TGF-ß phospho antibody array identifies altered SMAD2, PI3K/AKT/SMAD, and RAC signaling contribute to the pathogenesis of myxomatous mitral valve disease.

Background: TGFß signaling appears to contribute to the pathogenesis of myxomatous mitral valve disease (MMVD) in both dogs and humans. However, little is known about the extent of the downstream signaling changes that will then affect cell phenotype and function in both species. Objective: Identify changes in downstream signals in the TGFß pathway in canine MMVD and examine the effects of antagonism of one significant signal (SMAD2 was selected). Materials and methods: Canine cultures of normal quiescent valve interstitial cells (qVICs) and disease-derived activated myofibroblasts (aVICs) (n = 6) were examined for TGFß signaling protein expression using a commercial antibody array. Significant changes were confirmed, and additional proteins of interest downstream in the TGFß signaling pathway and markers of cell phenotype were examined (PRAS40, S6K, elF4E IRS-1, αSMA, and VIM), using protein immunoblotting. RT-PCR examined expression of gene markers of VIC activation (ACTA2, TAGLN, and MYH10; encoding the proteins αSMA, SM22, and Smemb, respectively). Attenuation of pSMAD2 in aVICs was examined using a combination of RNA interference technology (siRNA) and the SMAD7 (antagonizes SMAD2) agonist asiaticoside. Results: The antibody array identified significant changes (P < 0.05) in 19 proteins, of which six were phosphorylated (p). There was increased expression of pSMAD2 and pRAC1 and decreased expression of pmTOR, pERK1/2, and pAKT1. Expression of pPRAS40 and pIRS-1 was increased, as was the mTOR downstream transcription factor pS6K, with increased expression of peIF4E in aVICs, indicating negative feedback control of the PI3K/AKT/mTOR pathway. SMAD2 antagonism by siRNA and the SMAD7 agonist asiaticoside decreased detection of pSMAD by at least 50%, significantly decreased expression of the aVIC gene markers ACTA2, TAGLN, and MYH10, and pαSMA, pAKT2, and pERK1, but had no effect on pS6K, pERK2, or pVIM expression in aVICs. SMAD2 antagonism transitioned diseased aVICs to normal qVICs, while maintaining a mesenchymal phenotype (VIM+) while concurrently affecting non-canonical TGFß signaling. Conclusion: MMVD is associated with changes in both the canonical and non-canonical TGFß signaling pathway. Antagonism of SMAD2 transitions diseased-activated myofibroblasts back to a normal phenotype, providing data that will inform studies on developing novel therapeutics to treat MMVD in dogs and humans.

PFKFB3-driven vascular smooth muscle cell glycolysis promotes vascular calcification via the altered FoxO3 and lactate production.

A link between increased glycolysis and vascular calcification has recently been reported, but it remains unclear how increased glycolysis contributes to vascular calcification. We therefore investigated the role of PFKFB3, a critical enzyme of glycolysis, in vascular calcification. We found that PFKFB3 expression was upregulated in calcified mouse VSMCs and arteries. We showed that expression of miR-26a-5p and miR-26b-5p in calcified mouse arteries was significantly decreased, and a negative correlation between Pfkfb3 mRNA expression and miR-26a-5p or miR-26b-5p was seen in these samples. Overexpression of miR-26a/b-5p significantly inhibited PFKFB3 expression in VSMCs. Intriguingly, pharmacological inhibition of PFKFB3 using PFK15 or knockdown of PFKFB3 ameliorated vascular calcification in vD3 -overloaded mice in vivo or attenuated high phosphate (Pi)-induced VSMC calcification in vitro. Consistently, knockdown of PFKFB3 significantly reduced glycolysis and osteogenic transdifferentiation of VSMCs, whereas overexpression of PFKFB3 in VSMCs induced the opposite effects. RNA-seq analysis and subsequent experiments revealed that silencing of PFKFB3 inhibited FoxO3 expression in VSMCs. Silencing of FoxO3 phenocopied the effects of PFKFB3 depletion on Ocn and Opg expression but not Alpl in VSMCs. Pyruvate or lactate supplementation, the product of glycolysis, reversed the PFKFB3 depletion-mediated effects on ALP activity and OPG protein expression in VSMCs. Our results reveal that blockade of PFKFB3-mediated glycolysis inhibits vascular calcification in vitro and in vivo. Mechanistically, we show that FoxO3 and lactate production are involved in PFKFB3-driven osteogenic transdifferentiation of VSMCs. PFKFB3 may be a promising therapeutic target for the treatment of vascular calcification.

