Age-related post-error slowing and stimulus repetition effect in motor inhibition during a stop-signal task.

This study aims to investigate how older adults react to a failed-inhibition error while performing a stop-signal task. That is, whether elderly people would exhibit enlarged post-error slowing and whether such slowing revealed an adaptive process, maladaptive process, or a mixture of maladaptive followed by adaptive processes. This study also addresses if the post-error process might further interact with a stimulus repetition effect based on the memory retrieval explanation. A group of 34 younger adults (age range 20-30 years) and a group of 34 older adults (age range 60-80 years) were included for the analyses. The results of the current study supported a mixture model by showing that older adults exhibited a larger post-error slowing than younger adults, and their post-error slowing was initially accompanied by deceased accuracy that then increased on the subsequent trial. Furthermore, such post-error slowing on older adults only occurred in the trial condition where the stimulus was repeated from the previous trial suggesting a memory-based process (a form of negative priming) involved in post-error processes. The implication of the current finding is that older adults might maintain the ability to detect and monitor the response error, yet their post-error adjustment might require a much longer time to start functioning well after the initial detrimental orienting response to the error and the entire process was memory-based.

Between-module functional connectivity of the salient ventral attention network and dorsal attention network is associated with motor inhibition.

The ability to inhibit motor response is crucial for daily activities. However, whether brain networks connecting spatially distinct brain regions can explain individual differences in motor inhibition is not known. Therefore, we took a graph-theoretic perspective to examine the relationship between the properties of topological organization in functional brain networks and motor inhibition. We analyzed data from 141 healthy adults aged 20 to 78, who underwent resting-state functional magnetic resonance imaging and performed a stop-signal task along with neuropsychological assessments outside the scanner. The graph-theoretic properties of 17 functional brain networks were estimated, including within-network connectivity and between-network connectivity. We employed multiple linear regression to examine how these graph-theoretical properties were associated with motor inhibition. The results showed that between-network connectivity of the salient ventral attention network and dorsal attention network explained the highest and second highest variance of individual differences in motor inhibition. In addition, we also found those two networks span over brain regions in the frontal-cingulate-parietal network, suggesting that these network interactions are also important to motor inhibition.

Mixed method study of quality of life for children with trisomy 18 and 13 after cardiac surgery.

BACKGROUND AND OBJECTIVES: Cardiac surgical interventions for children with trisomy 18 and trisomy 13 remain controversial, despite growing evidence that definitive cardiac repair prolongs survival. Understanding quality of life for survivors and their families therefore becomes crucial. Study objective was to generate a descriptive summary of parental perspectives on quality of life, family impact, functional status, and hopes for children with trisomy 18 and trisomy 13 who have undergone heart surgery. METHODS: A concurrent mixed method approach utilising PedsQL™ 4.0 Generic Core Parent Report for Toddlers or the PedsQL™ Infant Scale, PedsQL™ 2.0 Family Impact Module, Functional Status Scale, quality of life visual analogue scale, and narrative responses for 10 children whose families travelled out of state to access cardiac surgery denied to them in their home state due to genetic diagnoses. RESULTS: Parents rated their child's quality of life as 80/100, and their own quality of life as 78/100 using validated scales. Functional status was rated 11 by parents and 11.6 by providers (correlation 0.89). On quality of life visual analogue scale, all parents rated their child's quality of life as "high" with mean response 92.7/100. Parental hopes were informed by realistic perspective on prognosis while striving to ensure their children had access to reaching their full potential. Qualitative analysis revealed a profound sense of the child's relationality and valued life meaning. CONCLUSION: Understanding parental motivations and perceptions on the child's quality of life has potential to inform care teams in considering cardiac interventions for children with trisomy 18 and trisomy 13.

Preplanning prediction of the left anterior descending artery maximum dose based on patient, dosimetric, and treatment planning parameters.

