To evaluate whether a relatively low CaMg dose (185 mg/kg/ day) is still able to retard the development of aortic calcification, a group of 12 male Wistar rats (250 g, Charles River, Lille, France) was chosen, in which CRF and vascularcalcification was induced by feeding a 0.75% adenine/2.5% protein diet (SSNIFF Spezialdia¨ten, Soest, Germany) for 4 weeks. Prior to CRF induction and after adenine withdrawal, rats were fed a 1.03% phosphorus diet (SSNIFF Spezialdia¨ten) for 2 weeks. One week after CRF induction, treatment with either vehicle (n = 14) or 185 mg/kg/day CaMg (n = 12) was started by oral gavage in a constant dose volume of 10 ml/kg. After 6 weeks of renal failure animals were sacrificed. Blood and urine samples were taken before CRF induction (week 0), before start of treatment (week 1), after adenine withdrawal (week 4) and at sacrifice (week 6). Rats were sacrificed by exsanguination through the retro-orbital plexus after anaesthesia with intraperitoneal injection of 60 mg/kg pentobarbital.
that the initiation of calcification requires an increased uptake of Ca/P by VSMCs, which leads to a pattern of cellular adaptations and damage that ultimately promote calcification (75). Indeed, vascular calcification’s most devastating manifestation transpires in CKD due to dysregulated mineral metabolism, the main pathologic characteristic within this patients, that conducts to long-term elevation of serum Ca/P levels (76). Vascular smooth muscle cells exposed to elevated Ca/P minerals, present loss of their contractibility, and upregulation of the expression of bone-related protein (osteochondrogenic expression) such as runt-related transcription factor 2 (Runx2), osteopontin, osteocalcin, alkaline phosphatase (ALP) ending up secreting calcifying EVs into the vessels ECM that promote mineralization sites and consequent calcification (47, 60).
The process of vascularcalcification involves the initial loss of VSMC phenotype followed by the development of osteogenic cells and subsequent calcification . Although we observed that BMP inhibition did not prevent loss of VSMC phenotype in the aortas of MGP -/- mice, BMP signaling was required for the induction of osteogenic cells. We next investigated whether calcification of MGP-deficient VSMCs is dependent on BMP signaling in vitro. Calcification was induced in isolated VSMCs by growing the cells in DMEM supplemented with 10% FCS and 2 mM sodium phosphate . Expression of MGP in MGP -/- VSMCs using an adenovirus vector reduced calcification by 70%, as detected by von Kossa stain, compared with adenovirus control-treated cells (Fig. 9A–B, F). Treatment of wild-type VSMCs with siRNA directed against MGP (siMGP) increased calcification compared to treatment with control scrambled siRNA (siSC) (Fig. 9C–D). These results indicate that, similar to in vivo findings, MGP expres- sion modulates the calcification occurring in cultured VSMCs treated with high phosphate- containing media. Treatment with LDN-193189 inhibited the calcification induced by siMGP in wild-type VSMCs by approximately 50% (P = 0.0003; Fig. 9D–E, 9G). Taken together, these observations suggest that MGP inhibits calcification of isolated VSMCs and that calcification of MGP-deficient VSMCs is, at least in part, dependent on BMP signal transduction.
In a landmark study using bone biopsies to estimate bone volume and multi-slice computed tomography to assess coronary artery calcification, low bone volume was found to be a signifi- cant risk factor for coronary calcification in HD patients. In addition, low bone volume has been recognized as an important risk factor for the occurrence of fractures in both the general population [27–29] and dialysis patients.[30, 31] Therefore, it is reasonable to assume that dial- ysis patients with evident VC are more likely to experience a fracture. Our results have clearly shown that the dialysis patients with aortic arch calcification had a higher risk of fractures (Table 3, Figs 2 and 3). Specifically, in uremic milieu, dialysis patients tended to consume more fetuin A to prevent VC and therefore, dialysis subjects with baseline VC tended to have a lower fetuin A level concurrently. In an outstanding study which has shown that fetuin A is trafficked and exocytosed via exosome release in MV bodies. Interestingly, MV bodies containing exo- somes were easily observed in vessels, especially in calcified MVs in dialysis patients. Factors that increase exosome release will promote vascularcalcification, specifically under the condi- tion of environmental calcium stress or fetuin A deficiency. Therefore, the consequent gen- eralized VC can be highly anticipated.