TGF-ß-induced PI3K/AKT/mTOR pathway controls myofibroblast differentiation and secretory phenotype of valvular interstitial cells through the modulation of cellular senescence in a naturally occurring in vitro canine model of myxomatous mitral valve disease.

PI3K/AKT/mTOR signalling contributes to several cardiovascular disorders. The aim of this study was to examine the PI3K/AKT/mTOR pathway in myxomatous mitral valve disease (MMVD). Double-immunofluorescence examined expression of PI3K and TGF-ß1 in canine valves. Valve interstitial cells (VICs) from healthy or MMVD dogs were isolated and characterized. Healthy quiescent VICs (qVICs) were treated with TGF-ß1 and SC-79 to induce activated myofibroblast phenotypes (aVICs). Diseased valve-derived aVICs were treated with PI3K antagonists and expression of RPS6KB1 (encoding p70 S6K) was modulated using siRNA and gene overexpression. SA-ß-gal and TUNEL staining were used to identify cell senescence and apoptosis, and qPCR and ELISA to examine for senescence-associated secretory phenotype. Protein immunoblotting was used to examine expression of phosphorylated and total proteins. TGF-ß1 and PI3K are highly expressed in mitral valve tissues. Activation of PI3K/AKT/mTOR and increased expression of TGF-ß are found in aVICs. TGF-ß transitions qVICs to aVICs by upregulation of PI3K/AKT/mTOR. Antagonism of PI3K/AKT/mTOR reverses aVIC myofibroblast transition by inhibiting senescence and promoting autophagy. Upregulation of mTOR/S6K induces transformation of senescent aVICs, with reduced capacity for apoptosis and autophagy. Selective knockdown of p70 S6K reverses cell transition by attenuating cell senescence, inhibiting apoptosis and improving autophagy. TGF-ß-induced PI3K/AKT/mTOR signalling contributes to MMVD pathogenesis and plays crucial roles in the regulation of myofibroblast differentiation, apoptosis, autophagy and senescence in MMVD.

p53 Regulates Mitochondrial Dynamics in Vascular Smooth Muscle Cell Calcification.

Arterial calcification is an important characteristic of cardiovascular disease. It has key parallels with skeletal mineralization; however, the underlying cellular mechanisms responsible are not fully understood. Mitochondrial dynamics regulate both bone and vascular function. In this study, we therefore examined mitochondrial function in vascular smooth muscle cell (VSMC) calcification. Phosphate (Pi)-induced VSMC calcification was associated with elongated mitochondria (1.6-fold increase, p < 0.001), increased mitochondrial reactive oxygen species (ROS) production (1.83-fold increase, p < 0.001) and reduced mitophagy (9.6-fold decrease, p < 0.01). An increase in protein expression of optic atrophy protein 1 (OPA1; 2.1-fold increase, p < 0.05) and a converse decrease in expression of dynamin-related protein 1 (DRP1; 1.5-fold decrease, p < 0.05), two crucial proteins required for the mitochondrial fusion and fission process, respectively, were noted. Furthermore, the phosphorylation of DRP1 Ser637 was increased in the cytoplasm of calcified VSMCs (5.50-fold increase), suppressing mitochondrial translocation of DRP1. Additionally, calcified VSMCs showed enhanced expression of p53 (2.5-fold increase, p < 0.05) and ß-galactosidase activity (1.8-fold increase, p < 0.001), the cellular senescence markers. siRNA-mediated p53 knockdown reduced calcium deposition (8.1-fold decrease, p < 0.01), mitochondrial length (3.0-fold decrease, p < 0.001) and ß-galactosidase activity (2.6-fold decrease, p < 0.001), with concomitant mitophagy induction (3.1-fold increase, p < 0.05). Reduced OPA1 (4.1-fold decrease, p < 0.05) and increased DRP1 protein expression (2.6-fold increase, p < 0.05) with decreased phosphorylation of DRP1 Ser637 (3.20-fold decrease, p < 0.001) was also observed upon p53 knockdown in calcifying VSMCs. In summary, we demonstrate that VSMC calcification promotes notable mitochondrial elongation and cellular senescence via DRP1 phosphorylation. Furthermore, our work indicates that p53-induced mitochondrial fusion underpins cellular senescence by reducing mitochondrial function.