PURPOSE: Maximum dose to the left anterior descending artery (LADmax) is an important physical constraint to reduce the risk of cardiovascular toxicity. We generated a simple algorithm to guide the positioning of the tangent fields to reliably maintain LADmax <10 Gy. METHODS AND MATERIALS: Dosimetric plans from 146 consecutive women treated prone to the left breast enrolled in prospective protocols of accelerated whole breast radiation therapy, with a concomitant daily boost to the tumor bed (40.5 Gy/15 fraction to the whole breast and 48 Gy to the tumor bed), provided the training set for algorithm development. Scatter plots and correlation coefficients were used to describe the bivariate relationships between LADmax and several parameters: distance from the tumor cavity to the tangent field edge, cavity size, breast separation, field size, and distance from the tangent field. A logistic sigmoid curve was used to model the relationship of LADmax and the distance from the tangent field. Furthermore, we tested this prediction model on a validation data set of 53 consecutive similar patients. RESULTS: A lack of linear relationships between LADmax and distance from cavity to LAD (-0.47), cavity size (-0.18), breast separation (-0.02), or field size (-0.28) was observed. In contrast, distance from the tangent field was highly negatively correlated to LADmax (-0.84) and was used in the models to predict LADmax. From a logistic sigmoid model we selected a cut-point of 2.46 mm (95% confidence interval, 2.19-2.74 mm) greater than which LADmax is <10 Gy (95% confidence interval, 9.30-10.72 Gy) and LADmean is <3.3 Gy. CONCLUSIONS: Placing the edge of the tangents at least 2.5 mm from the closest point of the contoured LAD is likely to assure LADmax is <10 Gy and LADmean is <3.3 Gy in patients treated with prone accelerated breast radiation therapy.

Surveillance epidemiology and end results analysis demonstrates improvement in overall survival for cervical cancer patients treated in the era of concurrent chemoradiotherapy.

BACKGROUND: In February 1999, the National Cancer Institute (NCI) issued a clinical alert based on five randomized trials that reported better overall survival (OS) with concurrent chemoradiotherapy (CCRT) than with surgery or radiation alone for locoregional cervical cancer. This study analyzes data from the surveillance epidemiology and end results (SEER) program to evaluate the improvement in survival in the era of CCRT. METHODS: The SEER database was queried for FIGO stages IB2-IVA cervical cancer patients treated with radiotherapy between 1995 and 2002. Patients diagnosed between 1999 and 2002 (CCRT era) were assumed to have received CCRT more frequently than patients diagnosed between 1995 and 1998 (RT era). Cases were stratified by period of diagnosis, age, and SEER region. OS and cause specific survival (CSS) were compared between the two time periods with chi-square log-rank tests. Multivariable Cox models were also used to compare OS and CSS between the two time periods, with adjustment for stratification variables and other covariates. RESULTS: The study included 3517 patients. Unadjusted OS and CSS were significantly improved in 1999-2002 compared with 1995-1998 (OS: p < 0.001, hazard ratio (HR): 0.81; CSS: p < 0.001, HR: 0.79). Significant improvements in OS and CSS were retained after adjustment for multiple variables (multivariable OS HR 0.78; CSS HR 0.76). CONCLUSION: Cervical cancer patients treated with radiotherapy after 1999 had improved OS and CSS compared with patients treated before 1999, likely reflecting increased usage of CCRT. This study adds to the population-level evidence supporting the adoption of CCRT as the standard of care for locoregional cervical cancer.

Radiation therapy improves survival in rectal small cell cancer - Analysis of Surveillance Epidemiology and End Results (SEER) data.

BACKGROUND: Small cell carcinoma of the rectum is a rare neoplasm with scant literature to guide treatment. We used the Surveillance Epidemiology and End Results (SEER) database to investigate the role of radiation therapy in the treatment of this cancer. METHODS: The SEER database (National Cancer Institute) was queried for locoregional cases of small cell rectal cancer. Years of diagnosis were limited to 1988-2010 (most recent available) to reduce variability in staging criteria or longitudinal changes in surgery and radiation techniques. Two month conditional survival was applied to minimize bias by excluding patients who did not survive long enough to receive cancer-directed therapy. Patient demographics between the RT and No_RT groups were compared using Pearson Chi-Square tests. Overall survival was compared between patients who received radiotherapy (RT, n = 43) and those who did not (No_RT, n = 28) using the Kaplan-Meier method. Multivariate Cox proportional hazards model was used to evaluate important covariates. RESULTS: Median survival was significantly longer for patients who received radiation compared to those who were not treated with radiation; 26 mo vs. 8 mo, respectively (log-rank P = 0.009). We also noted a higher 1-year overall survival rate for those who received radiation (71.1% vs. 37.8%). Unadjusted hazard ratio for death (HR) was 0.495 with the use of radiation (95% CI 0.286-0.858). Among surgery, radiotherapy, sex and age at diagnosis, radiation therapy was the only significant factor for overall survival with a multivariate HR for death of 0.393 (95% CI 0.206-0.750, P = 0.005). CONCLUSIONS: Using SEER data, we have identified a significant survival advantage with the use of radiation therapy in the setting of rectal small cell carcinoma. Limitations of the SEER data apply to this study, particularly the lack of information on chemotherapy usage. Our findings strongly support the use of radiation therapy for patients with locoregional small cell rectal cancer.

Outcomes of rectal cancer with liver oligometastases.

