetic protein 2 (BMP2), and Kelch-like ECH-associated protein 1 (Keap1) were also measured. RESULTS: Treatment with Pi significantly increased intracellular calcium deposition and ALP activity, which were suppressed by PQS in a concentration-dependent manner. During VSMCs calcification, PQS inhibited the mRNA and protein expression of Runx2 and BMP2. PQS treatment reduced intracellular ROS production and significantly upregulated Nrf2 transcriptional activity and the expression of Nrf2 and its target antioxidant gene HO-1. PQS suppressed the Pi-induced protein expression of Keap1, which is an endogenous inhibitor of Nrf2. Keap1 siRNA treatment induced Nrf2 expression and downregulated Runx2 expression in the presence of Pi and PQS. CONCLUSION: Taken together, these findings suggest that PQS could effectively inhibit VSMCs calcification by ameliorating oxidative stress and regulating osteogenic genes via the promotion of Nrf2 expression.

GLSP and GLSP-derived triterpenes attenuate atherosclerosis and aortic calcification by stimulating ABCA1/G1-mediated macrophage cholesterol efflux and inactivating RUNX2-mediated VSMC osteogenesis.

Background and Purpose: Atherosclerosis is the main pathophysiological foundation of cardiovascular disease, which was caused by inflammation and lipid metabolism disorder, along with vascular calcification. Aortic calcification leads to reduced plaque stability and eventually causes plaque rupture which leads to cardiovascular events. Presently, the drug to treat aortic calcification remains not to be available. Ganoderma lucidum spore powder (GLSP) is from Ganoderma lucidum which is a Traditional Chinese Medicine with the homology of medicine and food. It has multiple pharmacological effects, but no research on aortic calcification during atherosclerosis was performed. This study investigated the effects of GLSP on atherosclerosis and aortic calcification and revealed the underlying mechanism. Methods: In vivo, 8-week-aged male LDLR-/- mice were fed a high-fat diet to induce atherosclerosis along with aortic calcification. Simultaneously, the mice were treated with GLSP at the first week of HFD feeding to determine the protection against early and advanced atherosclerosis. Subsequently, the mice tissues were collected to evaluate the effects of GLSP on atherosclerosis, and aortic calcification, and to reveal the underlying mechanism. In vitro, we determined the major components of GLSP triterpenes by HPLC, and subsequently assessed the protective effects of these main active components on lipid metabolism, inflammation, and calcification in RAW264.7 and HASMC cells. Results: We observed GLSP attenuated plaque area and aortic calcification in the mice with early and advanced atherosclerosis. GLSP reduced the number of foam cells by improving ABCA1/G1-mediated cholesterol efflux in macrophages. In addition, GLSP protected against the aortic endothelium activation. Moreover, GLSP inhibited aortic calcification by inactivating RUNX2-mediated osteogenesis in HASMCs. Furthermore, we determined the major components of GLSP triterpenes, including Ganoderic acid A, Ganoderic acid B, Ganoderic acid C6, Ganoderic acid G, and Ganodermanontriol, and found that these triterpenes promoted ABCA1/G1-mediated cholesterol efflux and inhibited inflammation in macrophage, and inactivated RUNX2-mediated osteogenesis in VSMC. Conclusions: This study demonstrates that GLSP attenuates atherosclerosis and aortic calcification by improving ABCA1/G1-mediated cholesterol efflux and inactivating RUNX2-mediated osteogenesis in LDLR-/- mice. GLSP may be a potential drug candidate for the treatment of atherosclerosis and vascular calcification.

Beneficial effect of the short-chain fatty acid propionate on vascular calcification through intestinal microbiota remodelling.