Increased PHOSPHO1 and alkaline phosphatase expression during the anabolic bone response to intermittent parathyroid hormone delivery.

The administration of intermittent parathyroid hormone (iPTH) is anabolic to the skeleton. Recent studies with cultured osteoblasts have revealed that the expression of PHOSPHO1, a bone-specific phosphatase essential for the initiation of mineralisation, is regulated by PTH. Therefore, this study sought to determine whether the bone anabolic response to iPTH involves modulation of expression of Phospho1 and of other enzymes critical for bone matrix mineralisation. To mimic iPTH treatment, primary murine osteoblasts were challenged with 50 nM PTH for 6 h in every 48 h period for 8 days (4 cycles), 14 days (7 cycles) and 20 days (10 cycles) in total. The expression of both Phospho1 and Smpd3 was almost completely inhibited after 4 cycles, whereas 10 cycles were required to stimulate a similar response in Alpl expression. To explore the in vivo role of PHOSPHO1 in PTH-mediated osteogenesis, the effects of 14- and 28-day iPTH (80 µg/kg/day) administration was assessed in male wild-type (WT) and Phospho1-/- mice. The expression of Phospho1, Alpl, Smpd3, Enpp1, Runx2 and Trps1 expression was enhanced in the femora of WT mice following iPTH administration but remained unchanged in the femora of Phospho1-/- mice. After 28 days of iPTH administration, the anabolic response in the femora of WT was greater than that noted in Phospho1-/- mice. Specifically, cortical and trabecular bone volume/total volume, as well as cortical thickness, were increased in femora of iPTH-treated WT but not in iPTH-treated Phospho1-/- mice. Trabecular bone osteoblast number was also increased in iPTH-treated WT mice but not in iPTH-treated Phospho1-/-  mice. The increased levels of Phospho1, Alpl, Enpp1 and Smpd3 in WT mice in response to iPTH administration is consistent with their contribution to the potent anabolic properties of iPTH in bone. Furthermore, as the anabolic response to iPTH was attenuated in mice deficient in PHOSPHO1, this suggests that the osteoanabolic effects of iPTH are at least partly mediated via bone mineralisation processes.

Metformin protects against vascular calcification through the selective degradation of Runx2 by the p62 autophagy receptor.

Vascular calcification is associated with aging, type 2 diabetes, and atherosclerosis, and increases the risk of cardiovascular morbidity and mortality. It is an active, highly regulated process that resembles physiological bone formation. It has previously been established that pharmacological doses of metformin alleviate arterial calcification through adenosine monophosphate-activated protein kinase (AMPK)-activated autophagy, however the specific pathway remains elusive. In the present study we hypothesized that metformin protects against arterial calcification through the direct autophagic degradation of runt-related transcription factor 2 (Runx2). Calcification was blunted in vascular smooth muscle cells (VSMCs) by metformin in a dose-dependent manner (0.5-1.5 mM) compared to control cells (p < 0.01). VSMCs cultured under high-phosphate (Pi) conditions in the presence of metformin (1 mM) showed a significant increase in LC3 puncta following bafilomycin-A1 (Baf-A; 5 nM) treatment compared to control cells (p < 0.001). Furthermore, reduced expression of Runx2 was observed in the nuclei of metformin-treated calcifying VSMCs (p < 0.0001). Evaluation of the functional role of autophagy through Atg3 knockdown in VSMCs showed aggravated Pi-induced calcification (p < 0.0001), failure to induce autophagy (punctate LC3) (p < 0.001) and increased nuclear Runx2 expression (p < 0.0001) in VSMCs cultured under high Pi conditions in the presence of metformin (1 mM). Mechanistic studies employing three-way coimmunoprecipitation with Runx2, p62, and LC3 revealed that p62 binds to both LC3 and Runx2 upon metformin treatment in VSMCs. Furthermore, immunoblotting with LC3 revealed that Runx2 specifically binds with p62 and LC3-II in metformin-treated calcified VSMCs. Lastly, we investigated the importance of the autophagy pathway in vascular calcification in a clinical setting. Ex vivo clinical analyses of calcified diabetic lower limb artery tissues highlighted a negative association between Runx2 and LC3 in the vascular calcification process. These studies suggest that exploitation of metformin and its analogues may represent a novel therapeutic strategy for clinical intervention through the induction of AMPK/Autophagy Related 3 (Atg3)-dependent autophagy and the subsequent p62-mediated autophagic degradation of Runx2.