PURPOSE: In patients with oligometastatic colorectal cancer to the liver, long term survival is possible and a multi-modality treatment approach may be considered. This is a report of a single institution experience of oligometastatic rectal cancer patients after treatment of the primary tumor and pelvic lymph nodes with extended course chemoradiation therapy. METHODS: Between 2004 and 2013, 26 oligometastatic rectal cancer patients with liver metastases were treated with extended course chemoradiation at our institution followed by total mesorectal excision (TME). Amongst these there were 17 men and 9 women. The mean age at the time of diagnosis was 59.8 years, with a range from 36 to 87 years of age. Eleven patients had metastases in other sites in addition to liver, and one patient in our cohort had lung metastasis with no liver metastasis. Kaplan-Meier method was used to generate overall survival (OS), progression free survival (PFS), distant metastases (DM) and local control (LC). RESULTS: OS rates were 95%, and 70% at 12 and 24 months respectively, with a mean survival time of 40.5 months. PFS rates were 91% and 36% at 12 and 24 months respectively, with a mean PFS time of 23.1 months. LC rates were 91% and 66% at 12 and 24 months respectively. DM rates were 0% and 61% at 12 and 24 months respectively. Finally, when censoring deaths, progression of liver metastases and distant progression, Kaplan-Meier analysis demonstrated five events of local failure. CONCLUSIONS: This series demonstrated an OS of 70% at 24 months, with a mean survival of 40.5 months. Significantly, LC was only 66% despite the use of extended course chemoradiation and TME. This data suggests that many patients with oligometastatic rectal cancer will survive past 2 years, and that a substantial number will fail locally as well as distantly. Therefore, a multimodality approach is reasonable. Recent data suggests that a hypofractionated radiation regiment of 25 Gy in 5 Gy fractions allows an equivalent LC compared to extended course chemoradiation with 50.4 Gy in 1.8 Gy fractions. A short course of radiation may be more consistent with the goals of care of the oligometastatic rectal cancer patient who is at high risk of recurrence.

Feasibility of MR imaging/MR spectroscopy-planned focal partial salvage permanent prostate implant (PPI) for localized recurrence after initial PPI for prostate cancer.

PURPOSE: To assess the feasibility of magnetic resonance imaging (MRI)-planned partial salvage permanent prostate implant (psPPI) among patients with biopsy-proven local recurrence after initial PPI without evidence of distant disease. METHODS AND MATERIALS: From 2003-2009, 15 patients underwent MRI/magnetic resonance spectroscopy (MRS) planning for salvage brachytherapy (psPPI, I-125 [n=14; 144 Gy]; Pd-103 [n=1; 125 Gy]) without hormone therapy. Full dose was prescribed to areas of recurrence and underdosage, without entire prostate implantation. Limiting urethral and rectal toxicity was prioritized. Follow-up was from salvage date to prostate-specific antigen (PSA) concentration failure (Phoenix criteria = nadir + 2.0; ASTRO = 3 consecutive rises), recurrence, distant metastases, or last follow-up PSA level. Progression-free survival (PFS) was defined as no PSA failure or biopsy-proven recurrence without all-cause mortality. Toxicity was scored using Common Terminology Criteria for Adverse Events version 4.0. RESULTS: At salvage, median age was 68 years, and PSA concentration was 3.5 ng/mL (range, 0.9-5.6 ng/mL). Abnormal MRI/MRS findings were evident in 40% of patients. Biopsy-proven recurrences consisted of a single focus (80%) or 2 foci (20%). At recurrence, Gleason score was 6 (67%) or ≥7 (27%). Median interval between initial and salvage implantation was 69 months (range, 28-132 months). psPPI planning characteristics limited doses to the rectum (mean V100 = 0.5% [0.07 cc]) and urethra (V100 = 12% [0.3 cc]). At median follow-up (23.3 months; range, 8-88 months), treatment failure (n=2) resulted only in localized recurrence; both patients underwent second psPPI with follow-up PSA tests at 12 and 26 months, resulting in 0.6 and 0.7 ng/mL, respectively. American Society for Radiation Oncology PFS rates at 1, 2, and 3 years were 86.7%, 78.4%, and 62.7%, respectively, with 5 patients for whom treatment failed (n=3 with negative transrectal ultrasound-guided biopsy results). Phoenix PFS rates at 1, 2, and 3 years were 100%, 100%, and 71.4%. 73%, respectively; achieved PSA nadir of <0.5 ng/mL; and 47% of patients had a nadir of <0.1 ng/mL. Treatment-related toxicity was minimal, with no operative interventions, fistulas, or other grade ≥3 gastrointestinal (GI)/genitourinary (GU) toxicity. Thirteen percent had grade 1 GI and 33% had grade 2 GU toxicities. Postsalvage, 20% of patients had no erectile dysfunction, 67% of patients had medication-responsive erectile dysfunction, and 13% of patients had erectile dysfunction refractory to medication. CONCLUSIONS: Focal psPPI with MR-planning in highly selected patients is feasible with short-term control comparable to conventional salvage, with less toxicity. Longer follow-up is needed to confirm its impact on quality of life and treatment.

