Onset Of Calcification Research Articles

The evolution of carotid atherosclerotic plaque composition: a prospective study

Abstract Background The rupture of carotid atherosclerotic plaque is one of the major causes of ischemic stroke, and largely determined by the composition of the plaque. To date, most studies on carotid plaque composition focused on individual plaque components, whilst the interplay between different components within the same plaque may be a critical driving force for plaque evolution and rupture. Objectives To determine the interplay between carotid plaque components over time and describe the dynamics of plaque composition with a specific focus on age- and sex-specific changes in a population-based setting. Methods From a population-based cohort in Netherlands, 802 participants (mean age ± SD 68 ± 8 years, 43% women) with subclinical carotid atherosclerosis underwent repeat magnetic resonance imaging (MRI) of carotid plaque (first scan between 2007 and 2012, second scan between 2014 to 2017). We evaluated carotid plaque composition, including calcification, lipid-rich necrotic core (LRNC) and intraplaque hemorrhage (IPH). To assess the relationship between the incidence of one specific component and baseline plaque composition, plaques with this specific component present at the baseline were excluded. Generalized estimation equation models were used with adjustments for baseline plaque size and cardiovascular risk factors. We demonstrated the evolution of plaque composition along age and by sex. Results Baseline presence of calcification in carotid plaque is independently associated with a higher incidence of IPH (adjusted odds ratio: 2.25; 95% confidence interval: 1.40 to 3.63). No associations were found between baseline plaque composition and the new onset of calcification or LRNC. The proportion of complex plaques with multiple components significantly increased at follow-up (Figure 1a). The plaque composition dramatically evolved over time and the proportion of plaques with two or more components started with 10% around 55 years and increased to over 50% after 70 years of age (Figure 1b). Although the age distribution was comparable between men and women, men were more likely to have and develop plaques with multiple vulnerable plaque components such as IPH (Figure 2). Conclusions A higher incidence of IPH was found in plaques with baseline calcification. The pre-existing plaque composition strongly determines how the plaque will evolve over time. Given the interplay between individual components, the overall plaque composition exhibits significant age- and sex- specific characteristics.

Абдоминальный туберкулез у ребенка раннего возраста (клиническое наблюдение)

Abdominal tuberculosis is a specific tuberculous lesion of the abdominal organs. In children, abdominal tuberculosis is mostly secondary, occurring mainly in the form of mesadenitis and intestinal tuberculosis. In most cases (63–96%), the process is localized in the ileocecal valve. The article presents a case of untimely diagnosed abdominal tuberculosis with lymph node lesions of the peritoneum and intestines in a child with intrathoracic lymph node tuberculosis of all groups during densification and the calcification onset. The examination (computed tomography) revealed a lesion of the ileocecal valve, while histology of biopsy material taken during colonoscopy showed granulomatous colitis of mycobacterial nature. The leading clinical manifestation was malabsorption syndrome with increased protein-energy undernutrition. The disease pattern was the rare localization of a specific lesion — in the small intestine with the development of jejunal stenosis, which led to a significant disorder of enteral nutrition and the development of malabsorption syndrome with severe trophological insufficiency. The decisive factor in determining the cause of the malabsorption syndrome was laparotomy, which was performed in presented signs of acute intestinal obstruction. In our opinion, an indication for diagnostic laparoscopy in children with abdominal tuberculosis should be considered the enteral nutrition disorder and malabsorption syndrome, which cannot be corrected at the therapeutic process stage. KEYWORDS: primary generalized tuberculosis, mycobacterium tuberculosis, intestinal tuberculosis, mesadenitis, malabsorption syndrome, jejunal stenosis, computed tomography, histology. FOR CITATION: Poddubnaya L.V., Dubakova G.F., Geleskul T.S., Shilova E.P., Kolpakova T.A., Osipenko M.F., Nikonov S.D., Drobysheva V.P. Abdominal tuberculosis in a baby (case report). Russian Medical Inquiry. 2023;7(5):337–342 (in Russ.). DOI: 10.32364/2587-6821-2023- 7-5-13.