Increased PHOSPHO1 expression mediates cortical bone mineral density in renal osteodystrophy.

Patients with advanced chronic kidney disease (CKD) often present with skeletal abnormalities, a condition known as renal osteodystrophy (ROD). While tissue non-specific alkaline phosphatase (TNAP) and PHOSPHO1 are critical for bone mineralization, their role in the etiology of ROD is unclear. To address this, ROD was induced in both WT and Phospho1 knockout (P1KO) mice through dietary adenine supplementation. The mice presented with hyperphosphatemia, hyperparathyroidism, and elevated levels of FGF23 and bone turnover markers. In particular, we noted that in CKD mice, bone mineral density (BMD) was increased in cortical bone (P < 0.05) but decreased in trabecular bone (P < 0.05). These changes were accompanied by decreased TNAP (P < 0.01) and increased PHOSPHO1 (P < 0.001) expression in WT CKD bones. In P1KO CKD mice, the cortical BMD phenotype was rescued, suggesting that the increased cortical BMD of CKD mice was driven by increased PHOSPHO1 expression. Other structural parameters were also improved in P1KO CKD mice. We further investigated the driver of the mineralization defects, by studying the effects of FGF23, PTH, and phosphate administration on PHOSPHO1 and TNAP expression by primary murine osteoblasts. We found both PHOSPHO1 and TNAP expressions to be downregulated in response to phosphate and PTH. The in vitro data suggest that the TNAP reduction in CKD-MBD is driven by the hyperphosphatemia and/or hyperparathyroidism noted in these mice, while the higher PHOSPHO1 expression may be a compensatory mechanism. Increased PHOSPHO1 expression in ROD may contribute to the disordered skeletal mineralization characteristic of this progressive disorder.

The Role of Transforming Growth Factor-ß Signaling in Myxomatous Mitral Valve Degeneration.

Mitral valve prolapse (MVP) due to myxomatous degeneration is one of the most important chronic degenerative cardiovascular diseases in people and dogs. It is a common cause of heart failure leading to significant morbidity and mortality in both species. Human MVP is usually classified into primary or non-syndromic, including Barlow's Disease (BD), fibro-elastic deficiency (FED) and Filamin-A mutation, and secondary or syndromic forms (typically familial), such as Marfan syndrome (MFS), Ehlers-Danlos syndrome, and Loeys-Dietz syndrome. Despite different etiologies the diseased valves share pathological features consistent with myxomatous degeneration. To reflect this common pathology the condition is often called myxomatous mitral valve degeneration (disease) (MMVD) and this term is universally used to describe the analogous condition in the dog. MMVD in both species is characterized by leaflet thickening and deformity, disorganized extracellular matrix, increased transformation of the quiescent valve interstitial cell (qVICs) to an activated state (aVICs), also known as activated myofibroblasts. Significant alterations in these cellular activities contribute to the initiation and progression of MMVD due to the increased expression of transforming growth factor-ß (TGF-ß) superfamily cytokines and the dysregulation of the TGF-ß signaling pathways. Further understanding the molecular mechanisms of MMVD is needed to identify pharmacological manipulation strategies of the signaling pathway that might regulate VIC differentiation and so control the disease onset and development. This review briefly summarizes current understanding of the histopathology, cellular activities, molecular mechanisms and pathogenesis of MMVD in dogs and humans, and in more detail reviews the evidence for the role of TGF-ß.

PALMD regulates aortic valve calcification via altered glycolysis and NF-κB-mediated inflammation.

Recent genome-wide association and transcriptome-wide association studies have identified an association between the PALMD locus, encoding palmdelphin, a protein involved in myoblast differentiation, and calcific aortic valve disease (CAVD). Nevertheless, the function and underlying mechanisms of PALMD in CAVD remain unclear. We herein investigated whether and how PALMD affects the pathogenesis of CAVD using clinical samples from CAVD patients and a human valve interstitial cell (hVIC) in vitro calcification model. We showed that PALMD was upregulated in calcified regions of human aortic valves and calcified hVICs. Furthermore, silencing of PALMD reduced hVIC in vitro calcification, osteogenic differentiation, and apoptosis, whereas overexpression of PALMD had the opposite effect. RNA-Seq of PALMD-depleted hVICs revealed that silencing of PALMD reduced glycolysis and nuclear factor-κB (NF-κB)-mediated inflammation in hVICs and attenuated tumor necrosis factor α-induced monocyte adhesion to hVICs. Having established the role of PALMD in hVIC glycolysis, we examined whether glycolysis itself could regulate hVIC osteogenic differentiation and inflammation. Intriguingly, the inhibition of PFKFB3-mediated glycolysis significantly attenuated osteogenic differentiation and inflammation of hVICs. However, silencing of PFKFB3 inhibited PALMD-induced hVIC inflammation, but not osteogenic differentiation. Finally, we showed that the overexpression of PALMD enhanced hVIC osteogenic differentiation and inflammation, as opposed to glycolysis, through the activation of NF-κB. The present study demonstrates that the genome-wide association- and transcriptome-wide association-identified CAVD risk gene PALMD may promote CAVD development through regulation of glycolysis and NF-κB-mediated inflammation. We propose that targeting PALMD-mediated glycolysis may represent a novel therapeutic strategy for treating CAVD.