Prone accelerated partial breast irradiation after breast-conserving surgery: compliance to the dosimetry requirements of RTOG-0413.

PURPOSE: The dosimetric results from our institution's trials of prone accelerated partial breast irradiation are compared with the dosimetric requirements of RTOG-0413. METHODS AND MATERIALS: Trial 1 and Trial 2 are 2 consecutive trials of prone-accelerated partial breast irradiation. Eligible for both trials were stage I breast cancer patients with negative margins after breast-conserving surgery. The planning target tumor volume (PTV) was created by extending the surgical cavity 2.0 cm for Trial 1 and 1.5 cm for Trial 2, respectively. Contralateral breast, heart, lungs, and thyroid were contoured. Thirty Gray was delivered in five daily fractions of 6 Gy by a three-dimensional conformal radiation therapy technique in Trial 1 and were by image-guided radiation therapy/intensity-modulated radiation therapy in Trial 2. Dosimetric results from the trials are reported and compared with RTOG 0413 requirements. RESULTS: One hundred forty-six consecutive plans were analyzed: 67 left and 79 right breast cancers. The plans from the trials complied with the required>90% of prescribed dose covering 90% of PTV_EVAL (=generated from the PTV by cropping 0.5 cm from the skin edge and excluding the chest wall): V90% was 98.1±3.0% (with V100% and V95%, 89.4±12.8%, 96.4±5.1%, respectively). No significant difference between laterality was found (Student's t test). The dose constraints criteria of the RTOG-0413 protocol for ipsilateral and contralateral lung (V30<15% and Dmax<3%), heart (V5<40%), and thyroid (Dmax<3%) were satisfied because the plans showed an average V5% of 0.6% (range, 0-13.4) for heart, an average V30% of 0.6% (range, 0-9.1%) for ipsilateral lung, and <2% maximum dose to the thyroid. However, our partial breast irradiation plans demonstrated a higher dose to contralateral breast than that defined by RTOG constraints, with a median value of maximum doses of 4.1% (1.2 Gy), possibly as a result of contouring differences. CONCLUSIONS: Our technique for prone accelerated partial breast irradiation generally satisfied RTOG-0413 requirements.

Prone accelerated partial breast irradiation after breast-conserving surgery: five-year results of 100 patients.

PURPOSE: To report the 5-year results of a prospective trial of three-dimensional conformal external beam radiotherapy (3D-CRT) to deliver accelerated partial breast irradiation in the prone position. METHODS AND MATERIALS: Postmenopausal patients with Stage I breast cancer with nonpalpable tumors <2 cm, negative margins and negative nodes, positive hormone receptors, and no extensive intraductal component were eligible. The trial was offered only after eligible patients had refused to undergo standard whole-breast radiotherapy. Patients were simulated and treated on a dedicated table for prone setup. 3D-CRT was delivered at a dose of 30 Gy in five 6-Gy/day fractions over 10 days with port film verification at each treatment. Rates of ipsilateral breast failure, ipsilateral nodal failure, contralateral breast failure, and distant failure were estimated using the cumulative incidence method. Rates of disease-free, overall, and cancer-specific survival were recorded. RESULTS: One hundred patients were enrolled in this institutional review board-approved prospective trial, one with bilateral breast cancer. One patient withdrew consent after simulation, and another patient elected to interrupt radiotherapy after receiving two treatments. Ninety-eight patients were evaluable for toxicity, and, in 1 case, both breasts were treated with partial breast irradiation. Median patient age was 68 years (range, 53-88 years); in 55% of patients the tumor size was <1 cm. All patients had hormone receptor-positive cancers: 87% of patients underwent adjuvant antihormone therapy. At a median follow-up of 64 months (range, 2-125 months), there was one local recurrence (1% ipsilateral breast failure) and one contralateral breast cancer (1% contralateral breast failure). There were no deaths due to breast cancer by 5 years. Grade 3 late toxicities occurred in 2 patients (one breast edema, one transient breast pain). Cosmesis was rated good/excellent in 89% of patients with at least 36 months follow-up. CONCLUSIONS: Five-year efficacy and toxicity of 3D-CRT delivered in prone partial breast irradiation are comparable to other experiences with similar follow-up.

