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Introduction Bryozoans have been described as a minor phylum, and it is true that the combination of being inedible, small, difficult to identify, and currently found mainly in cryptic habitats [...], Bryozoans are colonial benthic marine invertebrate calcifiers, important and especially abundant and diverse in southern hemisphere shelf environments. Large heavily calcified colonies can be up to 50 years old, but most longer-lived bryozoans are limited to 10-20 y. Many smaller species are annual. Radial extension in flat encrusting bryozoans is generally on the order of 1-5 mm/y. Erect calcified species generally grow vertically 2-15 mm/y, though articulated species such as Cenaria may reach rates of 40 mm/y. Corresponding calcification rates are generally [10.sup.1]-[10.sup.2] mg/y, but there can be an order of magnitude variation in rate among years in high-latitude bryozoans. Multi-branched bryozoans produce up to 24 g of CaC[O.sub.3]/y. The carbonate produced by bryozoans varies from calcite to aragonite and mixtures of both. Skeletal carbonate mineralogy of bryozoans is complex and appears to be strongly genetically controlled. Global climate change, leading to increasing water temperatures, will generally increase marine bryozoan metabolic rates, and may increase Mg in calcite. On the other hand, decreasing pH (ocean acidification) causes corrosion, changes in mineralogy, and decreased survival. This review of bryozoan growth and calcification allows a general perspective, but also reveals gaps in our knowledge which need to be addressed.

Introduction Coralline algae (Corallinales and Sporolithales, Rhodo-phyta) are important components of marine communities worldwide, cementing coral reefs (Steneck and Adey, 1976) and quickly colonizing bare rock to provide habitat for [...], Ocean acidification caused by rising atmospheric C[O.sub.2] is predicted to negatively impact growth and calcification rates of coralline algae. Decreases in coralline abundance may have cascading effects on marine ecosystems and on carbon sequestration worldwide. In this study, we measured growth and calcification rates of three common species of articulated coralline algae (Bossiella plu-mosa. Calliarthron tuberculosum, and Corallina vancouv-eriensis) at an intertidal field site in British Columbia. Linear growth rates measured in the field were slow, al-though Bossiella grew significantly faster (0.22 cm [mon.sup.-1]) than Calliarthron and Corallina (0.17 and 0.15 cm [mon.sup.-1], respectively). Growth rates in the field were generally slower than growth rates in the laboratory. Suggesting that data generated in the laboratory may not be representative of natural field conditions. Growth rates did not decrease as fronds approached their maximum observed size, suggest-ing that maximum frond size might be determined not by intrinsic factors but by external factors such as wave-induced drag forces. Using growth data, we estimate that the largest observed Bossiella frond (20 cm2) and Calliarthron frond (40 [cm.sup.2]) were about 4 and 11 years old, respectively, and had deposited approximately 1 and 6 g CaCO3 in that time. Given the great abundance of coralline algae along the coast of British Columbia, deposition rates of Ca[CO.sub.3] are expected to play a significant but poorly characterized role in carbon sequestration.

The vast majority of ectothermic organisms grow larger when developing at cooler environmental temperatures, a pattern frequently referred to as the temperature-size rule (TSR). Assuming that this reaction norm has adaptive significance, life history theory predicts that converse patterns may evolve if favored by natural selection, namely if the costs associated with complying to the TSR outweigh the benefits. Calcifying ectotherms may comprise such an exception not following the TSR, because calcification is expected to be more costly at lower temperatures thus increasing associated costs. To test this hypothesis, we reared wild-caught juveniles of the intertidal gastropod Monetaria annulus and compared their shell sizes at the end of the juvenile stage between two rearing temperatures. Contrary to our prediction, M. annulus does follow the TSR, suggesting that increased calcification costs at lower temperatures are not high enough to break the TSR. Such plastic responses should be considered when interpreting geographical patterns such as latitudinal size clines, which may be caused at least partly by phenotypic plasticity., Introduction Most ectotherms mature at a larger size at lower developmental temperatures, a pattern of wide taxonomic prevalence which has been coined the temperature-size rule (TSR; Atkinson 1994). The TSR [...]