Türk Toplumunda 5-15 Yaş Grubu Çocuklarında Üçüncü Molar Dişlerin Gelişimlerinin Radyografik Olarak Değerlendirilmesi

Objective: In this study, it was aimed to determine the age of the onset of crown calcification of third molars in children aged 5-15 years in Turkey, and to evaluate the development status of third molars by age.Material and Methods: Panoramic radiographs of the first 1024 patients between the ages of 5 and 15 years were evaluated. The development (calcification) of third molars was classified according to the Demirjian method.Results: When the onset age of the stages for maxillary and mandibular third molars were compared, no statistically significant difference was found (p≥0.05). In addition, although no statistically significant difference was found between genders regarding the age of calcification onset of third molars, it was observed that teeth #28 and #48 developed in boys approximately one year before girls (p≥0.05). When the onset age of the stages for maxillary and mandibular third molars were compared, no statistically significant difference was found (p≥0.05). Concerning stage 5, in which the furcation zone of third molars begins to calcify, although not statistically significant, all the maxillary and mandibular third molars were seen earlier in girls than boys.Conclusion: It was found that the maxillary third molars on the right side developed earlier than mandibular third molars.

Abstract P3095: Correlating Spatial Wall Shear Stress With Transcriptomics In Pre-calcific Stages Of Bicuspid Aortic Valve Disease

Bicuspid aortic valve (BAV) is the most common congenital cardiovascular defect and characterized by formation of two, rather than three cusps. Approximately 50% of young adults with BAV prematurely develop calcification and significant stenosis, which is typically more severe in right/non-coronary (R/NC) fusion. Mechanisms underlying this process are unknown. However, based on BAV-induced narrowing of the valve orifice and obstruction of blood flow, it has been speculated that abnormal biomechanical stimuli play a major role. Despite this, validation studies correlating these stimuli to molecular changes that promote calcification in BAV are lacking. Our objective is to define biomechanical stimuli in young patients and mice with R/NC BAV prior to calcification onset, and further use the murine model to correlate biomechanical indices with transcriptomic changes indicative of mechanosensitive regulators of calcification.Fluid-structure interaction (FSI) models and quantified biomechanical indices (wall shear stress (WSS), von Mises stress) are being generated from echocardiography and MRI from young adults and NfatC1 cre(+) ;Exoc5 fl/+ mice with R/NC BAV, and age-matched tricuspid aortic valve controls. In parallel, tissue sections collected from diseased mice are being subject to spatial transcriptomics (10x Visium) to determine the localization of differentially expressed genes relative to abnormal biomechanical stimuli. FSI results from a 23 y/o BAV patient indicate that cusp fusion leads to higher WSS on the fibrosa surface compared to the control, and high von Mises stress at the fusion site, both of which are regions prone to calcification in humans. To date, we have identified aortic valve stenosis in 25% NfatC1 cre(+) ;Exoc5 fl/+ mice, and spatial transcriptomics in these mice is currently underway. To our knowledge these are the first FSI models to characterize biomechanical stimuli for a young adult with R/NC BAV prior to calcification. Our FSI studies in mice will validate the use of a mouse model to study human disease by comparison of biomechanical indices. Furthermore, parallel transcriptomic studies in BAV mice will allow us to correlate biomechanical stimuli with transcriptomic changes underlying premature calcification.

The Time-Dependent Role of Bisphosphonates on Atherosclerotic Plaque Calcification.

Atherosclerotic plaque calcification directly contributes to the leading cause of morbidity and mortality by affecting plaque vulnerability and rupture risk. Small microcalcifications can increase plaque stress and promote rupture, whereas large calcifications can stabilize plaques. Drugs that target bone mineralization may lead to unintended consequences on ectopic plaque calcification and cardiovascular outcomes. Bisphosphonates, common anti-osteoporotic agents, have elicited unexpected cardiovascular events in clinical trials. Here, we investigated the role of bisphosphonate treatment and timing on the disruption or promotion of vascular calcification and bone minerals in a mouse model of atherosclerosis. We started the bisphosphonate treatment either before plaque formation, at early plaque formation times associated with the onset of calcification, or at late stages of plaque development. Our data indicated that long-term bisphosphonate treatment (beginning prior to plaque development) leads to higher levels of plaque calcification, with a narrower mineral size distribution. When given later in plaque development, we measured a wider distribution of mineral size. These morphological alterations might be associated with a higher risk of plaque rupture by creating stress foci. Yet, bone mineral density positively correlated with the duration of the bisphosphonate treatment.

