Phosphate-induced Vascular Smooth Muscle Cell Research Articles

Abstract 146: Phosphodiesterase 10A Mediates Arterial Calcification Through A P38/MAPK-MMP3 Signaling

Background: Vascular calcification is prevalent in patients with atherosclerosis, chronic kidney disease (CKD), diabetes, and peripheral artery disease (PAD). When located in the arterial media, it is strongly associated with increased cardiovascular morbidity and mortality. The cyclic nucleotides cAMP and cGMP play important regulatory roles in a variety of human diseases that are controlled by distinct phosphodiesterase (PDE) isozymes. We have recently found that PDE10A is the most highly induced isoform in a rodent model of arterial calcification. The function and underlying mechanisms of PDE10A in medial artery calcification, however, remain unknown. Methods: Vascular smooth muscle cell (VSMC) calcification was mediated by a high phosphate media. Medial artery calcification was induced by vitamin D 3 injection and 5/6 nephrectomy calcification models. Vascular calcification was assessed by calcium assay and Von Kossa staining. Results: PDE10A was markedly increased in calcifying VSMCs in vitro , calcified arteries in vivo , and calcified human tibial arteries from patients with PAD. Knockdown or inhibition of PDE10A markedly attenuated phosphate-induced VSMC osteogenic transformation and calcification in VSMCs. Conversely, overexpression of PDE10A increased VSMC calcification. Deficiency and inhibition of PDE10A significantly decreased arterial calcification in vivo . Several labs including our own have demonstrated that matrix metallopeptidases (MMPs) are involved in vascular calcification. Using bioinformatics analysis and loss-of-function strategy, we found that MMP-3 could be regulated by PDE10A in calcifying VSMCs. Our further mechanistic studies showed that knockdown or inhibition of PDE10A decreased activated p38 MAPK and that inhibition of p38 MAPK attenuated MMP-3 upregulation under a calcifying condition. These data suggest that PDE10A mediates arterial calcification through a p38 MAPK-MMP-3 signaling pathway. Conclusion: PDE10A is a key regulator in the development of medial artery calcification. Our findings provide insight into the use of PDE10A inhibition strategies to reduce arterial calcification in patients with CKD and PAD.

Paraspeckle protein NONO attenuates vascular calcification by inhibiting bone morphogenetic protein 2 transcription

Vascular calcification is a pathological process commonly associated with atherosclerosis, chronic kidney disease, and diabetes. Paraspeckle protein NONO is a multifunctional RNA/DNA binding protein involved in many nuclear biological processes but its role in vascular calcification remains unclear. Here, we observed that NONO expression was decreased in calcified arteries of mice and patients with CKD. We generated smooth muscle-specific NONO-knockout mice and established three different mouse models of vascular calcification by means of 5/6 nephrectomy, adenine diet to induce chronic kidney failure, or vitamin D injection. The knockout mice were more susceptible to the development of vascular calcification relative to control mice, as verified by an increased calcification severity and calcium deposition. Likewise, aortic rings from knockout mice showed more significant vascular calcification than those from control mice ex vivo. In vitro, NONO deficiency aggravated high phosphate-induced vascular smooth muscle cell osteogenic differentiation and apoptosis, whereas NONO overexpression had a protective effect. Mechanistically, we demonstrated that the regulation of vascular calcification by NONO was mediated by bone morphogenetic protein 2 (BMP2). NONO directly bound to the BMP2 promoter using its C-terminal region, exerting an inhibitory effect on the transcription of BMP2. Thus, our study reveals that NONO is a novel negative regulator of vascular calcification, which inhibits osteogenic differentiation of vascular smooth muscle cell and vascular calcification via negatively regulating BMP2 transcription. Hence, NONO may provide a promising target for the prevention and treatment of vascular calcification.

Glucagon-Like Peptide Receptor Agonist Inhibits Angiotensin II-Induced Proliferation and Migration in Vascular Smooth Muscle Cells and Ameliorates Phosphate-Induced Vascular Smooth Muscle Cells Calcification.