Using C[O.sub.2] perturbation experiments, we examined the pre- and post-settlement growth responses of a dominant biofouling tubeworm (Hydroides elegans)to a range of pH. In three different experiments, embryos were reared to, or past, metamorphosis in seawater equilibrated to C[O.sub.2] values of about 480 (control), 980, 1,480, and 2,300 µatm resulting in pH values of around 8.1 (control), 7.9, 7.7, and 7.5, respectively. These three decreased pH conditions did not affect either embryo or larval development, but both larval calcification at the time of metamorphosis and early juvenile growth were adversely affected. During the 24-h settlement assay experiment, half of the metamorphosed larvae were unable to calcify tubes at pH 7.9 while almost no tubes were calcified at pH 7.7. Decreased ability to calcify at decreased pH may indicate that these calcifying tubeworms may be one of the highly threatened species in the future ocean., Introduction Carbon dioxide (C[O.sub.2]) produced by anthropogenic activity is absorbed by the ocean, reducing the seawater pH and carbonate chemistry that can adversely affect marine organisms, especially calcifiers, many of [...]

INTRODUCTION Patients with chronic kidney disease (CKD) have a high prevalence of vascular calcification, especially coronary artery calcification (CAC), a measure of subclinical atherosclerosis. (1) The calcification process begins in [...], BACKGROUND/OBJECTIVE: Recent epidemiological data have shown that abdominal fat accumulation is associated with increased risk of cardiovascular events in patients with chronic kidney disease (CKD). This study aimed to investigate the association between visceral adiposity and coronary artery calcification (CAC) in CKD patients. SUBJECTS/METHODS: Cross-sectional study with 65 nondialyzed CKD male patients (59 ± 9 years, CKD stages 3 and 4). Abdominal fat compartments were assessed by computed tomography (CT) at L4-L5 level. Visceral to subcutaneous (V/S) fat ratio was calculated. Visceral obesity was defined as a V/S fat ratio greater than the median value of the sample study (>0.55). CAC was detected by multi-slice CT. CAC scores were calculated with the Agatston method. RESULTS: CAC was present (calcium score >10AU) in 66% of patients. In the group with visceral obesity, the CAC score was significantly higher. This group had lower adiponectin and higher leptin levels compared to patients without visceral obesity. In the whole sample, higher V/S fat ratio was associated with CAC score, independently of age, body mass index, diabetes, ionized calcium, smoking or renal function. CONCLUSION: Our results show an association between visceral obesity and CAC in CKD patients, suggesting a deleterious effect of visceral fat in these patients. Increased visceral adiposity might enhance cardiovascular risk in this particular population. European Journal of Clinical Nutrition (2013) 67, 610-614;doi: 10.1038/ejcn.2013.66; published online 27 March 2013 Keywords: chronic kidney disease; coronary artery calcification; visceral obesity; adipokines

Introduction Cardiovascular events are the leading cause of death in patients suffering from chronic kidney disease (CKD), and patients with CKD are considered among the highest risk group for cardio-vascular [...], Klotho is a potent regulator of 1,25-hydroxyvitamin D3 [[[1,25(OH).sub.2]D.sub.3]] formation and calcium-phosphate metabolism. Klotho-hypomorphic mice (kl/kl mice) suffer from severe growth deficits, rapid aging, hyper-phosphatemia, hyperaldosteronism, and extensive vascular and soft tissue calcification. Sequelae of klotho deficiency are similar to those of end-stage renal disease. We show here that the mineralocorticoid receptor antagonist spironolactone reduced vascular and soft tissue calcification and increased the life span of kl/kl mice, without significant effects on [[1,25(OH).sub.2]D .sub.3], FGF23, calcium, and phosphate plasma concentrations. Spironolactone also reduced the expression of osteoinductive Pit1 and Tnfa mRNA, osteogenic transcription factors, and alkaline phosphatase (Alpl) in calcified tissues of kl/kl mice. In human aortic smooth muscle cells (HAoSMCs), aldosterone dose-dependently increased PIT1 mRNA expression, an effect paralleled by increased expression of osteogenic transcription factors and enhanced ALP activity. The effects of aldosterone were reversed by both spironolactone treatment and PIT1 silencing and were mitigated by FGF23 cotreatment in HAoSMCs. In conclusion, aldosterone contributes to vascular and soft tissue calcification, an effect due, at least in part, to stimulation of spironolactone-sensitive, PIT1-dependent osteoinductive signaling.