Vascularcalcification (VC) is a common occurrence in patients affected with chronic diseases including diabetes, chronic kidney disease (CKD), or atherosclerosis. VC is also a hallmark of rare genetic diseases including pseudoxanthoma elasticum (PXE), generalized arterial calcification of infancy (GACI), Keutel syndrome, and progeria (1). Although the pathogenesis and clinical significance of VC are dependent on the etiology, the endpoint is invariably the formation of hydroxyapatite (HA) deposits in the arterial wall. Over the last two decades, studies have identified a number of calcification inhibitors in the healthy vessel wall that act to protect the vascular smooth muscle cells (VSMCs) from calcification. These factors act by either directly interfering with molecular pathways and/or sequestering hydroxyapatite components impairing their assembly and deposition. Their actions also depend on the stage of crystal formation and environmental context. Tremendous efforts have been put into the understanding of the mechanisms involved in the activity of these endogenous inhibitors that represent attractive factors with therapeutic relevance to VC treatment.
MGP and GRP are vitamin K-dependent protein (VKDP) synthetized by VSMCs in vascular tissues. Fetuin-A is a liver-derived blood cysteine protease inhibitor uptake from circulation by VSMCs. MGP and fetuin-A are longstanding recognized vascular cal- cification inhibitors [90, 91]. GRP was more recently shown to function both as a calcification inhibitor and an anti-inflammatory agent in the cardiovascular and articular systems [92-94]. Functional in vivo and in vitro models have established the importance of these inhibitors in vascularcalcification, with a pre- ponderant role at tissue and systemic levels. Knockout mice for MGP (MGP-/-) result in massive vascularcalcification affecting the main arteries and death within 8 weeks of birth . Restoration of MGP expression in VSMCs from MGP-/- rescued the arterial calcification phenotype . Fetuin-A deficient mice combined with a calcification-sensitive mouse strain or a mineral and vitamin D rich diet, results in progressive and lethal calcification of soft tissues, including kidneys, skin, heart and vasculature . The role of GRP as inferred by animal models is still being explored. Similarly to fetuin-A, GRP knockout mice (GRP-/-) without additional challenging conditions such as aging or disease, present a normal phenotype in terms of skeletal development . However, after destabilization of the medial meniscus, GRP-/- mice develop a severe osteoarthritis phenotype clearly indicating a chondro- protective effect for GRP . Also, VSMCs from GRP-/- mice exposed to calcifying conditions show increased mineralization and expression of osteo- chondrogenic markers . In addition, using a human ex vivo model of VC, γ-carboxylated GRP was shown to inhibit calcification and osteochondrogenic differentiation . These studies confirm a preponderant role for GRP as an inhibitor of VC. This is also in line with functional studies in zebrafish suggesting an essential role of GRP in skeletal development and calciﬁcation . It should be noted that the use of animal models has several shortcomings in direct translation to the human situation. In the case of GRP, several differences in terms of protein and gene expression patterns and different isoforms in mice and human, might imply different functional mechanisms [88, 102, 103]. Of note, despite the impressive phenotype of MGP-/- mice, loss-of- function mutations in the human MGP gene, known as the Keutel syndrome, results in non-lethal abnormal soft tissue calcifications , suggesting that additional or compensatory mechanisms of pathological mineralization inhibition might exist in human.
Vascular calcifications were assessed by the simple vascularcalcification score (SVCS), a semi- quantitative score developed by us and evaluated in plain radiographs of pelvis and hands . Pelvis films were divided into four sections by two imaginary lines: a horizontal line over the upper limit of both femoral heads and a median vertical line over the vertebral column. A hori- zontal line divided hand films over the upper limit of the metacarpal bones. In each section, the presence of any type of vascularcalcification lining the vessel walls, either in a linear or irregular pattern, was rated as 1 and its absence as 0. Final score was the sum of calcifications found in all sections and ranged from 0 to 8. In the same diagnostic centre, vascular calcifica- tions were simultaneously assessed in 42 patients by the Agatston score, using Multislice Com- puted Tomography (MSCT). MSCT scans were performed with the four-slice technique on the model Somatom Volume Zoom (Siemens AG, Erlangen, Germany). Slices of 2.5 mm thickness were acquired under the following conditions: 120 kVp, 130 mAs, and 0.5 gantry rotation time. All images were transferred to a workstation and analysed with calcium scoring software (HeartView CT, Siemens AG, Erlangen, Germany).