Glucagon-like peptide-1 receptor agonist (GLP-1RA), which is a therapeutic agent for the treatment of type 2 diabetes mellitus, has a beneficial effect on the cardiovascular system. To examine the protective effects of GLP-1RAs on proliferation and migration of vascular smooth muscle cells (VSMCs), A-10 cells exposed to angiotensin II (Ang II) were treated with either exendin-4, liraglutide, or dulaglutide. To examine the effects of GLP-1RAs on vascular calcification, cells exposed to high concentration of inorganic phosphate (Pi) were treated with exendin-4, liraglutide, or dulaglutide. Ang II increased proliferation and migration of VSMCs, gene expression levels of Ang II receptors AT1 and AT2, proliferation marker of proliferation Ki-67 (Mki-67), proliferating cell nuclear antigen (Pcna), and cyclin D1 (Ccnd1), and the protein expression levels of phospho-extracellular signal-regulated kinase (p-Erk), phospho-c-JUN N-terminal kinase (p-JNK), and phospho-phosphatidylinositol 3-kinase (p-Pi3k). Exendin-4, liraglutide, and dulaglutide significantly decreased the proliferation and migration of VSMCs, the gene expression levels of Pcna, and the protein expression levels of p-Erk and p-JNK in the Ang II-treated VSMCs. Erk inhibitor PD98059 and JNK inhibitor SP600125 decreased the protein expression levels of Pcna and Ccnd1 and proliferation of VSMCs. Inhibition of GLP-1R by siRNA reversed the reduction of the protein expression levels of p-Erk and p-JNK by exendin-4, liraglutide, and dulaglutide in the Ang II-treated VSMCs. Moreover, GLP-1 (9-36) amide also decreased the proliferation and migration of the Ang II-treated VSMCs. In addition, these GLP-1RAs decreased calcium deposition by inhibiting activating transcription factor 4 (Atf4) in Pi-treated VSMCs. These data show that GLP-1RAs ameliorate aberrant proliferation and migration in VSMCs through both GLP-1Rdependent and independent pathways and inhibit Pi-induced vascular calcification.

Endothelial Cell Dysfunction Promotes Phosphate-Induced Vascular Smooth Muscle Cell Calcification

Endothelial Cell Dysfunction Promotes Phosphate-Induced Vascular Smooth Muscle Cell Calcification

Role of prolyl hydroxylase/HIF-1 signaling in vascular calcification.

Morbidity and mortality of chronic kidney disease (CKD) patients are largely associated with vascular calcification, an actively regulated process in which vascular smooth muscle cells (VSMCs) change into cells similar to osteocytes/chondrocytes, known as trans-differentiation. Cellular and systemic response to low oxygen (hypoxia) is regulated by the prolyl hydroxylase/hypoxia-inducible factor (HIF)-1 pathway. Recent studies highlighted that hypoxia-mediated activation of HIF-1 induces trans-differentiation of VSMCs into bone-forming type through an increase in osteo-/chondrogenic genes. Inhibition of the HIF-1 pathway abolished osteochondrogenic differentiation of VSMCs. Hypoxia strongly enhanced elevated phosphate-induced VSMC osteogenic trans-differentiation and calcification. HIF-1 was shown to be essential for phosphate enhanced VSMC calcification. O2-dependent degradation HIF-1 is triggered by the prolyl hydroxylase domain proteins (PHD). Prolyl hydroxylase inhibitors, daprodustat and roxadustat, increase high phosphate-induced VC in VSMCs, stabilizing HIF-1α and activating the HIF-1 pathway in these cells. Whether the use of these PHD inhibitors to treat anemia in CKD patients will favor the development and progression of vascular calcification remains to be explored.

Abstract 291: Phosphodiesterase 10A Regulates Medial Artery Calcification

Vascular calcification results from deposition of calcium hydroxyapatite crystals in the vessel wall. It is highly prevalent in patients with chronic kidney disease (CKD), diabetes, and peripheral artery disease (PAD). In lower extremity arteries, elevated calcification levels are associated with an increased risk of ischemic events including amputation. The cyclic nucleotides cAMP and cGMP, controlled by distinct cyclic nucleotide phosphodiesterase (PDE) isozymes, play important regulatory roles in a variety of human diseases. Using a qPCR PDE array, we found that PDE10A was the most highly induced isoform in a rodent model of arterial calcification. PDE10A was also markedly increased in calcifying vascular smooth muscle cells (VSMCs) in vitro , calcified arteries from rodents with CKD, and calcified human tibial arteries. Knockdown or inhibition of PDE10A markedly attenuated high phosphate-induced VSMC osteogenic transformation and calcification in both rat and human VSMCs. Importantly, deficiency of PDE10A significantly decreased arterial calcification in both ex vivo aortic ring and in vivo vitamin D 3 medial calcification models. Several labs including our own have previously demonstrated that matrix metallopeptidases (MMPs) are involved in vascular calcification. Using a loss-of-function strategy and bioinformatics analysis, we found that MMP-3 expression was regulated by PDE10A in calcifying VSMCs. Our further mechanistic studies showed that PDE10A could regulate vascular calcification by controlling p38 MAPK signaling and MMP-3 activity through cGMP/PKG signaling. These findings suggest that PDE10A plays a critical role in the development of medial artery calcification, and that targeting it may provide a novel therapeutic strategy to reduce medial calcification in patients with PAD and CKD with the ultimate goal of preventing major limb amputation.