I tested the hypothesis that high pC[O.sub.2] (76.6 Pa and 87.2 Pa vs. 42.9 Pa) has no effect on the metabolism of juvenile massive Porites spp. after 11 days at 28 °C and 545 µmol quanta [m.sup.-2] [s.sup.-1]. The response was assessed as aerobic dark respiration, skeletal weight (i.e., calcification), biomass, and chlorophyll fluorescence. Corals were collected from the shallow (3-4 m) back reef of Moorea, French Polynesia (17°28.614'S, 149°48.917'W), and experiments conducted during April and May 2011. An increase in pC[O.sub.2] to 76.6 Pa had no effect on any dependent variable, but 87.2 Pa pC[O.sub.2] reduced area-normalized (but not biomass-normalized) respiration 36 %, as well as maximum photochemical efficiency ([F.sub.v]/[F.sub.m]) of open RCIIs and effective photochemical efficiency of RCIIs in actinic light (ΔF/[F'.sub.m]); neither biomass, calcification, nor the energy expenditure coincident with calcification (J [g.sup.-1]) was effected. These results do not support the hypothesis that high pC[O.sub.2] reduces coral calcification through increased metabolic costs and, instead, suggest that high pC[O.sub.2] causes metabolic depression and photochemical impairment similar to that associated with bleaching. Evidence of a pC[O.sub.2] threshold between 76.6 and 87.2 Pa for inhibitory effects on respiration and photochemistry deserves further attention as it might signal the presence of unpredictable effects of rising pC[O.sub.2]., Introduction Amidst efforts to evaluate the effects of ocean acidification (OA) on the calcification of scleractinian corals (Erez et al. 2011), much of which has revealed negative implications (Doney et [...]

Introduction Ionized serum calcium levels are critical for the correct functioning of multiple vital cellular processes. Accordingly, the regulation of calcium homeostasis is directed at maintaining serum calcium levels within [...], Serum calcium levels are tightly controlled by an integrated hormone-controlled system that involves active vitamin D [1,25[(OH).sub.2]D], which can elicit calcium mobilization from bone when intestinal calcium absorption is decreased. The skeletal adaptations, however, are still poorly characterized. To gain insight into these issues, we analyzed the consequences of specific vitamin D receptor (Vdr) inactivation in the intestine and in mature osteoblasts on calcium and bone homeostasis. We report here that decreased intestinal calcium absorption in intestine-specific Vdr knockout mice resulted in severely reduced skeletal calcium levels so as to ensure normal levels of calcium in the serum. Furthermore, increased 1,25[(OH).sub.2]D levels not only stimulated bone turnover, leading to osteopenia, but also suppressed bone matrix mineralization. This resulted in extensive hyperosteoidosis, also surrounding the osteocytes, and hypomineralization of the entire bone cortex, which may have contributed to the increase in bone fractures. Mechanistically, osteoblastic VDR signaling suppressed calcium incorporation in bone by directly stimulating the transcription of genes encoding mineralization inhibitors. Ablation of skeletal Vdr signaling precluded this calcium transfer from bone to serum, leading to better preservation of bone mass and mineralization. These findings indicate that in mice, maintaining normocalcemia has priority over skeletal integrity, and that to minimize skeletal calcium storage, 1,25[(OH).sub.2]D not only increases calcium release from bone, but also inhibits calcium incorporation in bone.