BACKGROUND: Vascular calcification is a major cause of the high morbidity and mortality of cardiovascular diseases and is closely associated with the intestinal microbiota. Short-chain fatty acids (SCFAs) are derived from the intestinal microbiota and can also regulate intestinal microbiota homeostasis. However, it remains unclear whether exogenous supplementation with propionate, a SCFA, can ameliorate vascular calcification by regulating the intestinal microbiota. This study was conducted to explore the roles of propionate and the intestinal microbiota in the process of vascular calcification. METHODS: In total, 92 patients were enrolled consecutively as the observational cohort to analyse the relationship between SCFAs and vascular calcification in both blood and faecal samples. A rat model of vascular calcification was induced by vitamin D3 and nicotine (VDN) to validate the effect of propionate. Differences in the intestinal microbiota were analysed by 16S ribosomal RNA gene sequencing. Faecal microbiota transplantation and Akkermansia muciniphila transplantation experiments were performed to evaluate the functions of the intestinal microbiota. RESULTS: The results of the observational cohort study revealed that the levels of SCFAs (particularly propionate) in both blood and faecal samples independently correlated negatively with calcification scores (P < 0.01). To verify the activities of propionate, it was provided to VDN-treated rats, and oral or rectal propionate delivery reshaped the intestinal microbiota, resulted in elevated SCFA production, improved intestinal barrier function and alleviated inflammation, ultimately ameliorating vascular calcification. Furthermore, we demonstrated that transplantation of the propionate-modulated intestinal microbiota induced beneficial outcomes similar to those with oral or rectal propionate administration. Interestingly, linear discriminant analysis (LDA) effect size (LEfSe) revealed that oral or rectal propionate administration and propionate-modulated intestinal microbiota transplantation both enriched primarily Akkermansia. Subsequently, we demonstrated that Akkermansia supplementation could ameliorate VDN-induced vascular calcification in rats. CONCLUSIONS: Propionate can significantly ameliorate vascular calcification in VDN-treated rats, and this effect is mediated by intestinal microbiota remodelling. The findings in our study indicate that the intestinal tract-vessel axis is a promising target for alleviating vascular calcification. Video Abstract.

Reversal Of Arterial Disease by modulating Magnesium and Phosphate (ROADMAP-study): rationale and design of a randomized controlled trial assessing the effects of magnesium citrate supplementation and phosphate-binding therapy on arterial stiffness in moderate chronic kidney disease.

BACKGROUND: Arterial stiffness and calcification propensity are associated with high cardiovascular risk and increased mortality in chronic kidney disease (CKD). Both magnesium and phosphate are recognized as modulators of vascular calcification and chronic inflammation, both features of CKD that contribute to arterial stiffness. In this paper, we outline the rationale and design of a randomized controlled trial (RCT) investigating whether 24 weeks of oral magnesium supplementation with or without additional phosphate-binding therapy can improve arterial stiffness and calcification propensity in patients with stage 3-4 CKD. METHODS: In this multi-center, placebo-controlled RCT, a total of 180 participants with an estimated glomerular filtration rate of 15 to 50 ml/min/1.73 m2 without phosphate binder therapy will be recruited. During the 24 weeks intervention, participants will be randomized to one of four intervention groups to receive either magnesium citrate (350 mg elemental magnesium/day) or placebo, with or without the addition of the phosphate binder sucroferric oxyhydroxide (1000 mg/day). Primary outcome of the study is the change of arterial stiffness measured by the carotid-femoral pulse wave velocity over 24 weeks. Secondary outcomes include markers of calcification and inflammation, among others calcification propensity (T50) and high-sensitivity C-reactive protein. As explorative endpoints, repeated 18F-FDG and 18F-NaF PET-scans will be performed in a subset of participants (n = 40). Measurements of primary and secondary endpoints are performed at baseline, 12 and 24 weeks. DISCUSSION: The combined intervention of magnesium citrate supplementation and phosphate-lowering therapy with sucroferric oxyhydroxide, in stage 3-4 CKD patients without overt hyperphosphatemia, aims to modulate the complex and deregulated mineral metabolism leading to vascular calcification and arterial stiffness and to establish to what extent this is mediated by T50 changes. The results of this combined intervention may contribute to future early interventions for CKD patients to reduce the risk of CVD and mortality. TRIAL REGISTRATION: Netherlands Trial Register, NL8252 (registered December 2019), EU clinical Trial Register 2019-001306-23 (registered November 2019).

Dendrobium officinale polysaccharide inhibits vascular calcification via anti-inflammatory and anti-apoptotic effects in chronic kidney disease.