Zinc Ameliorates the Osteogenic Effects of High Glucose in Vascular Smooth Muscle Cells

In diabetic patients, medial vascular calcification is common and associated with increased cardiovascular mortality. Excessive glucose concentrations can activate the nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-kB) and trigger pro-calcific effects in vascular smooth muscle cells (VSMCs), which may actively augment vascular calcification. Zinc is able to mitigate phosphate-induced VSMC calcification. Reduced serum zinc levels have been reported in diabetes mellitus. Therefore, in this study the effects of zinc supplementation were investigated in primary human aortic VSMCs exposed to excessive glucose concentrations. Zinc treatment was found to abrogate the stimulating effects of high glucose on VSMC calcification. Furthermore, zinc was found to blunt the increased expression of osteogenic and chondrogenic markers in high glucose-treated VSMCs. High glucose exposure was shown to activate NF-kB in VSMCs, an effect that was blunted by additional zinc treatment. Zinc was further found to increase the expression of TNFα-induced protein 3 (TNFAIP3) in high glucose-treated VSMCs. The silencing of TNFAIP3 was shown to abolish the protective effects of zinc on high glucose-induced NF-kB-dependent transcriptional activation, osteogenic marker expression, and the calcification of VSMCs. Silencing of the zinc-sensing receptor G protein-coupled receptor 39 (GPR39) was shown to abolish zinc-induced TNFAIP3 expression and the effects of zinc on high glucose-induced osteogenic marker expression. These observations indicate that zinc may be a protective factor during vascular calcification in hyperglycemic conditions.

Hdac9 inhibits medial artery calcification through down-regulation of Osterix

BackgroundsMedial artery calcification (MAC) significantly contributes to the increased cardiovascular death in patients with chronic kidney disease (CKD). Previous genome-wide association studies have shown that various genetic variants of the histone deacetylase Hdac9 are associated with cardiovascular disease, but the role of Hdac9 in MAC under CKD conditions remains unclear. MethodsHigh phosphate-induced vascular smooth muscle cell (VSMC) calcification and MAC in mice administered with vitamin D3 (vD) were used in the present study. Alizarin red staining, calcium quantitative assay, qPCR, western blotting and histology were performed. ResultsHdac9 expression was significantly down-regulated during high phosphate-induced vascular smooth muscle cell (VSMC) calcification and MAC in mice administered with vitamin D3 (vD). Furthermore, high phosphate treatment inhibited phosphorylation of Akt, and pharmacological inhibition of Akt signaling reduced Hdac9 expression in cultured VSMCs. Knockdown of Hdac9 significantly enhanced calcium deposition in VSMCs. Conversely, adenovirus mediated-overexpression of Hdac9 inhibited high phosphate induced VSMC in vitro calcification. Our subsequent mechanistic studies revealed that the anti-calcific effect of Hdac9 was mediated through down-regulation of osteoblast-specific transcription factor Osterix. ConclusionThese data suggest that Hdac9 is a novel inhibitor of MAC and may represent a potential therapeutic target for MAC in CKD patients.