BACKGROUND: Secondhand smoke (SHS) consists of fine particulate matter, carcinogens, and various toxins that affect large parts of the population. SHS increases the risk for acute cardiovascular events and may [...]

RhoA/Rho kinases (ROCK) play a critical role in vascular smooth muscle cell (VSMC) actin cytoskeleton organization, differentiation, and function and are implicated in the pathogenesis of cardiovascular disease. We have previously determined that an important step in the regulation of calcification is fetuin-A endocytosis, a process that is dependent on changes in the cytoskeleton, which, in turn, is known to be affected by the RhoA/ ROCK signaling pathway. In the present study, bovine VSMC (BVSMC) were treated with the ROCK inhibitor Y-27632 or transfected with ROCK small interfering (si) RNA to knock down ROCK expression. Both conditions resulted in reduced actin stress fibers and increased Cy5-labeled fetuin-A uptake. Inhibition of ROCK by Y-27632 or siRNA also significantly increased BVSMC alkaline phosphatase (ALP) activity and calcification of BVSMC and rat aorta organ cultures. Cells were then incubated in calcification media in the presence or absence of Y-27632 and matrix vesicles (MV) isolated by collagenase digestion. These MV, isolated from BVSMC incubated with Y-27632, had increased ALP activity and increased ability of MV to subsequently calcify collagen by 66%. In contrast, activation of RhoA, which is upstream of ROCK, by transfecting plasmids encoding the dominant active Rho GTPase mutant (Rho-L63) led to decreased fetuin-A uptake and reduced calcification in BVSMC. These results demonstrate that the RhoA/ROCK signaling pathway is an important negative regulator of vascular calcification. vascular calcification; endocytosis; cytoskeleton; Rho GTPase doi: 10.1152/ajprenal.00730.2009.

Mammalian urine contains a range of macromolecule proteins that play critical roles in renal stone formation, among which Tamm-Horsfall protein (THP) is by far the most abundant. While THP is a potent inhibitor of crystal aggregation in vitro and its ablation in vivo predisposes one of the two existing mouse models to spontaneous intrarenal calcium crystallization, key controversies remain regarding the role of THP in nephrolithiasis. By carrying out a long-range follow-up of more than 250 THP-null mice and their wild-type controls, we demonstrate here that renal calcification is a highly consistent phenotype of the THP-null mice that is age and partially gene dosage dependent, but is gender and genetic background independent. Renal calcification in THP-null mice is progressive, and by 15 mo over 85% of all the THP-null mice develop spontaneous intrarenal crystals. The crystals consist primarily of calcium phosphate in the form of hydroxyapatite, are located more frequently in the interstitial space of the renal papillae than intratubularly, particularly in older animals, and lack accompanying inflammatory cell infiltration. The interstitial deposits of hydroxyapatite observed in THP-null mice bear strong resemblances to the renal crystals found in human kidneys bearing idiopathic calcium oxalate stones. Compared with 24-h urine from the wild-type mice, that of THP-null mice is supersaturated with brushite (calcium phosphate), a stone precursor, and has reduced urinary excretion of citrate, a stone inhibitor. While less frequent than renal calcinosis, renal pelvic and ureteral stones and hydronephrosis occur in the aged THP-null mice. These results provide direct in vivo evidence indicating that normal THP plays an important role in defending the urinary system against calcification and suggest that reduced expression and/or decreased function of THP could contribute to nephrolithiasis. knockout mice; nephrolithiasis; interstitial calcification; supersaturation; urolithiasis doi: 10.1152/ajprenal.00243.2010.