Remodeling of structurally reinforced (TPU+PCL/PCL)-Hep electrospun small-diameter bilayer vascular grafts interposed in rat abdominal aortas.

As thermoplastic polyurethane (TPU) elastomers possess good biocompatibility and mechanical properties similar to those of native vascular tissues, they were intended to be co-electrospun with poly(ε-caprolactone) (PCL) onto the outer surface of PCL electrospun small-diameter single-layer vascular grafts (SLVGs) in this study, combining with surface heparinization. In this work, a kind of structurally reinforced TPU+PCL/PCL small-diameter bilayer vascular graft (BLVG) was fabricated via layer-by-layer electrospinning followed by the heparinization of PCL via EDC/NHS chemistry. The resulting (TPU+PCL/PCL)-Hep BLVGs presented excellent mechanical strength and higher compliance, and sustainably released heparin exhibited enhanced anti-coagulation activity. During 6-month implantation in 18 rat abdominal aortas, these vascular prostheses induced the remodeling and regeneration of neovascular tissues, and promoted ECM deposition. Compared to heparinized PCL (PCL-Hep) SLVGs, the formation of aneurysm was completely inhibited and the onset of calcification was significantly delayed in (TPU+PCL/PCL)-Hep BLVGs. Not only vascular cell makers co-expressed by CD206+ cells were identified, but also a high content of elastin was evidenced due to the improvement of mechanical strength and compliance. These results indicated the feasibility and efficacy of inhibiting the aneurysm formation and boosting the vascular remodeling by incorporating TPU into PCL-Hep small-diameter artificial vascular grafts.

Generalized Arterial Calcification of Infancy Type 1 (GACI1): Identification of a Novel Pathogenic Variant (c.1715T>C (p.Leu572Ser)).

Generalized Arterial Calcification of Infancy (GACI) is a rare disease inherited in a recessive manner, with severe and diffuse early onset of calcifications along the internal elastic lamina in large and medium size arteries. The diagnosis results are from clinical manifestations, imaging, histopathologic exams, and genetic tests. GACI is predominantly caused by biallelic pathogenic variant in the ENPP1 gene (GACI1, OMIM#208000) and, to a lesser extent, by pathogenic variants in the ABCC6 gene (GACI2, OMIM#614473). We present a novel variation in the ENPP1 gene identified in a patient clinically diagnosed with GACI and confirmed by genetic investigation and autopsy as GACI type 1. The sequence analysis of the patient’s ENPP1 gene detected two heterozygous variants c.1412A>G (p.Tyr471Cys) and c.1715T>C (p.Leu572Ser). The variant c.1715T>C (p.Leu572Ser) has not been described yet in the literature and in mutation databases. A genetic analysis was also carried out for the parents of the newborn; the heterozygous pathogenic variant c.1412A>G (p.Tyr471Cys) was detected in the mother’s ENPP1 gene, and a sequence analysis of the father’s ENPP1 gene revealed the novel heterozygous variant c.1715T>C (p.Leu572Ser). Our results showed that the variant c.1715T>C (p.Leu572Ser) may have a pathogenic role in the development of GACI type1 (GACI1, OMIM#208000), at least when associated with the pathogenic c.1412A>G (p.Tyr471Cys) variant. The identification of novel mutations potentially enabled genotype/phenotype associations that will ultimately have an impact on clinical management and prognosis for the disease.