Vascular calcification is very common in patients with chronic kidney disease (CKD), but so far, there is no effective treatment. Dendrobium officinale polysaccharide (DOP), a natural component of Chinese herbal medicine, has been shown to exert anti-inflammatory and anti-apoptotic activity. Inflammation and apoptosis play an essential role in the progression of vascular calcification. However, the exact role and molecular mechanisms of DOP in vascular calcification remain unclear. In this study, we investigated the effects of DOP on vascular calcification using vascular smooth muscle cells (VSMCs), arterial rings, and CKD rats. Alizarin red staining and gene expression analysis revealed that DOP inhibited calcification and osteogenic differentiation of rat VSMCs in a dose-dependent manner. Similarly, ex vivo studies revealed that DOP inhibited the calcification of rat arterial rings. Furthermore, the administration of DOP alleviated vascular calcification in CKD rats. Moreover, DOP treatment suppressed VSMC inflammation and apoptosis. Finally, DOP treatment upregulated mRNA and protein levels of heme oxygenase-1 (HMOX-1); both pharmacological inhibition of HMOX-1 by the HMOX-1 inhibitor zinc protoporphyrin-9ZnPP9 and knockdown of HMOX-1 by siRNA markedly abrogated the suppression of inflammation and osteogenic differentiation of VSMCs by DOP. Collectively, these results suggest that DOP alleviates vascular calcification in CKD by suppressing apoptosis and inflammation via HMOX-1 activation. These results may provide a promising treatment for vascular calcification in CKD.

The Ameliorative Effect of Berberine on Vascular Calcification by Inhibiting Endoplasmic Reticulum Stress.

ABSTRACT: Vascular calcification (VC), which currently cannot be prevented or treated, is an independent risk factor for cardiovascular events. We aimed to investigate the ameliorative effect of berberine on VC via the activation of Akt signaling and inhibition of endoplasmic reticulum stress (ERS). The VC model was induced by high-dose Vitamin D 3 in rats and beta-glycerophosphate in primary vascular smooth muscle cells of rat aortas, which were evaluated by Alizarin red staining to determine the calcium content and alkaline phosphatase activity. ERS was determined by the levels of GRP78 and CHOP, whereas that of the Akt signaling pathway was determined by the levels of phosphorylated Akt and GSK3ß. VC was significantly ameliorated by berberine treatment in vivo and in vitro, and the inhibition of ERS and the activation of the Akt/GSK3 signaling pathway. In the vascular smooth muscle cells of primary rats, tunicamycin, an ERS activator, blocked the ameliorative effect of berberine on VC and ERS, but not the activation of Akt/GSK3. The ameliorative effects of berberine on VC, ERS, and the Akt signaling pathway were all prevented by inhibitor IV. Four-phenylbutyric acid, an ERS inhibitor, can restore the ameliorative effect of berberine on VC and ERS that was blocked by inhibitor IV. Our results are the first to demonstrate the ameliorative effect of VC that was mediated by the activation of the Akt signaling pathway and inhibition of ERS. These results may provide a new pharmaceutical candidate for the prevention and treatment of VC.

Lanthanum hydroxide inhibits vascular calcification by regulating the HIF-1 pathway.

The main reason for the high incidence of cardiovascular disease in chronic kidney disease (CKD) patients with vascular calcification (VC) is also the main cause of death in CKD patients. Lanthanum hydroxide (LH) has an inhibitory effect on VC in chronic renal failure; however, the mechanism of its inhibition is poorly defined. Here, we used network pharmacology analysis and found that hypoxia-inducible factor (HIF) is related to VC. In a CKD rat model induced by adenine combined with high phosphorus (1.2%), LH improved the survival rate and inhibited the occurrence and development of VC. In an in vitro study, we found that lanthanum chloride inhibited the occurrence of VC induced by high phosphorus and reduced the production of reactive oxygen species. This study thus revealed that LH can inhibit the occurrence and development of VC by inhibiting the activation of HIF-1.

Vitamin D and Calcium Supplementation Accelerate Vascular Calcification in a Model of Pseudoxanthoma Elasticum.

Arterial calcification is a common feature of pseudoxanthoma elasticum (PXE), a disease characterized by ABCC6 mutations, inducing a deficiency in pyrophosphate, a key inhibitor of calcium phosphate crystallization in arteries. METHODS: we analyzed whether long-term exposure of Abcc6-/- mice (a murine model of PXE) to a mild vitamin D supplementation, with or without calcium, would impact the development of vascular calcification. Eight groups of mice (including Abcc6-/- and wild-type) received vitamin D supplementation every 2 weeks, a calcium-enriched diet alone (calcium in drinking water), both vitamin D supplementation and calcium-enriched diet, or a standard diet (controls) for 6 months. Aorta and kidney artery calcification was assessed by 3D-micro-computed tomography, Optical PhotoThermal IR (OPTIR) spectroscopy, scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy (SEM-EDS) and Yasue staining. RESULTS: at 6 months, although vitamin D and/or calcium did not significantly increase serum calcium levels, vitamin D and calcium supplementation significantly worsened aorta and renal artery calcification in Abcc6-/- mice. CONCLUSIONS: vitamin D and/or calcium supplementation accelerate vascular calcification in a murine model of PXE. These results sound a warning regarding the use of these supplementations in PXE patients and, to a larger extent, patients with low systemic pyrophosphate levels.