Measurements of primary production (PP), calcification (CF), and coccolithophore abundance were made during late summer (July--August 2007) in the Iceland Basin. Low numbers of coccolithophore cells and detached coccoliths (< 1 x [10.sup.3] cells [mL.sup.1] and 1-15 x [10.sup.3] coccoliths [mL.sup.-1], respectively) indicated a non-bloom community, with Emiliania huxleyi as the dominant coccolithophore in terms of abundance, coccolithophore organic biomass, and cell calcite. PP ranged from 0.1 to 2 mmol C [m.sup.-3] [d.sup.-1], while CF ranged from 10 to 250 [micro]mol C [m.sup.-3] [d.sup.-1], with both typically decreasing with depth. Coccolithophores were estimated to contribute 10-20% toward total chlorophyll a, phytoplankton carbon, and PP within the euphotic zone. In these non-bloom conditions, ~30-60% of the total calcite in the water column was present as detached coccoliths rather than whole cells. Both cell numbers and variability in cell-normalized CF controlled the magnitude of total CF, and hence both physiological limits to cell CF and growth, as well as mortality factors, need to be taken into account when examining oceanic coccolithophore communities. Combining cell-normalized CF with an estimate of coccolith calcite gave coccolith production rates (0.4-1.8 [h.sup.-1]) similar to those reported in the literature for laboratory cultures of E. huxleyi. None of the factors currently associated with coccolithophore blooms (irradiance, mixed-layer depth, nitrate, phosphate, or calcite saturation) showed a clear correlation with community or cellular CF. Hence, although mortality is likely to control cell numbers, other factors such as trace metal (iron) availability may influence coccolithophore physiology in the central Iceland Basin during late summer. doi: 10.4319/10.2010.55.4.1601

Vascular calcification is a multifaceted process involving gain of calcification inducers and loss of calcification inhibitors. One such inhibitor is inorganic pyrophosphate ([PP.sub.i]), and regulated generation and homeostasis of extracellular [PP.sub.i] is a critical determinant of soft-tissue mineralization. We recently described an autocrine mechanism of extracellular [PP.sub.i] generation in cultured rat aortic vascular smooth muscle cells (VSMC) that involves both ATP release coupled to the ectophosphodiesterase/pyrophosphatase ENPP1 and efflux of intracellular [PP.sub.i] mediated or regulated by the plasma membrane protein ANK. We now report that increased cAMP signaling and elevated extracellular inorganic phosphate ([P.sub.i]) act synergistically to induce calcification of these VSMC that is correlated with progressive reduction in ability to accumulate extracellular [PP.sub.i]. Attenuated [PP.sub.i] accumulation was mediated in part by cAMP-dependent decrease in ANK expression coordinated with cAMP-dependent increase in expression of TNAP, the tissue nonselective alkaline phosphatase that degrades [PP.sub.i]. Stimulation of cAMP signaling did not alter ATP release or ENPP1 expression, and the cAMP-induced changes in ANK and TNAP expression were not sufficient to induce calcification. Elevated extracellular [P.sub.i] alone elicited only minor calcification and no significant changes in ANK, TNAP, or ENPP1. In contrast, combined with a cAMP stimulus, elevated [P.sub.i] induced decreases in the ATP release pathway(s) that supports ENPP1 activity: this resulted in markedly reduced rates of [PP.sub.i] accumulation that facilitated robust calcification. Calcified VSMC were characterized by maintained expression of multiple SMC differentiation marker proteins including smooth muscle (SM) [alpha]-actin, SM22[alpha], and calponin. Notably, addition of exogenous ATP (or [PP.sub.i] per se) rescued cAMP + phosphate-treated VSMC cultures from progression to the calcified state. These observations support a model in which extracellular [PP.sub.i] generation mediated by both ANK- and ATP release-dependent mechanisms serves as a critical regulator of VSMC calcification. ectonucleotide pyrophosphatase/phosphodiesterase-1; ANK; adenosine 3',5'-cyclic monophosphate; ATP release doi:10.1152/ajpcell.00419.2009.