5‐Fluorouracil Induces Vascular Calcification in Rat Aortic Smooth Muscle Cells

Background 5-Fluorouracil (5-FU) is a commonly prescribed chemotherapy used to treat malignancies of the gastrointestinal tract, breast, and head/neck. Despite its high clinical efficacy in treating cancer, it is associated with late occurring cardiotoxicity, specifically increases in arterial stiffness along with a heightened risk for developing heart failure and cardiovascular disease. Because increases in arterial stiffness and vessel calcification can occur concurrently and are associated with cardiovascular disease outcomes, we determined if 5-FU treated aortic smooth muscle cells adopted a calcified phenotype. We hypothesized 1) aortic smooth muscle cells resected from 5-FU treated rats would have a faster calcification onset when challenged with inorganic phosphate media and 2) a single in vitro dose of 5-FU in healthy cells would result in a calcified phenotype. Methods Primary aortic smooth muscle cell culture (ASMC) was performed via resection of rat thoracic aorta from 6-month-old Sprague Dawley rats (n= 8) that were treated with a clinically relevant continuous infusion of 5-FU (50mg/kg and 270mg/kg) or saline equivalent volume. After treatment and resection, ASMC were cultured using explant methods until cells were identifiable via smooth muscle actin staining. Cells derived from each group were cultured with normal media (NM) or media including a pathological concentration (3.2 mmol) of inorganic phosphate media (PI) for 5, 8, or 10 days to determine differences in calcification onset between conditions. In a second experiment, healthy rat aortic smooth muscle cells were incubated in NM or media containing 7x10-3 5-FU (NM + 5-FU) to determine if a single dose, in vitro, directly results in calcification. Calcification was quantified using an Alizarin Red staining assay and colorimetric calcium assay. Data are expressed as Alizarin Red Stain concentration (mM) and total calcium (mg/dL). Results In experiment 1, 5-FU+PI presented with significantly higher concentration of Alizarin Red at days 8 (.043 vs .031; p=.048) and 10 (.068 vs .017mM; p<0.0001) compared to 5-FU+NM whereas no differences were found between CON+PI and CON+NM at any timepoint (all p>0.05). These data were confirmed via Calcium assay in both 5-FU (.346 vs.235 mg/dl; p =0.0039) and CON (.293 vs .269 mg/dl; p=0.69) at the 8-day timepoint. Experiment 2 indicated that healthy rat aortic smooth muscle cells treated directly with 5-FU for 72 h expressed significantly greater calcium absorbance compared to CON (5-FU = .3347 mg/dl, CON = .2362 mg/dl; p=0.02). Conclusion After treatment with 5-FU, ASMCs from rat thoracic aorta explants are prone to a calcification phenotype compared to saline control treated cells. This predisposition with treatment with 5-FU is an ongoing investigation into the possible molecular mechanisms involved in oxidative stress signaling.

Ectopic mineralization in heart valves: new insights from in vivo and in vitro procalcific models and promising perspectives on noncalcifiable bioengineered valves.

Ectopic calcification of native and bioprosthetic heart valves represents a major public health problem causing severe morbidity and mortality worldwide. Valve procalcific degeneration is known to be caused mainly by calcium salt precipitation onto membranes of suffering non-scavenged cells and dead-cell-derived products acting as major hydroxyapatite nucleators. Although etiopathogenesis of calcification in native valves is still far from being exhaustively elucidated, it is well known that bioprosthesis mineralization may be primed by glutaraldehyde-mediated toxicity for xenografts, cryopreservation-related damage for allografts and graft immune rejection for both. Instead, mechanical valves, which are free from calcification, are extremely thrombogenic, requiring chronic anticoagulation therapies for transplanted patients. Since surgical substitution of failed valves is still the leading therapeutic option, progressive improvements in tissue engineering techniques are crucial to attain readily available valve implants with good biocompatibility, proper functionality and long-term durability in order to meet the considerable clinical demand for valve substitutes. Bioengineered valves obtained from acellular non-valvular scaffolds or decellularized native valves are proving to be a compelling alternative to mechanical and bioprosthetic valve implants, as they appear to permit repopulation by the host's own cells with associated tissue remodelling, growth and repair, besides showing less propensity to calcification and adequate hemodynamic performances. In this review, insights into valve calcification onset as revealed by in vivo and in vitro procalcific models are updated as well as advances in the field of valve bioengineering.

Early skeletal colonization of the coral holobiont by the microboring Ulvophyceae Ostreobium sp.

Ostreobium sp. (Bryopsidales, Ulvophyceae) is a major microboring alga involved in tropical reef dissolution, with a proposed symbiotic lifestyle in living corals. However, its diversity and colonization dynamics in host’s early life stages remained unknown. Here, we mapped microborer distribution and abundance in skeletons of the branching coral Pocillopora damicornis from the onset of calcification in primary polyps (7 days) to budding juvenile colonies (1 and 3 months) growing on carbonate and non-carbonate substrates pre-colonized by natural biofilms, and compared them to adult colonies (in aquarium settings). Primary polyps were surprisingly already colonized by microboring filaments and their level of invasion depended on the nature of settlement substrate and the extent of its pre-colonization by microborers. Growth of early coral recruits was unaffected even when microborers were in close vicinity to the polyp tissue. In addition to morphotype observations, chloroplast-encoded rbcL gene sequence analyses revealed nine new Ostreobium clades (OTU99%) in Pocillopora coral. Recruits and adults shared one dominant rbcL clade, undetected in larvae, but also present in aquarium seawater, carbonate and non-carbonate settlement substrates, and in corals from reef settings. Our results show a substratum-dependent colonization by Ostreobium clades, and indicate horizontal transmission of Ostreobium-coral associations.