Ameliorative effect of allicin on vascular calcification via inhibiting endoplasmic reticulum stress.

OBJECTIVES: Vascular calcification (VC) is an independent predictor for cardiovascular events and mortality. However, there are currently no effective methods to reverse or prevent it. The present study aimed to determine the ameliorative effect of allicin on VC. METHODS: VC model of rats was induced by high-dose vitamin D3, which was valued by Alizarin Red staining, calcium contents, and alkaline phosphatase in the aorta. Systolic blood pressure, pulse pressure, and pulse wave velocity were measured to determine aortic stiffness. Protein levels were detected by Western blot. RESULTS: Allicin treatment rescued aortic VC and stiffness. The increased protein levels of RUNX2 and BMP2, two markers of osteoblastic phenotype of vascular smooth muscle cells, in the calcified aorta were attenuated by allicin, whereas the decreased levels of calponin and SM22α induced by calcification were improved. Allicin treatment significantly attenuated the increased protein levels of GRP78, GRP94, and CHOP, which are key markers of endoplasmic reticulum stress, in the calcified aorta. The activation of PERK/eIF2α/ATF4 cascades was also prevented by allicin. CONCLUSIONS: Allicin could ameliorate aortic VC and stiffness. The ameliorative effect of allicin on VC might be mediated by inhibiting PERK/eIF2α/ATF4 cascades. Our results might provide a new proof for VC treatment.

Oral supplementation of inorganic pyrophosphate in pseudoxanthoma elasticum.

Pseudoxanthoma elasticum (PXE; OMIM 264800) is a rare heritable multisystem disorder, characterized by ectopic mineralization affecting elastic fibres in the skin, eyes and the cardiovascular system. Skin findings often lead to early diagnosis of PXE, but currently, no specific treatment exists to counteract the progression of symptoms. PXE belongs to a group of Mendelian calcification disorders linked to pyrophosphate metabolism, which also includes generalized arterial calcification of infancy (GACI) and arterial calcification due to CD73 deficiency (ACDC). Inactivating mutations in ABCC6, ENPP1 and NT5E are the genetic cause of these diseases, respectively, and all of them result in reduced inorganic pyrophosphate (PPi ) concentration in the circulation. Although PPi is a strong inhibitor of ectopic calcification, oral supplementation therapy was initially not considered because of its low bioavailability. Our earlier work however demonstrated that orally administered pyrophosphate inhibits ectopic calcification in the animal models of PXE and GACI, and that orally given Na4 P2 O7 is absorbed in humans. Here, we report that gelatin-encapsulated Na2 H2 P2 O7  has similar absorption properties in healthy volunteers and people affected by PXE. The sodium-free K2 H2 P2 O7 form resulted in similar uptake in healthy volunteers and inhibited calcification in Abcc6-/- mice as effectively as its sodium counterpart. Novel pyrophosphate compounds showing higher bioavailability in mice were also identified. Our results provide an important step towards testing oral PPi in clinical trials in PXE, or potentially any condition accompanied by ectopic calcification including diabetes, chronic kidney disease or ageing.

[Tanshinone IIa attenuates vascular calcification through inhibition of NF-κB and ß-catenin signaling pathways].

Tanshinone IIa is a key ingredient extracted from the traditional Chinese medicine Salvia miltiorrhiza (Danshen), and is widely used to treat various cardiovascular diseases. Vascular calcification is a common pathological change of cardiovascular tissues in patients with chronic kidney disease, diabetes, hypertension and atherosclerosis. However, whether Tanshinone IIa inhibits vascular calcification and the underlying mechanisms remain largely unknown. This study aims to investigate whether Tanshinone IIa can inhibit vascular calcification using high phosphate-induced vascular smooth muscle cell and aortic ring calcification model, and high dose vitamin D3 (vD3)-induced mouse models of vascular calcification. Alizarin red staining and calcium quantitative assay showed that Tanshinone IIa significantly inhibited high phosphate-induced vascular smooth muscle cell and aortic ring calcification. qPCR and Western blot showed that Tanshinone IIa attenuated the osteogenic transition of vascular smooth muscle cells. In addition, Tanshinone IIa also significantly inhibited high dose vD3-induced mouse aortic calcification and aortic osteogenic transition. Mechanistically, Tanshinone IIa inhibited the activation of NF-κB and ß-catenin signaling in normal vascular smooth muscle cells. Similar to Tanshinone IIa, inhibition of NF-κB and ß-catenin signaling using the chemical inhibitors SC75741 and LF3 attenuated high phosphate-induced vascular smooth muscle cell calcification. These results suggest that Tanshinone IIa attenuates vascular calcification at least in part through inhibition of NF-κB and ß-catenin signaling, and Tanshinone IIa may be a potential drug for the treatment of vascular calcification.