Am J Physiol Heart Circ Physiol 298: H5-H15, 2010. First Published October 23, 2009; doi: 10:1152/ajpheart.00824.2009--Recent epidemiologic studies have revealed the risk factors associated for vascular atherosclerosis, including the male sex, smoking, hypertension, and elevated serum cholesterol, similar to the risk factors associated with the development of AV stenosis. An increasing number of models of experimental hypercholesterolemia demonstrate features of atherosclerosis in the AV, which are similar to the early stages of vascular atherosclerotic lesions. Experimental and clinical studies demonstrate that the hypercholesterolemic AV develops an atherosclerotic lesion which is proliferative and expresses high levels of osteoblast bone markers which mineralize over time to form bone. Calcification, the end-stage process of the disease, is necessary to understand as a prognostic indicator in the modification of this cellular process before it is too late. In summary, these findings suggest that medical therapies may have a potential role in patients in the early stages of this disease process to slow the progression to severe aortic stenosis and to delay the timing of the need for surgery. The translation of these experimental studies to clinical practice will be important to understand the potential for medical therapy for this disease process. valvular heart disease; lipids; pathophysiology atherosclerosis; experimental models

With the realization that many calcified skeletons form by processes involving a precursor phase of amorphous calcium carbonate (ACC), a new paradigm for mineralization is emerging. There is evidence the Mg content in biogenic ACC is regulated by carboxylated (acidic) proteins and other macromolecules, but the physical basis for such a process is unknown. We test the hypothesis that ACC compositions express a systematic relationship to the chemistry of carboxyl-rich biomolecules. A series of inorganic control experiments were conducted to establish the dependence of Mg/Ca ratios in ACC on solution composition. We then determined the influence of a suite of simple carboxylated organic acids on Mg content. Molecules with a strong affinity for binding Ca compared with Mg promote the formation of Mg-enriched ACC that is compositionally equivalent to high-magnesium calcites and dolomite. Measurements show Mg/Ca ratios are controlled by a predictable dependence upon the binding properties of the organic molecules. The trend appears rooted in the conformation and electrostatic potential topology of each molecule, but dynamic factors also may be involved. The dependence suggests a physical basis for reports that specific sequences of calcifying proteins are critical to modulating mineralization. Insights from this study may provide a plausible explanation for why some biogenic carbonates and carbonaceous cements often contain higher Mg signatures than those that are possible by classical crystal growth processes. The findings reiterate the controls of microenvironment on mineralization and suggest an origin of compositional offsets, or vital effects, long recognized by the paleoclimate community. biomineralization | dolomite | calcite | signatures | proteins doi/10.1073/pnas.0906741106

Diel cycles of photosynthesis, respiration, and calcification were studied in situ on two hermatypic corals, Acropora eurystoma and Faviafavus, using a submerged respirometer deployed at 5-m depth in the Gulf of Eilat, Israel. A fast-repetition-rate fluorometer (FRRF) was placed in front of one coral in each incubation. Net photosynthesis ([P.sub.N]) and calcification were positively correlated with photosynthetically available radiation (PAR). A hysteresis phenomenon occurred in both photosynthesis and calcification relative to PAR: rates were higher in the afternoon than in the morning. This hysteresis is reversed (counterclockwise) compared to that observed in other photosynthetic organisms (plants and algae); however, the quantum yield ([DELTA]F' : Fm') and the rate of photosynthetic electron transport per PSII reaction center ([P.sub.f]) showed a regular (clockwise) hysteresis. The photosynthetic compensation point ([I.sub.c]) was significantly higher in the morning than in the afternoon, ~900 and ~300 [micro]mol photons [m.sup.-2] [s.sup.-1], respectively. We suggest that PN, reversed hysteresis, and lower morning compensation points are caused by higher respiration in the morning than in the afternoon. This may be a result of higher predation rates by the corals during the night, when plankton is more abundant, followed by higher respiration during the digestion of the prey. Calcification counterclockwise hysteresis was observed during high-PAR periods (>400 and 200 [micro]mol photons [m.sup.-2] [s.sup.-1] for A. eurystoma and F. favus, respectively). We suggest that high morning respiration may induce a decrease in internal tissue pH, causing lower calcification rates.