Aortic Calcification Onset and Progression: Association With the Development of Coronary Atherosclerosis.

BackgroundThoracic aortic calcification (TAC) and coronary artery calcification (CAC) are markers of subclinical atherosclerosis and are associated with incident major cardiovascular events. We investigated major determinants for incidence and progression of TAC and the association between TAC and CAC incidence and progression.Methods and ResultsIn a population‐based cohort study, 3270 participants (aged 45–74 years, 53.1% women) received cardiac computed tomography at baseline and after a mean follow‐up of 5.1±0.3 years for quantification of calcification of the ascending (ATAC) and descending thoracic aorta (DTAC) and CAC. Multivariable relative risk regression analysis was used to investigate associations of cardiovascular risk factors with incident TAC, of baseline TAC with incident CAC, and of baseline CAC with incident TAC. Of 1243 participants with baseline TAC of 0, 517 (41.6%) revealed incident TAC after 5 years. Incidence of descending TAC was higher (34.5%) than ascending TAC (23.3%). Incident TAC after 5 years was associated with age (relative risk 1.26 [95% CI 1.21–1.33], per 5 years), blood pressure (relative risk 1.06 [95% CI 1.03–1.10], per 10 mm Hg), low‐density lipoprotein cholesterol (relative risk 1.08 [95% CI 1.04–1.12], per 20 mg/dL), and smoking (relative risk 1.28 [95% CI 1.07–1.53]). Among the 1185 participants without CAC at baseline, the risk of developing CAC was 28.3% when baseline TAC was present compared with 22.2% among those without baseline TAC (excess risk 6.1% [95% CI 1.2–11.0%]). The point estimate of excess risk for incident CAC was higher for ascending TAC (10.8% [95% CI 4.8–16.7%]) and low for descending TAC (1.8% [95% CI −3.2% to 6.7%]). Excess risk for developing ascending and descending TAC with present baseline CAC was 16.4% (95% CI 12.7–20.0%) and 15.6% (95% CI 10.8–20.4%), respectively.ConclusionTAC and CAC share similar major determinants for incident calcification. Participants with TAC, especially ascending TAC, are at elevated risk for development of CAC.

Assessing Calcification Onset in Aortic Valve Interstitial Cells

Aortic valve stenosis (AVS) is one of the most common heart valve diseases, and surgical intervention remains the only viable treatment option. Thus, there is a need for novel and innovative treatments. One approach is to study the biological pathogenesis of this disease in hopes of finding new targets for drug therapy. Although this may be a viable option, it faces some methodological concerns. Many studies attempted to address the underlying mechanisms of this disease using aortic valves from bovine models. Although these may be viable models in certain diseased conditions, this may not be the case when studying calcification in AVS. Thus, the aim of our study was to assess the significance of drawing conclusions from bovine models to humans in the context of AVS, and investigate the role of alkaline phosphatase (ALP), an enzyme that increases calcium mineralization and deposition in aortic valve calcification. We also wanted to identify any differences in calcification when using different osteogenic media. We used human and bovine valve interstitial cells (HAVICs and BAVICs, respectively), which are the most commonly used when study calcification in AVS, and cultured them in osteogenic media or DMEM as control media. We found that ALP activity differs widely between the two models, with bovine samples having approximately ten times more ALP activity. Our data also suggests that the degree of calcification and ALP activity differs between the different osteogenic media used. Careful consideration should be taken when experimenting with bovine valves and drawing conclusions to human AVS, as they may not exhibit the same mechanisms of action. Furthermore, it may be important to identify a single standard osteogenic medium to use when studying calcification in AVS.

Effects of Elevated Temperature on the Shell Density of the Large Benthic Foraminifera Amphistegina lobifera.