Targeting androgen receptor in macrophages inhibits phosphate-induced vascular smooth muscle cell calcification by decreasing IL-6 expression

Vascular calcification (VC) is a common complication of chronic kidney disease (CKD). However, its mechanisms remain unclear. VC, similar to atherosclerosis, is an inflammatory disease. Vascular smooth muscle cells (VSMCs) play a key role in VC progression. The androgen receptor (AR) in monocytes/macrophages plays an important role in inflammatory diseases. Here, we define the role of macrophage (MФ) AR in inorganic phosphate-induced VSMC calcification. Our results show that the conditioning medium (CM) of silencing AR in macrophages inhibits inorganic phosphate-induced human aortic smooth muscle cell (HASMC) calcification, and alleviates the transdifferentiation of HASMCs into osteoblasts for the protein expression of osteoblasts marker Runt-related transcription factor-2 (Runx2) in HASMCs decreased while that of smooth muscle cell marker SM22α increased. The effect of AR on HASMC calcification might mainly be mediated by the inflammatory cytokine IL-6. Silencing AR in monocytes/macrophages can dramatically decrease IL-6 expression. We also investigated how macrophage AR regulates IL-6. ChIP and luciferase assays indicate that AR directly binds to the ARE sequence in the promoter of the IL-6 gene to accelerate transcription and expression. To our knowledge, this is the first investigation that has established the correlation between AR and VC and identified the contribution of AR in the calcification of VSMCs. In addition, this study describes a novel target for therapeutic intervention in VC.

Klotho/FGF23 axis mediates high phosphate-induced vascular calcification in vascular smooth muscle cells via Wnt7b/β-catenin pathway.

Vascular calcification (VC) plays as a critical role on cardiovascular disease (CVD) and acts as a notable risk factor in cardiovascular system. Vascular smooth muscle cells (VSMCs) calcification can be triggered by high phosphate treatment; however, the explicit mechanism remains unclear. In the present study, we isolated VSMCs from primary rat artery, applied β-GP (β-glycerophosphate) for inducing VSMCs calcification in vitro to explore the mechanism of phosphate-induced calcification in VSMCs. Alizarin red staining was performed to assess the mineralization in VSMCs. Calcium deposition experiment was taken to evaluate the calcium content. ALP staining was determined to assess the ALP activity. The recombinant adenoviruses were constructed for the overexpression of Klotho and FGF23, respectively. qRT-PCR and western blot analysis were subjected to measure the expression of Klotho/FGF23 and correlated genes among Wnt7b/β-catenin pathway. We found that the calcium content was obviously increased and Alizarin red staining was positive in calcification group exposure with high phosphate in a time-dependent manner. The expression of Klotho and FGF23 was significantly decreased in the calcification group. However, overexpression of Klotho and FGF23 markedly reversed VSMCs calcification stimulating with high phosphate treatment. Moreover, Wnt7b/β-catenin inhibitor DKK1 could partly attenuate the effect of high phosphate on calcified VSMCs. These findings demonstrated that Klotho/FGF23 axis could modulate high phosphate-induced VSMCs calcification via Wnt7b/β-catenin signaling pathway. Our findings unravel that Klotho/FGF23- Wnt7b/β-catenin axis functions as a crucial role in the VSMCs calcification.

Hyaluronan negatively regulates vascular calcification involving BMP2 signaling

Vascular calcification is a highly regulated biological process similar to bone formation involving osteogenic differentiation of vascular smooth muscle cells (VSMCs). Hyaluronan (HA), a major structural component of the extracellular matrix in cartilage, has been shown to inhibit osteoblast differentiation. However, whether HA affects osteogenic differentiation and calcification of VSMCs remains unclear. In the present study, we used in vitro and ex vivo models of vascular calcification to investigate the role of HA in vascular calcification. Both high and low molecular weight HA treatment significantly reduced calcification of rat VSMCs in a dose-dependent manner, as detected by alizarin red staining and calcium content assay. Ex vivo study further confirmed the inhibitory effect of HA on vascular calcification. Similarly, HA treatment decreased ALP activity and expression of bone-related molecules including Runx2, BMP2 and Msx2. By contrast, inhibition of HA synthesis by 4-methylumbelliferone (4MU) promoted calcification of rat VSMCs. In addition, adenovirus-mediated overexpression of HA synthase 2 (HAS2), a major HA synthase in VSMCs, also inhibited calcification of VSMCs, whereas CRISPR/Cas9-mediated HAS2 knockout promoted calcification of rat A10 cells. Furthermore, we found that BMP2 signaling was inhibited in VSMCs after HA treatment. Recombinant BMP2 enhanced high calcium and phosphate-induced VSMC calcification, which can be blocked by HA treatment. Taken together, these findings suggest that HA inhibits vascular calcification involving BMP2 signaling.Hyaluronan (HA) is known to inhibit osteoblast differentiation, but its importance in vascular calcification needed clarification. In this study, the authors identify HA as a novel negative regulator of osteogenic differentiation of vascular smooth muscle cells via bone morphogenetic protein 2 signaling. These results indicate that HA may be a novel potential therapeutic agent for the treatment of vascular calcification.