Increasing atmospheric C[O.sub.2] concentration affects calcification in most planktonic calcifiers. Both reduced or stimulated calcification under high C[O.sub.2] have been reported in the widespread coccolithophore Emiliania huxleyi. This might affect the response of cells to photosynthetically active radiation (PAR; 400 700 nm) and ultraviolet radiation (UVR; 280-400 nm) by altering the thickness of the coccolith layer. Here we show that in the absence of UVR, the calcification rates in E. huxleyi decrease under lowered pH levels ([pH.sub.NBS] of 7.9 and 7.6; pC[O.sub.2] of 81 and 178 Pa or 804 and 1759 ppmv, respectively) leading to thinned coccolith layers, whereas photosynthetic carbon fixation was slightly enhanced at pH 7.9 but remained unaffected at pH 7.6. Exposure to UVR (UV-A 19.5 W [m.sup.2], UV-B 0.67 W [m.sup.-2]) in addition to PAR (88.5 W [m.sup.-2]), however, results in significant inhibition of both photosynthesis and calcification, and these rates are further inhibited with increasing acidification. The combined effects of UVR and seawater acidification resulted in the inhibition of calcification rates by 96% and 99% and that of photosynthesis by 6% and 15%, at pH 7.9 and 7.6, respectively. This differential inhibition of calcification and photosynthesis leads to significant reduction of the ratio of calcification to photosynthesis. Seawater acidification enhanced the transmission of harmful UVR by about 26% through a reduction of the coccolith layer of 31%. Our data indicate that the effect of a high-C[O.sub.2] and low-pH ocean on E. huxleyi (because of reduced calcification associated with changes in the carbonate system) enhances the detrimental effects of UVR on the main pelagic calcifier.

Medial artery calcification, which does not accompany lipid or cholesterol deposit, preferentially occurs in elderly population, but its underlying mechanisms remain unclear. In the present study, we investigated the potential role of senescent vascular smooth muscle cells (VSMCs) in the formation of senescence-associated medial calcification. Replicative senescence was induced by the extended passages (until passages 11-13) in human primary VSMCs, and cells in early passage (passage 6) were used as control young cells. VSMC calcification was markedly enhanced in the senescent cells compared with that in the control young cells. We identified that genes highly expressed in osteoblasts, such as alkaline phosphatase (ALP) and type I collagen, were significantly upregulated in the senescent VSMCs, suggesting their osteoblastic transition during the senescence. Knock-down of either ALP or type I collagen significantly reduced the calcification in the senescent VSMCs. Of note, runt-related transcription factor-2 (RUNX-2), a core transcriptional factor that initiates the osteoblastic differentiation, was also upregulated in the senescent VSMCs. Knockdown of RUNX-2 significantly reduced the ALP expression and calcification in the senescent VSMCs, suggesting that RUNX-2 is involved in the senescence-mediated osteoblastic transition. Furthermore, immunohistochemistry of aorta from the [klotho.sup.-/-] aging mouse model demonstrated in vivo emergence of osteoblast-like cells expressing RUNX-2 exclusively in the calcified media. We also found that statin and Rho-kinase inhibitor effectively reduced the VSMC calcification by inhibiting [P.sub.i]-induced apoptosis and potentially enhancing matrix Gla protein expression in the senescent VSMCs. These findings strongly suggest an important role of senescent VSMCs in the pathophysiology of senescence-associated medial calcification, and the inhibition of osteoblastic transition could be a new therapeutic approach for the prevention of senescence-associated medial calcification. runt-related transcription factor-2; statin doi: 10.1152/ajpheart.00455.2009.