This study investigated the effects of elevated temperature on shell density and Mg-ATPase activity of Amphistegina lobifera. This species is abundant in shallow reef habitats, and can be vulnerable to daily physicochemical fluctuations. To assess potential responses and acclimation mechanisms of A.lobifera to changing temperature conditions, we performed a blocked-design experiment exposing specimens collected from different reef sites (inshore and offshore) to three temperature treatments (Control: 24°C, +2°C: 26°C and +5°C: 29°C) for 30days. The final size and shell density of inshore reef foraminifera were unaffected by elevated temperature, and the enzyme activity in these individuals showed that they were able to acclimate to new temperature conditions. In contrast, offshore A.lobifera were more sensitive to changes in temperature, and heat stress caused growth impairment and inhibited Mg-ATPase activity. However, newly added chambers were not affected. These results suggested that Mg-ATPase plays an important role in regulating intracellular Mg2+ ions, but has little influence in the onset of calcification in A.lobifera. Moreover, it suggests that even though A.lobifera can regulate intracellular functions, local habitat seems to play a crucial role in determining how foraminifera respond to environmental changes.

Valve Endothelial Cell-Derived Tgfβ1 Signaling Promotes Nuclear Localization of Sox9 in Interstitial Cells Associated With Attenuated Calcification.

Aortic valve disease, including calcification, affects >2% of the human population and is caused by complex interactions between multiple risk factors, including genetic mutations, the environment, and biomechanics. At present, there are no effective treatments other than surgery, and this is because of the limited understanding of the mechanisms that underlie the condition. Previous work has shown that valve interstitial cells within the aortic valve cusps differentiate toward an osteoblast-like cell and deposit bone-like matrix that leads to leaflet stiffening and calcific aortic valve stenosis. However, the mechanisms that promote pathological phenotypes in valve interstitial cells are unknown. Using a combination of in vitro and in vivo tools with mouse, porcine, and human tissue, we show that in valve interstitial cells, reduced Sox9 expression and nuclear localization precedes the onset of calcification. In vitro, Sox9 nuclear export and calcific nodule formation is prevented by valve endothelial cells. However, in vivo, loss of Tgfβ1 in the endothelium leads to reduced Sox9 expression and calcific aortic valve disease. Together, these findings suggest that reduced nuclear localization of Sox9 in valve interstitial cells is an early indicator of calcification, and therefore, pharmacological targeting to prevent nuclear export could serve as a novel therapeutic tool in the prevention of calcification and stenosis.

Quantitative proteomics and bioinformatic analysis provide new insight into protein function during avian eggshell biomineralization.

The calcitic avian eggshell protects the developing embryo and, moreover, ensures that the nutritious table egg remains free of pathogens. The eggshell is formed by nucleation upon a fibrous scaffold (the eggshell membranes) followed by an interaction between the growing mineral crystals and the shell organic matrix. This interaction leads to a highly ordered shell microstructure and texture which contribute to its exceptional mechanical properties. Shell mineralization occurs in three distinct phases of calcification (initiation, growth and termination), which are associated with distinct populations of matrix proteins that are secreted into the acellular uterine fluid as modulators of the process. The recent development of high-throughput methods has led to the identification of many proteins in the shell, but little is known concerning their role in shell formation. In order to determine precisely the importance of particular proteins relative to eggshell mineralization, this project used qualitative and quantitative proteomics of the uterine fluid constituents, coupled with bioinformatic analysis, to predict the functional role of proteins secreted at each of the three main stages of shell calcification. Besides its relevance to food production and to hen reproduction, eggshell calcification is furthermore a relevant model for studying calcium carbonate biomineralization on a two-dimensional membrane support. Better understanding of this process will provide insight into the fabrication of ceramics at ambient pressure and temperature.