Ucma/GRP inhibits phosphate-induced vascular smooth muscle cell calcification via SMAD-dependent BMP signalling

Vascular calcification (VC) is the process of deposition of calcium phosphate crystals in the blood vessel wall, with a central role for vascular smooth muscle cells (VSMCs). VC is highly prevalent in chronic kidney disease (CKD) patients and thought, in part, to be induced by phosphate imbalance. The molecular mechanisms that regulate VC are not fully known. Here we propose a novel role for the mineralisation regulator Ucma/GRP (Upper zone of growth plate and Cartilage Matrix Associated protein/Gla Rich Protein) in phosphate-induced VSMC calcification. We show that Ucma/GRP is present in calcified atherosclerotic plaques and highly expressed in calcifying VSMCs in vitro. VSMCs from Ucma/GRP−/− mice showed increased mineralisation and expression of osteo/chondrogenic markers (BMP-2, Runx2, β-catenin, p-SMAD1/5/8, ALP, OCN), and decreased expression of mineralisation inhibitor MGP, suggesting that Ucma/GRP is an inhibitor of mineralisation. Using BMP signalling inhibitor noggin and SMAD1/5/8 signalling inhibitor dorsomorphin we showed that Ucma/GRP is involved in inhibiting the BMP-2-SMAD1/5/8 osteo/chondrogenic signalling pathway in VSMCs treated with elevated phosphate concentrations. Additionally, we showed for the first time evidence of a direct interaction between Ucma/GRP and BMP-2. These results demonstrate an important role of Ucma/GRP in regulating osteo/chondrogenic differentiation and phosphate-induced mineralisation of VSMCs.

Sphingosine 1-phosphate activation of ERM contributes to vascular calcification

Vascular calcification is the deposition of mineral in the artery wall by vascular smooth muscle cells (VSMCs) in response to pathological stimuli. The process is similar to bone formation and is an independent risk factor for cardiovascular disease. Given that ceramide and sphingosine 1-phosphate (S1P) are involved in cardiovascular pathophysiology and biomineralization, their role in VSMC matrix mineralization was investigated. During phosphate-induced VSMC mineralization, endogenous S1P levels increased accompanied by increased sphingosine kinase (SK) activity and increased mRNA expression of SK1 and SK2. Consistent with this, mineralization was increased by exogenous S1P, but decreased by C2-ceramide. Mechanistically, exogenous S1P stimulated ezrin-radixin-moesin (ERM) phosphorylation in VSMCs and ERM phosphorylation was increased concomitantly with endogenous S1P during mineralization. Moreover, inhibition of acid sphingomyelinase and ceramidase with desipramine prevented increased S1P levels, ERM activation, and mineralization. Finally, pharmacological inhibition of ERM phosphorylation with NSC663894 decreased mineralization induced by phosphate and exogenous S1P. Although further studies will be needed to verify these findings in vivo, this study defines a novel role for the SK-S1P-ERM pathways in phosphate-induced VSMC matrix mineralization and shows that blocking these pathways with pharmacological inhibitors reduces mineralization. These results may inform new therapeutic approaches to inhibit or delay vascular calcification.

Fibulin-3 Attenuates Phosphate-Induced Vascular Smooth Muscle Cell Calcification by Inhibition of Oxidative Stress