Cardiomyocytes disrupt pyrimidine biosynthesis in nonmyocytes to regulate heart repair.

Various populations of cells are recruited to the heart after cardiac injury, but little is known about whether cardiomyocytes directly regulate heart repair. Using a murine model of ischemic cardiac injury, we demonstrate that cardiomyocytes play a pivotal role in heart repair by regulating nucleotide metabolism and fates of nonmyocytes. Cardiac injury induced the expression of the ectonucleotidase ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), which hydrolyzes extracellular ATP to form AMP. In response to AMP, cardiomyocytes released adenine and specific ribonucleosides that disrupted pyrimidine biosynthesis at the orotidine monophosphate (OMP) synthesis step and induced genotoxic stress and p53-mediated cell death of cycling nonmyocytes. As nonmyocytes are critical for heart repair, we showed that rescue of pyrimidine biosynthesis by administration of uridine or by genetic targeting of the ENPP1/AMP pathway enhanced repair after cardiac injury. We identified ENPP1 inhibitors using small molecule screening and showed that systemic administration of an ENPP1 inhibitor after heart injury rescued pyrimidine biosynthesis in nonmyocyte cells and augmented cardiac repair and postinfarct heart function. These observations demonstrate that the cardiac muscle cell regulates pyrimidine metabolism in nonmuscle cells by releasing adenine and specific nucleosides after heart injury and provide insight into how intercellular regulation of pyrimidine biosynthesis can be targeted and monitored for augmenting tissue repair.

Mitochondrial Dysfunction: Cause or Consequence of Vascular Calcification?

Mitochondria are crucial bioenergetics powerhouses and biosynthetic hubs within cells, which can generate and sequester toxic reactive oxygen species (ROS) in response to oxidative stress. Oxidative stress-stimulated ROS production results in ATP depletion and the opening of mitochondrial permeability transition pores, leading to mitochondria dysfunction and cellular apoptosis. Mitochondrial loss of function is also a key driver in the acquisition of a senescence-associated secretory phenotype that drives senescent cells into a pro-inflammatory state. Maintaining mitochondrial homeostasis is crucial for retaining the contractile phenotype of the vascular smooth muscle cells (VSMCs), the most prominent cells of the vasculature. Loss of this contractile phenotype is associated with the loss of mitochondrial function and a metabolic shift to glycolysis. Emerging evidence suggests that mitochondrial dysfunction may play a direct role in vascular calcification and the underlying pathologies including (1) impairment of mitochondrial function by mineral dysregulation i.e., calcium and phosphate overload in patients with end-stage renal disease and (2) presence of increased ROS in patients with calcific aortic valve disease, atherosclerosis, type-II diabetes and chronic kidney disease. In this review, we discuss the cause and consequence of mitochondrial dysfunction in vascular calcification and underlying pathologies; the role of autophagy and mitophagy pathways in preventing mitochondrial dysfunction during vascular calcification and finally we discuss mitochondrial ROS, DRP1, and HIF-1 as potential novel markers and therapeutic targets for maintaining mitochondrial homeostasis in vascular calcification.

Zinc ameliorates human aortic valve calcification through GPR39 mediated ERK1/2 signalling pathway.