Abstract 201: BMP-pSmad1/5/8 Pathway Activation in a Novel Mouse Model of Calcific Aortic Valve Disease

Calcific Aortic Valve Disease (CAVD) affects &gt;2% of the population over the age of 65, for whom the current standard of care is valve replacement surgery. To date, there are no pharmacologic-based therapies that prevent the progression or inhibit the development of CAVD, thus highlighting the necessity for new therapeutic approaches. Despite its clinical significance, the pathogenic mechanisms that drive the development of CAVD, and that could serve as potential therapeutic targets, remain unknown. We have recently identified Klotho-deficient mice, an established model of premature aging, as a novel model of CAVD that exhibit aortic valve calcification on the fibrosa side of the hinge region, closely mimicking human CAVD pathology. Unlike previous models, calcification occurs independent of inflammation and valve thickening in these mice, supporting a distinct mechanism of age-related calcification common in elderly patients. In bone, BMP-mediated osteogenic gene induction is essential for the process of calcification. Notably, pSmad1/5/8 activation, indicative of active BMP signaling, is observed prior to the onset of calcification and later is localized with calcific nodule formation in klotho-deficient mice with CAVD. Our hypothesis is that activation of BMP-pSmad1/5/8 signaling pathway promotes osteogenic gene induction and aortic valve mineralization in CAVD. Osteogenic factors, Runx2 and Osteopontin, are significantly increased in the aortic valves of klotho-deficient mice, suggesting an osteogenic-like mechanism of disease. Likewise, pSmad1/5/8 activation precedes osteogenic gene induction in these mice. Moreover, increased BMP2/4 ligand expression is detected prior to the onset of disease, as well as during calcific nodule formation, thus supporting an active role for the BMP-pSmad1/5/8 signaling cascade during aortic valve calcification. Our ongoing work includes BMP pathway inhibition studies to determine if this is an effective therapeutic strategy for the treatment of CAVD in the klotho-deficient mice. Altogether our data support a role for the BMP-pSmad1/5/8 signaling cascade as a critical mechanism in the initial onset and progression of aortic valve calcification in a novel mouse model of CAVD.

Associations between aspirin and other non-steroidal anti-inflammatory drugs and aortic valve or coronary artery calcification: The Multi-Ethnic Study of Atherosclerosis and the Heinz Nixdorf Recall Study

BackgroundThe association between non-steroidal anti-inflammatory drugs (NSAIDs) and the incidence of valvular and arterial calcification is not well established despite known associations between these drugs and cardiovascular events. ObjectiveTo compare the association between the baseline use of aspirin with other NSAID class medications with the incidence and prevalence of aortic valve calcification (AVC) and coronary artery calcification (CAC). MethodsThe relationship of NSAID use to AVC and CAC detected by computed tomography was assessed in 6814 participants within the Multi-Ethnic Study of Atherosclerosis (MESA) using regression modeling. Results were adjusted for age, sex, ethnicity, study site, anti-hypertensive medication use, education, income, health insurance status, diabetes, smoking, exercise, body mass index, blood pressure, serum lipids, inflammatory markers, fasting glucose, statin medication use, and a simple diet score. Medication use was assessed by medication inventory at baseline which includes the use of non-prescription NSAIDs. MESA collects information on both incident and prevalent calcification. The 4814 participants of the Heinz Nixdorf Recall (HNR) Study, a German prospective cohort study with similar measures of calcification, were included in this analysis to enable replication. ResultsMean age of the MESA participants was 62 years (51% female). After adjustment for possible confounding factors, a possible association between aspirin use and incident AVC (Relative Risk(RR): 1.60; 95%Confidence Interval (CI): 1.19–2.15) did not replicate in the HNR cohort (RR: 1.06; 95%CI: 0.87–1.28). There was no significant association between aspirin use and incident CAC in the MESA cohort (RR 1.08; 95%CI: 0.91–1.29) or in the HNR cohort (RR 1.24; 95%CI: 0.87–1.77). Non-aspirin NSAID use was not associated with either AVC or CAC in either cohort. There were no associations between regular cardiac dose aspirin and incident calcification in either cohort. ConclusionBaseline NSAID use, as assessed by medication inventory, appears to have no protective effect regarding the onset of calcification in either coronary arteries or aortic valves.