Background/Aims: Fibulin-3, an extracellular matrix glycoprotein, inhibits vascular oxidative stress and remodeling in hypertension. Oxidative stress is prevalent in chronic kidney disease (CKD) patients and is an important mediator of osteo-/chondrogenic transdifferentiation and calcification of vascular smooth muscle cells (VSMCs) during hyperphosphatemia. Therefore, the present study explored the effects of Fibulin-3 on phosphate-induced vascular calcification. Methods: Experiments were performed in primary human aortic smooth muscle cells (HAoSMCs) treated with control or with phosphate without or with additional treatment with recombinant human Fibulin-3 protein or with hydrogen peroxide as an exogenous source of oxidative stress. Results: Treatment with calcification medium significantly increased calcium deposition in HAoSMCs, an effect significantly blunted by additional treatment with Fibulin-3. Moreover, phosphate-induced alkaline phosphatase activity and mRNA expression of osteogenic and chondrogenic markers MSX2, CBFA1, SOX9 and ALPL were all significantly reduced by addition of Fibulin-3. These effects were paralleled by similar regulation of oxidative stress in HAoSMCs. Phosphate treatment significantly up-regulated mRNA expression of the oxidative stress markers NOX4 and CYBA, down-regulated total antioxidant capacity and increased the expression of downstream effectors of oxidative stress PAI-1, MMP2 and MMP9 as well as BAX/BLC2 ratio in HAoSMCs, all effects blocked by additional treatment with Fibulin-3. Furthermore, the protective effects of Fibulin-3 on phosphate-induced osteogenic and chondrogenic markers expression in HAoSMCs were reversed by additional treatment with hydrogen peroxide. Conclusions: Fibulin-3 attenuates phosphate-induced osteo-/ chondrogenic transdifferentiation and calcification of VSMCs, effects involving inhibition of oxidative stress. Up-regulation or supplementation of Fibulin-3 may be beneficial in reducing the progression of vascular calcification during hyperphosphatemic conditions such as CKD.

Role of Cytosolic Serine Hydroxymethyl Transferase 1 (SHMT1) in Phosphate-Induced Vascular Smooth Muscle Cell Calcification

Background/Aims: Hyperphosphatemia promotes medial vascular calcification, at least partly, by induction of osteo-/chondrogenic transdifferentiation of vascular smooth muscle cells (VSMCs). The complex signaling pathways regulating this process are still incompletely understood. The present study investigated the role of cytosolic serine hydroxymethyl transferase 1 (SHMT1) in phosphate-induced vascular calcification. Methods: Endogenous expression of SHMT1 was suppressed by silencing in primary human aortic smooth muscle cells (HAoSMCs) followed by treatment without and with phosphate or antioxidants. Results: In HAoSMCs, SHMT1 mRNA expression was up-regulated by phosphate. Silencing of SHMT1 alone was sufficient to induce osteo-/chondrogenic transdifferentiation of HAoSMCs, as shown by increased tissue-nonspecific alkaline phosphatase (ALPL) activity and osteogenic markers MSX2, CBFA1 and ALPL mRNA expression. Furthermore, phosphate-induced ALPL mRNA expression and activity as well as calcification were augmented in SHMT1 silenced HAoSMCs as compared to negative control siRNA transfected HAoSMCs. Silencing of SHMT1 decreased total antioxidant capacity and up-regulated NADH/NADPH oxidase system components NOX4 and CYBA mRNA expression in HAoSMCs, effects paralleled by increased mRNA expression of matrix metalloproteinase MMP2 as well as BAX/BCL2 ratio. More importantly, additional treatment with antioxidants TEMPOL or TIRON blunted the increased osteogenic markers mRNA expression in SHMT1 silenced HAoSMCs. Conclusion: Silencing of SHMT1 promotes osteo-/chondrogenic signaling in VSMCs, at least in part, by inducing cellular oxidative stress. It thus aggravates phosphate-induced calcification of VSMCs. The present findings support a regulatory role of SHMT1 in vascular calcification during conditions of hyperphosphatemia such as chronic kidney disease.

Hypoxia-inducible factor-1 plays a role in phosphate-induced vascular smooth muscle cell calcification

Medial vascular calcification is a common complication of chronic kidney disease (CKD). Although elevated inorganic phosphate stimulates vascular smooth muscle cell (VSMC) osteogenic transdifferentiation and calcification, the mechanisms involved in their calcification during CKD are not fully defined. Because hypoxic gene activation is linked to CKD and stimulates bone cell osteogenic differentiation, we used invivo and invitro rodent models to define the role of hypoxic signaling during elevated inorganic phosphate-induced VSMC calcification. Cell mineralization studies showed that elevated inorganic phosphate rapidly induced VSMC calcification. Hypoxia strongly enhanced elevated inorganic phosphate-induced VSMC calcification and osteogenic transdifferentiation, as seen by osteogenic marker expression. Hypoxia-inducible factor-1 (HIF-1), thekey hypoxic transcription factor, was essential for enhanced VSMC calcification. Targeting HIF-1 expression inmurine VSMC blocked calcification in hypoxia with elevated inorganic phosphate while HIF-1 activators, including clinically used FG-4592/Roxadustat, recreated a procalcifying environment. Elevated inorganic phosphate rapidly activated HIF-1, even in normal oxygenation; an effect mediated by HIF-1α subunit stabilization. Thus, hypoxia synergizes with elevated inorganic phosphate to enhance VSMC osteogenic transdifferentiation. Our work identifies HIF-1 as an early CKD-related pathological event, prospective marker, and potential target against vascular calcification in CKD-relevant conditions.