AIMS: Calcific aortic valve disease (CAVD) is the most common heart valve disease in the Western world. It has been reported that zinc is accumulated in calcified human aortic valves. However, whether zinc directly regulates CAVD is yet to be elucidated. The present study sought to determine the potential role of zinc in the pathogenesis of CAVD. METHODS AND RESULTS: Using a combination of a human valve interstitial cell (hVIC) calcification model, human aortic valve tissues, and blood samples, we report that 20 µM zinc supplementation attenuates hVIC in vitro calcification, and that this is mediated through inhibition of apoptosis and osteogenic differentiation via the zinc-sensing receptor GPR39-dependent ERK1/2 signalling pathway. Furthermore, we report that GPR39 protein expression is dramatically reduced in calcified human aortic valves, and there is a significant reduction in zinc serum levels in patients with CAVD. Moreover, we reveal that 20 µM zinc treatment prevents the reduction of GPR39 observed in calcified hVICs. We also show that the zinc transporter ZIP13 and ZIP14 are significantly increased in hVICs in response to zinc treatment. Knockdown of ZIP13 or ZIP14 significantly inhibited hVIC in vitro calcification and osteogenic differentiation. CONCLUSIONS: Together, these findings suggest that zinc is a novel inhibitor of CAVD, and report that zinc transporter ZIP13 and ZIP14 are important regulators of hVIC in vitro calcification and osteogenic differentiation. Zinc supplementation may offer a potential therapeutic strategy for CAVD.

Osteoblast-specific deficiency of ectonucleotide pyrophosphatase or phosphodiesterase-1 engenders insulin resistance in high-fat diet fed mice.

Supraphysiological levels of the osteoblast-enriched mineralization regulator ectonucleotide pyrophosphatase or phosphodiesterase-1 (NPP1) is associated with type 2 diabetes mellitus. We determined the impact of osteoblast-specific Enpp1 ablation on skeletal structure and metabolic phenotype in mice. Female, but not male, 6-week-old mice lacking osteoblast NPP1 expression (osteoblast-specific knockout [KO]) exhibited increased femoral bone volume or total volume (17.50% vs. 11.67%; p < .01), and reduced trabecular spacing (0.187 vs. 0.157 mm; p < .01) compared with floxed (control) mice. Furthermore, an enhanced ability of isolated osteoblasts from the osteoblast-specific KO to calcify their matrix in vitro compared to fl/fl osteoblasts was observed (p < .05). Male osteoblast-specific KO and fl/fl mice showed comparable glucose and insulin tolerance despite increased levels of insulin-sensitizing under-carboxylated osteocalcin (195% increase; p < .05). However, following high-fat-diet challenge, osteoblast-specific KO mice showed impaired glucose and insulin tolerance compared with fl/fl mice. These data highlight a crucial local role for osteoblast NPP1 in skeletal development and a secondary metabolic impact that predominantly maintains insulin sensitivity.

Ex vivo 18F-fluoride uptake and hydroxyapatite deposition in human coronary atherosclerosis.

Early microcalcification is a feature of coronary plaques with an increased propensity to rupture and to cause acute coronary syndromes. In this ex vivo imaging study of coronary artery specimens, the non-invasive imaging radiotracer, 18F-fluoride, was highly selective for hydroxyapatite deposition in atherosclerotic coronary plaque. Specifically, coronary 18F-fluoride uptake had a high signal to noise ratio compared with surrounding myocardium that makes it feasible to identify coronary mineralisation activity. Areas of 18F-fluoride uptake are associated with osteopontin, an inflammation-associated glycophosphoprotein that mediates tissue mineralisation, and Runt-related transcription factor 2, a nuclear protein involved in osteoblastic differentiation. These results suggest that 18F-fluoride is a non-invasive imaging biomarker of active coronary atherosclerotic mineralisation.

Expression of Calcification and Extracellular Matrix Genes in the Cardiovascular System of the Healthy Domestic Sheep (Ovis aries).

The maintenance of a healthy cardiovascular system requires expression of genes that contribute to essential biological activities and repression of those that are associated with functions likely to be detrimental to cardiovascular homeostasis. Vascular calcification is a major disruption to cardiovascular homeostasis, where tissues of the cardiovascular system undergo ectopic calcification and consequent dysfunction, but little is known about the expression of calcification genes in the healthy cardiovascular system. Large animal models are of increasing importance in cardiovascular disease research as they demonstrate more similar cardiovascular features (in terms of anatomy, physiology and size) to humans than do rodent species. We used RNA sequencing results from the sheep, which has been utilized extensively to examine calcification of prosthetic cardiac valves, to explore the transcriptome of the heart and cardiac valves in this large animal, in particular looking at expression of calcification and extracellular matrix genes. We then examined genes implicated in the process of vascular calcification in a wide array of cardiovascular tissues and across multiple developmental stages, using RT-qPCR. Our results demonstrate that there is a balance between genes that promote and those that suppress mineralization during development and across cardiovascular tissues. We show extensive expression of genes encoding proteins involved in formation and maintenance of the extracellular matrix in cardiovascular tissues, and high expression of hematopoietic genes in the cardiac valves. Our analysis will support future research into the functions of implicated genes in the development of valve calcification, and increase the utility of the sheep as a large animal model for understanding ectopic calcification in cardiovascular disease. This study provides a foundation to explore the transcriptome of the developing cardiovascular system and is a valuable resource for the fields of mammalian genomics and cardiovascular research.