Surface seawaters are supersaturated with respect to calcite, but high concentrations of magnesium prevent spontaneous nucleation and growth of crystals. Foraminifera are the most widespread group of calcifying organisms and generally produce calcite with a low Mg content, indicating that they actively remove [Mg.sup.2+] from vacuolized seawater before calcite precipitation. However, one order of foraminifera has evolved a calcification pathway, by which it produces calcite with a very high Mg content, suggesting that these species do not alter the Mg/Ca ratio of vacuolized seawater considerably. The cellular mechanism that makes it possible to precipitate calcite at high Mg concentrations, however, has remained unknown. Here we demonstrate that they are able to elevate the pH at the site of calcification by at least one unit above seawater pH and, thereby, overcome precipitation-inhibition at ambient Mg concentrations. A similar result was obtained for species that precipitate calcite with a low Mg concentration, suggesting that elevating the pH at the site of calcification is a widespread strategy among foraminifera to promote calcite precipitation. Since the common ancestor of these two groups dates back to the Cambrian, our results would imply that this physiological mechanism has evolved over half a billion years ago. Since foraminifera rely on elevating the intracellular pH for their calcification, our results show that ongoing ocean acidification can result in a decrease of calcite production by these abundant calcifyers. benthic foraminifera | foraminiferal evolution | ocean acidification

The role of atherosclerotic calcification in plaque rupture remains controversial. In previous analyses using finite element model analysis, circumferential stress was reduced by the inclusion of a calcium deposit in a representative human anatomical configuration. However, a recent report, also using finite element analysis, suggests that microscopic calcium deposits increase plaque stress. We used mathematical models to predict the effects of rigid and liquid inclusions (modeling a calcium deposit and a lipid necrotic core, respectively) in a distensible material (artery wall) on mechanical failure under uniaxial and biaxial loading in a range of configurations. Without inclusions, stress levels were low and uniform. In the analytical model, peak stresses were elevated at the edges of a rigid inclusion. In the finite element model, peak stresses were elevated at the edges of both inclusions, with minimal sensitivity to the wall distensibility and the size and shape of the inclusion. Presence of both a rigid and a soft inclusion enlarged the region of increased wall stress compared with either alone. In some configurations, the rigid inclusion reduced peak stress at the edge of the soft inclusion but simultaneously increased peak stress at the edge of the rigid inclusion and increased the size of the region affected. These findings suggest that the presence of a calcium deposit creates local increases in failure stress, and, depending on relative position to any neighboring lipid pools, it may increase peak stress and the plaque area at risk of mechanical failure. plaque rupture; vulnerable plaque; atherosclerosis; vascular calcification

The purpose of the present study was to test the hypothesis that extraskeletal calcification regresses in uremic rats after reduction in phosphorus intake and treatment with calcimimetics. Extraosseous calcification was induced in five to six nephrectomized rats fed a high-phosphorus (1.2%) diet who received calcitriol (80 ng/kg ip) every other day for a period of 14 days. Next, dietary phosphorus was reduced to 0.6%, and rats were treated with vehicle (n = 20), calcitriol [80 ng/kg ip/48 h (n = 20)], or the calcimimetic AMG 641 [1.5 mg/kg sc/48 h (n = 20)]. Aortic and soft-tissue calcium and phosphorus content was evaluated after 14 and 28 days. At 28 days, reduction of phosphorus intake resulted in a significant decrease in tissue mineral content in vehicle- and AMG 641-treated rats but not in rats receiving calcitriol. Aortic calcium and phosphorus was lower in rats treated with AMG 641 (96.7 [+ or -] 26.4 mg/g) than in rats receiving vehicle (178.3 [+ or -] 38.6 mg/g). An infiltrate of phagocytic cells expressing the calciumsensing receptor was identified in areas surrounding loci of calcification. Additional studies in parathyroidectomized rats demonstrated that AMG 641 increased the urinary excretion of calcium (6.2 [+ or -] 0.6 vs. 3.1 [+ or -] 0.5 mg/day, vehicle) (P < 0.001). In conclusion, experimentally induced extraosseous calcification in uremic rats can be partially resolved by reducing phosphorus intake; the addition of calcimimetics may accelerate the regression process through mechanisms potentially involving a direct stimulatory effect on mineral phagocytic cells plus an increase in urinary calcium excretion. calcitriol; hyperparathyroidism; vascular calcification