The so-called Gla proteins, which con- tain γ-carboxyglutamic acid, include osteo- calcin and MGP. These proteins are ex- pressed in different human tissues, mainly bone and vascular cells, and are mediators and inhibitors of osteoid formation (7). Os- teocalcin is a Gla protein synthesized mainly by osteoblasts and, when carboxylated, it binds to hydroxyapatite in bone, leading to bone mineralization. However, osteocalcin does not seem to play a dominant role in the process of vascularcalcification (22). On the other hand, experimental studies on MGP- knockout mice have shown the formation of extensive and lethal arterial calcifications, a finding confirming the inhibitory role of this protein in vascularcalcification. These ani- mals also presented osteopenia, fractures, short stature, and erratic mineralization of the growth plates (23). Recent evidence in- dicates that this protein inhibits mesenchy- mal differentiation into osteogenic cell lines by blocking the action of BMP, a potent factor of bone maturation. The absence of this inhibition leads to the differentiation of vascular mesenchyme into bone cells, thus increasing calcification (24).
Chronic kidney disease (CKD) is estimated to affect more than 10% of the global population and represents an increasing health and economic burden for the society [1,2]. Cardiovascular disease (CVD) is the most important complication of CKD and the primary cause of death in these patients . In addition to traditional risk factors, most patients with CKD display abnormal mineral metabolism (MM) with underlying hormonal dysregulation, defined as chronic kidney disease-mineral and bone disorder (CKD-MBD) . CKD-MBD involves changes in mineral ion homeostasis, bone quality and turnover, cardiovascular and soft tissue calcifications, which highly contribute for cardiovascular complications [4,5]. Vascularcalcification (VC) is associated with significant morbidity and mortality and a strong predictor of cardiovascular risk in CKD patients [6,7]. The prevalence of VC and the risk of CVD are shown to increase as glomerular filtration rate (GFR) declines in CKD patients [3,8]. In fact, bone MM abnormalities start during the first stages of CKD, long before renal replacement therapy is required . Cardiovascular calcification is a highly-controlled and regulated process of calcium phosphate mineral deposition in the intima and media layers of the vessel wall and in cardiac valves. Epidemiologically, CKD, diabetes mellitus and atherosclerosis are the clinical conditions that most contribute towards development of vascular and valves calcification . Increased vascular stiffness is an established independent predictor of cardiovascular morbidity and mortality [11,12], and aortic calcification has been positively associated with arterial stiffness in the healthy and CKD populations [13,14]. Increased pulse pressure (PP) is one of the most evident hemodynamic consequences of increased vascular stiffness, and has been suggested as correlated with arterial calcification and cardiovascular events in non-CKD, dialysis and non-dialysis patients [15,16]. Although the relevance of vascularcalcification assessment is recognized in clinical practice, most reliable quantitative methods are still radiographic or echographic related, with many shortcomings, such as cost and time consumption, particular in the case of computed tomography methods, radiation exposure, operator dependency and lack of standardized scores . Therefore, the development of biomarkers for early detection of VC are crucial for the prevention of CVD outcomes in CKD patients, allowing preventive measures to reduce the development and progression of VC, left ventricular hypertrophy and arterial stiffness.