PHOSPHO1 is a skeletal regulator of insulin resistance and obesity.

BACKGROUND: The classical functions of the skeleton encompass locomotion, protection and mineral homeostasis. However, cell-specific gene deletions in the mouse and human genetic studies have identified the skeleton as a key endocrine regulator of metabolism. The bone-specific phosphatase, Phosphatase, Orphan 1 (PHOSPHO1), which is indispensable for bone mineralisation, has been recently implicated in the regulation of energy metabolism in humans, but its role in systemic metabolism remains unclear. Here, we probe the mechanism underlying metabolic regulation by analysing Phospho1 mutant mice. RESULTS: Phospho1-/- mice exhibited improved basal glucose homeostasis and resisted high-fat-diet-induced weight gain and diabetes. The metabolic protection in Phospho1-/- mice was manifested in the absence of altered levels of osteocalcin. Osteoblasts isolated from Phospho1-/- mice were enriched for genes associated with energy metabolism and diabetes; Phospho1 both directly and indirectly interacted with genes associated with glucose transport and insulin receptor signalling. Canonical thermogenesis via brown adipose tissue did not underlie the metabolic protection observed in adult Phospho1-/- mice. However, the decreased serum choline levels in Phospho1-/- mice were normalised by feeding a 2% choline rich diet resulting in a normalisation in insulin sensitivity and fat mass. CONCLUSION: We show that mice lacking the bone mineralisation enzyme PHOSPHO1 exhibit improved basal glucose homeostasis and resist high-fat-diet-induced weight gain and diabetes. This study identifies PHOSPHO1 as a potential bone-derived therapeutic target for the treatment of obesity and diabetes.

Hdac9 inhibits medial artery calcification through down-regulation of Osterix.

BACKGROUNDS: Medial 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. METHODS: High 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. RESULTS: Hdac9 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. CONCLUSION: These data suggest that Hdac9 is a novel inhibitor of MAC and may represent a potential therapeutic target for MAC in CKD patients.

Disease Severity-Associated Gene Expression in Canine Myxomatous Mitral Valve Disease Is Dominated by TGFß Signaling.

Myxomatous mitral valve disease (MMVD) is the most common acquired canine cardiovascular disease and shares many similarities with human mitral valvulopathies. While transcriptomic datasets are available for the end-stage disease in both species, there is no information on how gene expression changes as the disease progresses, such that it cannot be stated with certainty if the changes seen in end-stage disease are casual or consequential. In contrast to humans, the disease in dogs can be more readily examined as it progresses, and this allows an opportunity for insight into disease pathogenesis relevant to both species. The aim of this study was to identify changes in valve gene expression as canine MMVD advances over an entire life-time, from normal (grade 0) to severely affected (grade 4), and differences in gene expression comparing normal and disease areas of the same valve. Transcriptomic profiling identified 1002 differentially expressed genes (DEGs) across all four disease grades when compared with normal valves with the greatest number of DEGs in grade 3 (673) and grade 4 (507). DEGs were associated with a large number of gene families, including genes encoding cytoskeletal filaments, peptidases, extra-cellular matrix (ECM) proteins, chemokines and integrins. Gene enrichment analysis identified significant grade-dependent changes in gene clustering, with clusters trending both up and down as disease progressed. Significant grade-dependent changes in hallmark disease gene expression intensity were identified, including ACTA2, HTR2B, MMP12, and CDKN2A. Gene Ontology terms were dominated by terms for ECM and inflammation with TGFß1, TNF, IFGN identified as the top up-stream regulators in both whole and dissected diseased valve samples. These data show that while disease progression in MMVD is associated with increasing numbers of DEGs, TGFß appears to be the dominant signaling pathway controlling pathogenesis irrespective of disease severity.