Case 296: Phlebosclerotic Colitis.

History A 55-year-old woman without systemic underlying disease, such as diabetes mellitus, inflammatory bowel disease, autoimmune disease, or chronic kidney disease, presented with generalized dull abdominal pain of 1-week duration. She had ingested herbal medicine for physical conditioning for several years. Laboratory findings, including biochemistry, electrolyte levels, and complete blood count, were all within normal limits, except for elevated serum C-reactive protein level (7.719 mg/dL; normal range, <1 mg/dL). The patient underwent initial evaluation with conventional abdominal radiography. She underwent subsequent evaluation with noncontrast CT of the abdomen and colonoscopy.

Resveratrol Ameliorates Aortic Calcification in Ovariectomized Rats via SIRT1 Signaling.

Postmenopausal women are at an increased risk of vascular calcification which is defined as the pathological deposition of minerals in the vasculature, and is strongly linked with increased cardiovascular disease risk. Since estrogen-replacement therapy is associated with increased cancer risk, there is a strong need for safer therapeutic approaches. In this study we aimed to investigate the protective and therapeutic effects of the phytoestrogen resveratrol against vascular calcification in ovariectomized rats, a preclinical model of postmenopause. Furthermore, we aimed to compare the effects of resveratrol to those of estrogen and to explore the mechanisms underpinning those effects. Treatment with resveratrol or estrogen ameliorated aortic calcification in ovariectomized rats, as shown by reduced calcium deposition in the arterial wall. Mechanistically, the effects of resveratrol and estrogen were mediated via the activation of SIRT1 signaling. SIRT1 protein expression was downregulated in the aortas of ovariectomized rats, and upregulated in rats treated with resveratrol or estrogen. Moreover, resveratrol and estrogen reduced the levels of the osteogenic markers: runt-related transcription factor 2 (RUNX2), osteocalcin and alkaline phosphatase (ALP) which have been shown to play a role during vascular calcification. Additionally, the senescence markers (p53, p16 and p21) which were also reported to play a role in the pathogenesis of vascular calcification, were reduced upon treatment with resveratrol and estrogen. In conclusion, the phytoestrogen resveratrol may be a safer alternative to estrogen, as a therapeutic approach against the progression of vascular calcification during postmenopause.

Spermidine inhibits vascular calcification in chronic kidney disease through modulation of SIRT1 signaling pathway.

Vascular calcification is a common pathologic condition in patients with chronic kidney disease (CKD) and aging individuals. It has been established that vascular calcification is a gene-regulated biological process resembling osteogenesis involving osteogenic differentiation. However, there is no efficient treatment available for vascular calcification so far. The natural polyamine spermidine has been demonstrated to increase life span and protect against cardiovascular disease. It is unclear whether spermidine supplementation inhibits vascular calcification in CKD. Alizarin red staining and quantification of calcium content showed that spermidine treatment markedly reduced mineral deposition in both rat and human vascular smooth muscle cells (VSMCs) under osteogenic conditions. Additionally, western blot analysis revealed that spermidine treatment inhibited osteogenic differentiation of rat and human VSMCs. Moreover, spermidine treatment remarkably attenuated calcification of rat and human arterial rings ex vivo and aortic calcification in rats with CKD. Furthermore, treatment with spermidine induced the upregulation of Sirtuin 1 (SIRT1) in VSMCs and resulted in the downregulation of endoplasmic reticulum (ER) stress signaling components, such as activating transcription factor 4 (ATF4) and CCAAT/enhancer-binding protein homologous protein (CHOP). Both pharmacological inhibition of SIRT1 by SIRT1 inhibitor EX527 and knockdown of SIRT1 by siRNA markedly blocked the inhibitory effect of spermidine on VSMC calcification. Consistently, EX527 abrogated the inhibitory effect of spermidine on aortic calcification in CKD rats. We for the first time demonstrate that spermidine alleviates vascular calcification in CKD by upregulating SIRT1 and inhibiting ER stress, and this may develop a promising therapeutic treatment to ameliorate vascular calcification in CKD.