Vascularcalcification (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,
(PWA) using applanation tonometry has emerged as a technique for assessing vascular function and has been applied as an important research tool in CKD. PWA provides several vascular indices such as pulse wave velocity (PWV), augmentation index, ejection duration index, and SERV. PWV has been largely used in patients with CKD and mostly reflects arterial stiffness. Arterial stiffness causes an increase in afterload on the left ventricle resulting in left ventricular hypertrophy and reduced coronary perfusion. Several studies have demonstrated an association between vascularcalcification and increased arterial stiffness in patients with CKD. 2 Furthermore,
Previous research on vascularcalcification has mainly focused on the vascular intima and media. However, we show here that vascularcalcification may also occur in the adventitia. The purpose of this work is to help elucidate the pathogenic mechanisms underlying vascu- lar calcification. The calcified lesions were examined by Von Kossa staining in ApoE −/− mice which were fed high fat diets (HFD) for 48 weeks and human subjects aged 60 years and older that had died of coronary heart disease, heart failure or acute renal failure. Explant cultured fibroblasts and smooth muscle cells (SMCs)were obtained from rat adven- titia and media, respectively. After calcification induction, cells were collected for Alizarin Red S staining. Calcified lesions were observed in the aorta adventitia and coronary artery adventitia of ApoE-/-mice, as well as in the aorta adventitia of human subjects examined. Explant culture of fibroblasts, the primary cell type comprising the adventitia, was success- fully induced for calcification after incubation with TGF-β1 (20 ng/ml) + mineralization media for 4 days, and the phenotype conversion vascular adventitia fibroblasts into myofibroblasts was identified. Culture of SMCs, which comprise only a small percentage of all cells in the adventitia, in calcifying medium for 14 days resulted in significant calcification.Vascular cal- cification can occur in the adventitia. Adventitia calcification may arise from the fibroblasts which were transformed into myofibroblasts or smooth muscle cells.
serum calcitriol levels as a consequence of kidney damage induce increased PTH secretion. PTH promotes bone turnover as well as calcium and phosphate release from bone tissue , . Also, in CKD patients, inhibitors like fetuin-A are decreased. Thus, spontaneous HAP nucleation cannot be prevented and crystals are deposited in vascular walls of small arterioles of the skin and subcutaneous fat tissue . Vascularcalcification occurs as intimal calcification of atherosclerotic plaques or within the medial arteries. Medial calcification, also known as Mönckeberg sclerosis is most common in patients with CKD . Here, vascular smooth muscle cells (VSMC) undergo a phenotypic change into the osteochondrocytic lineage contributing to HAP deposition . It was demonstrated that under different cell stressing conditions occurring in atherosclerosis and CKD, including oxidative stress, elevated inflammation markers, hypophosphatemia and elastin degradation, Cbfa-1 expression is induced . Cbfa-1, also known as Runx2, is described to be major osteoblastic transcription factor and thus the main factor in osteochondrogenic transdifferentiation of vascular smooth muscle cells . In addition to the mechanisms described above, the lack of inhibitory proteins plays a crucial role and leads to formation and deposition of HAP crystals. Namely the most important proteins are OPN, MGP and fetuin-A .
that hepatic and peripheral carboxylation have unique vitamin K dependence. Vitamin K2 mainly affects periph- eral carboxylation so that supplementation prevents arterial calcification while vitamin K1 mostly affects hepatic carboxylation and its supplementation does not prevent vascularcalcification. Phylloquinone (vitamin K1) is found in green leafy vegetables. The exact source of vitamin K2 is controversial. Some authors claim that it comes either from enterocyte conversion of vitamin K1 or from indigenous intestinal bacteria. 5 Others state
cMGP antagonizes BMP and is consequently linked to signaling networks regulating inflammation  and inducing VSMC differentiation  and apoptosis . Thus, warfarin potentially affects plaque phenotype more profoundly than solely accelerating plaque calcification. We observed that warfarin treatment did not affect BMP-2 and -4 expression but increased collagen type-II and ALK expression concurring MGP regulated chrondrocytic trans- differentiation of VSMC [4,43]. Furthermore, warfarin caused increased plaque apoptosis and loss of VSMC, both have been linked to calcification [25,44] and progression towards unstable plaque [45,46]. Thus, the loss of VSMCs underneath the plaque seen in our model may link to the observed increased number of outward remodeled plaques. In addition, it was recently shown that warfarin treated rats displayed increased MMP-9 activity in the vasculature which related to elastin degradation and vascularcalcification . Outward remodeled plaques have been linked to vulnerability of the plaque to rupture .