Consequences of Bony Free Flap’s Pedicle Calcification After Jaw Reconstruction

Most of the time, discovery of free flap pedicle calcification is fortuitous. We describe this phenomenon, try to elucidate its consequences, and discuss about prevention. We studied the effect of radiotherapy on the onset of calcification. We describe the first case of pedicle calcification with a scapula free flap. We performed a retrospective study among the 162 bony free flaps harvested from 2003 to 2011. Follow-up included clinical examination, radiography, and head and neck computed tomography. We collected data including patients' age during free flap surgery, genre, primary disease, disease location, connected artery, ossification delay, clinical signs, flap vitality, and, eventually, calcification removal. We tested the effect of radiotherapy on the onset of calcification using a log-rank test. We retrieved data of 15 patients with pedicle ossification after bony free flap (9.2%). Fibula free flap was present in 14 patients and scapula free flap was present in 1 patient. We noticed vascular pedicle ossification in 42% of our maxillary reconstructions and in 7% of our mandibular reconstructions. The maxillary-to-mandibular ratio is 5:10. Pedicle ossification was discovered between 92 and 366 days after surgery. We performed radiography for diagnosis. Clinical signs were reported in 6 patients. We removed ossified pedicle in 3 patients (20%) owing to pain, trismus, or hard cheek swelling. No free flap failure was noted among the 15 calcified pedicles. There was no statistically significant difference in the effect of radiotherapy on the onset of calcification (P = 0.126). Ossification of the vascular pedicle is uncommon and probably underdiagnosed. Complications are rare, and surgical ossification removal should be reserved for patients with symptoms.

OC.09.1 CLINICAL, MORPHOLOGICAL AND FUNCTIONAL ASPECTS OF PATIENTS SUFFERING FROM PANCREATITIS ASSOCIATED WITH MUTATIONS OF CFTR AND SPINK1 GENES

OC.09.1 CLINICAL, MORPHOLOGICAL AND FUNCTIONAL ASPECTS OF PATIENTS SUFFERING FROM PANCREATITIS ASSOCIATED WITH MUTATIONS OF CFTR AND SPINK1 GENES

Osteoclastic bone resorption directly activates osteoblast function

Osteoclasts, the multinucleated cells that resorb bone, originate from cell cycle-arrested quiescent osteoclast precursors [1]. Mesenchymal osteoblastic cells are involved in osteoclast differentiation. Osteoclast precursors express RANK (a receptor of RANKL), recognize RANKL expressed by osteoblasts through cell-cell interaction and differentiate into osteoclasts in the presence of M-CSF. OPG, produced mainly by osteoblasts, is a soluble decoy receptor for RANKL. Deficiency of OPG in mice induces osteoporosis caused enhanced bone resorption. Elevated osteoblastic activity was suppressed by bisphosphonate administration in OPG-deficient mice. These results suggest that bone formation is accurately coupled with bone resorption. Collagen sponge disks containing BMP-2 were implanted into the dorsal muscle pouches in OPG-deficient mice. TRAP-positive osteoclasts and ALP-positive osteoblasts were observed in BMP-2-disks preceding the onset of calcification for one week. OPG and soluble RANK inhibited BMP-2-induced osteoclast formation but not the appearance of ALP-positive cells in OPG-deficient mice. We then examined how osteoblasts are involved in osteoclastogenesis other than RANKL expression, using RANKL-deficient mice. RANKL-deficient mice showed severe osteopetrosis due to loss of osteoclasts. Injection of RANKL into RANKL-deficient mice induced many osteoclasts in bone but not soft tissues [2]. These results suggest that osteoblasts determine the place of osteoclastogenesis from haemopoietic stem cells in bone. We next explored roles of osteoclasts in ectopic bone formation induced by BMP using op/op and c-fos-deficient osteopetrotic mice. The ectopic bones formed in op/op mice showed extremely rough surfaces, whereas those in wild-type mice showed smooth ones. Bone mineral density of BMP-induced ectopic bone in op/op mice was about 2-times higher than that in wild-type mice. TRAP-positive osteoclasts exhibit in outer of the ectopic bone in the wild-type mice. In op/op mice, although osteoclasts strongly exhibit in inside of the BMP-induced ectopic bone, TRAP-positive osteoclasts did not exhibit in outer of the BMP-induced ectopic bone. Furthermore, the accentuation of the BMP-induced ectopic bone formation did not exist in osteopetrotic c-Fos-deficient mice. In c-Fos-deficient mice, which are completely osteoclasts deficiency, the accentuation of the BMP-induced ectopic bone formation did not exist. Furthermore, there is no RANK-positive osteoclast progenitors in bone derived from c-Fos-deficient mice. These results suggest that RANK-positive osteoclast progenitors are positively regulate the signal of bone formation. In summary, osteoclastic bone resorption directly activates osteoblast function and osteoclasts are involved in normal bone morphogenesis.