Mammalian target of rapamycin signaling inhibition ameliorates vascular calcification via Klotho upregulation

Vascular calcification (VC) is a major risk factor for cardiovascular mortality in chronic renal failure (CRF) patients, but the pathogenesis remains partially unknown and effective therapeutic targets should be urgently explored. Here we pursued the therapeutic role of rapamycin in CRF-related VC. Mammalian target of rapamycin (mTOR) signal was activated in the aortic wall of CRF rats. As expected, oral rapamycin administration significantly reduced VC by inhibiting mTOR in rats with CRF. Further in vitro results showed that activation of mTOR by both pharmacological agent and genetic method promoted, while inhibition of mTOR reduced, inorganic phosphate-induced vascular smooth muscle cell (VSMC) calcification and chondrogenic/osteogenic gene expression, which were independent of autophagy and apoptosis. Interestingly, the expression of Klotho, an antiaging gene that suppresses VC, was reduced in calcified vasculature, whereas rapamycin reversed membrane and secreted Klotho decline through mTOR inhibition. When mTOR signaling was enhanced by either mTOR overexpression or deletion of tuberous sclerosis 1, Klotho mRNA was further decreased in phosphate-treated VSMCs, suggesting a vital association between mTOR signaling and Klotho expression. More importantly, rapamycin failed to reduce VC in the absence of Klotho by using either siRNA knockdown of Klotho or Klotho knockout mice. Thus, Klotho has a critical role in mediating the observed decrease in calcification by rapamycin in vitro and in vivo.

Inhibition of Phosphate-Induced Vascular Smooth Muscle Cell Osteo-/Chondrogenic Signaling and Calcification by Bafilomycin A1 and Methylamine

Background/Aims: Excessive phosphate concentrations trigger vascular calcification, an active process promoted by osteoinduction of vascular smooth muscle cells (VSMCs) with increased expression and activity of transcription factor RUNX2 (Core-binding factor α1, CBFA1), alkaline phosphatase (ALPL), TGFß1, transcription factor NFAT5, and NFAT5-sensitive transcription factor SOX9. The osteoinductive signaling and vascular calcification of hyperphosphatemic klotho-hypomorphic mice could be reversed by treatment with NH₄Cl, effects involving decrease of TGFß1 and inhibition of NFAT5-dependent osteoinductive signaling. Known effects of NH₄Cl include alkalinization of acidic cellular compartments. The present study explored whether osteo-/chondrogenic signaling could be influenced by alkalinization of acidic cellular compartments following inhibition of the vacuolar H⁺ ATPase with bafilomycin A1 or following dissipation of the pH gradient across the membranes of acidic cellular compartments with methylamine. Methods: Primary human aortic smooth muscle cells (HAoSMCs) were treated with high phosphate to trigger osteo-/chondrogenic signaling and calcification in the absence or presence of bafilomycin A1 or methylamine. Calcium content was determined using a QuantiChrom Calcium assay, ALP activity by a colorimetric assay and transcript levels by quantitative RT-PCR. Results: High phosphate increased significantly the calcium deposition, CBFA1 and ALPL mRNA expression as well as alkaline phosphatase activity in HAoSMCs, all effects ameliorated by both, bafilomycin A1 and methylamine. High phosphate further significantly up-regulated the mRNA levels of TGFB1, NFAT5 and SOX9, effects significantly blunted by additional treatment with bafilomycin A1 or methylamine. Treatment of HAoSMCs with human TGFß1 protein or high phosphate up-regulated NFAT5, SOX9, CBFA1 and ALPL mRNA expression to similarly high levels which could not be further increased by combined treatment with high phosphate and TGFß1. Bafilomycin A1 failed to reverse the osteo-/chondrogenic signaling triggered by high phosphate together with TGFß1. Conclusions: Inhibition of the vacuolar H⁺ ATPase or dissipation of the pH gradient across the membranes of acidic cellular compartments both disrupt osteo-/chondrogenic signaling and calcium deposition in VSMCs, observations supporting the hypothesis that vascular calcification requires acidic cellular compartments.