Despite the fact that OPG has been proven to participate in multiple aspects of vascularcalcification [15,17], the molecular mechanism(s) underlying its function remains exclusive. Whereas previous work has focused on the investigation of OPG on the osteoclastogensis in osteoclast development, our current study focused on the regulation of OPG on osteoblastic differentiation of VSMCs and the signaling pathway involved in this function. Our results show that not only is OPG an important inhibitor of osteoblastic conversion in VSMCs, but that it also inhibits VSMC calcification by blocking the Notch1- RBP-Jκ-dependent signaling pathway. These findings provide Figure 3. Expression of Notch1 and RBP-Jκ in VSMCs. (A) mRNA level of Notch1 and RBP-Jκ were measured by real- time RT-PCR in VSMCs cultured in different media. (B) Protein level of Notch1 and RBP-Jκ were measured by Western blotting in VSMCs cultured in different media. Notch1 and RBP- Jκ significantly increased by 3 to 4 folds in VSMC cultured in osteogenic medium compared to control at both mRNA and protein levels, suggesting that the Notch1-RBP-Jκ signaling pathway is activated in osteogenic conversion of VSMCs. DAPT reduced the expression of Notch1 by 50% and RBP-Jκ by 30% in VSMCs compared to osteogenic cells at bothe mRNA and protein levels. Similarly, OPG reduced the expression of Notch1 and RBP-Jκ compared to VSMCs in the osteogenic medium in a dose dependent manner. *, P<0.05 vs control, #, P<0.05 vs osteogenic medium, and &, P<0.05 vs OPG 0.1ng/ml. n=3.
Our data are in agreement with and extend the report of Kim et al. (13), who recently demonstrated that LA inhibited in vitro and vitamin D3-induced in vivo vascularcalcification by recovering mitochondrial function and restoring the Gas6/Axl/Akt survival pathway. On the other hand, our results are at variance with findings by Lalaoui et al. (12) and Yamada et al. (29). Lalaoui observed no effect of LA in a rat model of warfarin-induced medial elastocalcinosis, and Yamada et al. reported that tempol was able to reduce arterial calcification by ,33% in uremic rats. Differences in species, doses and time of antioxidant administration are possible reasons for these contrasting data. Furthermore, specific aspects of each calcification model should be considered. Warfarin inhibits carboxylation-driven activation of the Matrix Gla protein, a natural inhibitor of vascularcalcification, and thus exploits a specific pathway of the calcification process per se. In comparison, our vitD model explores several mechanisms present in complex human vascular disease that drive medial calcification, in particular, inflammation and oxida- tive stress. In the warfarin model used by Lalaoui, there was a small elevation of aortic superoxide, while hydrogen peroxide levels were not assessed. In the present study, there was a marked increase in oxidant generation, and while tempol inhibited superoxide generation, it could not prevent elastocalcinosis. These opposing effects of tempol suggest that distinct redox signaling path- ways have divergent effects on vascularcalcification. Comparable considerations can be made for the research by Yamada et al. (29). Overall, combining the production and elimination of diverse intermediates such as super- oxide and hydrogen peroxide under the label of ‘‘ROS’’
Results: A total of 26 issues and nearly 400 manuscripts were evaluated, regarding all levels of scientific evidence, comprehending editorials, original and review articles, consensus and/or scientific forums, case reports, letters to the editor and final paper abstracts. The texts dealt with all large thematic areas of angiology and vascular surgery, besides bioethics, sociology, philosophy and technological innovations. However, despite a progressive paradigm shift, Brazilian authors are still far from ideal with regard to appreciation of the Brazilian scientific production, besides that of J Vasc Bras. This behavior needs to be reconsidered.
O NO é formado na parede vascular tanto pela cNOS como pela iNOS. A quantidade de NO produzida pode determinar se ele é protetório ou tóxico. Embora pequenas quantidades sejam necessárias para a homeostasia, grandes quantidades, como aquelas produzidas na ativação da iNOS são citotóxicas. Porém, a produção de grandes quantidades de NO pode ser importante na defesa contra invasores celulares, tumores celulares e ainda em lesões vasculares com perda endotelial 13,37-39 .