Smad3 plays an inhibitory role in phosphate-induced vascular smooth muscle cell calcification

Arterial medial calcification is a major complication in patients with chronic kidney disease and diabetes. It has been hypothesized that a high concentration of inorganic phosphate (Pi) induces calcification in vascular smooth muscle cells (vSMCs). However, the role of transforming growth factor-β (TGF-β)/Smad3 signaling in Pi-induced vascular calcification remains controversial. The aim of this study was to investigate the possible involvement of Smad3 in Pi-induced vascular calcification. We compared the degree of Pi-induced vSMC calcification between vSMCs isolated from wild-type (Smad3+/+) and Smad3-deficient (Smad3−/−) mice. We found that vSMCs from Smad3+/+ mice had less calcium (Ca) than those from Smad3−/− mice when they were exposed to high concentrations of Pi and Ca (Pi+Ca). The phosphorylation of Smad3 was induced in Smad3+/+ vSMCs by exposure to Pi+Ca. The concentration of extracellular pyrophosphate (ePPi) was lower in Smad3−/− vSMCs than in Smad3+/+ vSMCs and was significantly increased in Smad3+/+ vSMCs by treatment with TGF-β1. Also, the addition of a small amount of PPi to culture medium significantly decreased the deposition of Ca in both Smad3+/+ and Smad3−/− vSMCs. Ectonucleotide phosphatase/phosphodiesterase1 (Enpp1) was decreased at the mRNA, protein, and enzymatic activity levels in Smad3−/− vSMCs compared with Smad3+/+ vSMCs. A ChIP assay showed that phosphorylated Smad3 directly binds to the Enpp1 gene. Furthermore, the calcification of aortic segments was attenuated by treatment with TGF-β1 only in Smad3+/+ mice. Taken together, we conclude that Pi-induced vSMC calcification is suppressed by Smad3 via an increase in ePPi.

Overexpression of C1q/Tumor Necrosis Factor–Related Protein-3 Promotes Phosphate-Induced Vascular Smooth Muscle Cell Calcification Both In Vivo and In Vitro

Vascular calcification is highly correlated with increased cardiovascular morbidity and mortality. C1q/tumor necrosis factor-related protein-3 (CTRP3) is a newly identified adipokine that plays important roles in cardiovascular system. Here, we investigated the role of CTRP3 in vascular calcification and its underlying mechanism. Adenine-induced chronic renal failure rat model was used to mimic the process of arterial medial calcification. The level of CTRP3 was elevated in serum and abdominal aorta of chronic renal failure rats. Periadventitial gene delivery of CTRP3 significantly accelerated the calcification of abdominal aorta and arterial ring. In cultured vascular smooth muscle cells (VSMCs), CTRP3 increased β-glycerophosphate-induced calcium deposition and alkaline phosphatase activity. Although CTRP3 alone was not sufficient to induce calcification in VSMCs, it upregulated the expression of osteogenic marker genes including runt-related transcription factor 2 (Runx2), bone morphogenetic protein 2, and osteopontin. CTRP3 further enhanced β-glycerophosphate-induced downregulation of smooth muscle α-actin and smooth muscle 22α, while augmenting osteogenic marker expression in VSMCs induced by β-glycerophosphate. In contrast, knockdown of CTRP3 in VSMCs potently suppressed β-glycerophosphate-induced calcification. Mechanistically, knockdown of Runx2 inhibited CTRP3-promoted VSMC calcification. CTRP3 increased extracellular signal-regulated kinase 1/2 phosphorylation and reactive oxygen species production. Preincubation with U0126, an extracellular signal-regulated kinase 1/2 upstream kinase inhibitor, had no effect on CTRP3-induced reactive oxygen species production. However, pretreatment with N-acetyl-l-cysteine, a reactive oxygen species scavenger, suppressed CTRP3-induced extracellular signal-regulated kinase 1/2 phosphorylation. Both N-acetyl-l-cysteine and U0126 significantly inhibited CTRP3-induced upregulation of Runx2 and calcified nodule formation. CTRP3 promotes vascular calcification by enhancing phosphate-induced osteogenic transition of VSMC through reactive oxygen species-extracellular signal-regulated kinase 1/2-Runx2 pathway.