The PI3K/Akt/mTOR pathway mediates retinal progenitor cell survival under hypoxic and superoxide stress.

Oxygen (O2) tension has emerged as a major regulator of stem cell (SC) biology. Low O2 concentrations that are toxic to mature cells can confer advantage to stem and early progenitors, while superoxide stress remains a constant threat in aerobic biology and may be partially avoided through sequestration of SCs in the relatively hypoxic stem or regenerative niche. Using primary retina-derived retinal progenitor cells (RPCs) and the R28 progenitor cell line in vitro, we show that RPCs are sensitive to hydrogen peroxide (H2O2) induced damage and resistant to moderate levels of low oxygen stress (1% O2). Under hypoxic conditions, multipotent RPCs upregulate Epo receptors, and Epo, along with insulin, protects against both superoxide- and severe hypoxia- (0.25% O2) induced apoptosis through activation of the canonical PI3K/Akt/mTOR pathway. This survival advantage is sensitive to inhibitors of PI3K and mTOR. We further demonstrate phosphorylation of the p70S6 ribosomal kinase, a downstream mediator of PI3K/Akt/mTOR and translational activator. Overall, these data confirm that RPCs are sensitive to superoxide stress and resistant to hypoxia and that this resistance is mediated in part by Epo. They further suggest that manipulation of RPCs ex vivo prior to ocular delivery, or the in vivo delivery of exogenous survival factors at the time of cell implantation, could enhance the success of regenerative therapies aimed to restore sight.

Topographic enhancement mapping of the cancer-associated breast stroma using breast MRI.

In animal and laboratory models, cancer-associated stroma, or elements of the supporting tissue surrounding a primary tumor, has been shown to be necessary for tumor evolution and progression. However, little is understood or studied regarding the properties of intact stroma in human cancer in vivo. In addition, for breast cancer patients, the optimal volume of local tissue to treat surrounding a primary tumor is not clear. Here, we performed an interdisciplinary study of normal-appearing breast tissue using breast magnetic resonance imaging (MRI), correlative histology and array comparative genomic hybridization to identify a cancer-associated stroma in humans. Using a novel technique for segmenting breast fibroglandular tissue, quantifiable topographic percent enhancement mapping of the stroma surrounding invasive breast cancer was found to be significantly elevated within 2 cm of the tumor edge. This region was also found to harbor increased microvessel density, and genomic changes that were closely associated with host normal breast tissue. These findings indicate that a cancer-associated stroma may be identified and characterized in human breast cancer using non-invasive imaging techniques. Identification of a cancer-associated stroma may be further developed to help guide local therapy to reduce recurrence and morbidity in breast cancer patients.

beta1 integrin targeting to enhance radiation therapy.

PURPOSE: Cell adhesion to extracellular matrix (ECM) proteins is mediated by the integrin family and has been known to modify radiation sensitivity and resistance in several cell types, including cancer cells. In particular, beta1 integrin signaling has been implicated in the progression and metastasis of various cancers and has been shown to facilitate resistance to radiation therapy. CONCLUSION: In this mini-review, we provide a brief overview of integrin targeting in radiation therapy. We specifically focus on the updated findings of beta1 integrin-mediated signaling pathways after exposure to ionising radiation (IR) using in vitro and in vivo experimental models, which could represent promising therapeutic targets for breast cancer.

Induction of calcification by serum depletion in cell culture: a model for focal calcification in aortas related to atherosclerosis.

BACKGROUND: Since aortic calcification has been shown to initiate in the lower zone of well-thickened plaques (LZP) adjacent to the aortic media of rabbits fed supplemental cholesterol diets, a restricted supply of serum to vascular cells could play a role in vascular calcification. This study was designed to use a cell culture model to support this hypothesis. RESULTS: Rabbit aortic smooth muscle cells were grown to confluence in a culture media containing 10 % fetal bovine serum (FBS). The confluent cells were then exposed to the media for 2 hrs with or without serum at a Ca x P ion product range of 4.5-9.4 mM2. In contrast to the cells cultured in the presence of FBS, confluent cells in its absence displayed marked mineral-positive alizarin red staining and infrared absorption of mineral phosphate. A kinetic parameter C1/2 was used to designate the concentration of serum or its protein constituents needed to reduce the deposition of Ca and P by half. The C1/2 for FBS and rabbit serum was 0.04-0.07 % The C1/2 value for rabbit serum proteins was 13.5 mug/ml corresponding to the protein concentration in 0.06 % of serum. This C1/2 was markedly smaller than 86.2 mug/ml for bovine serum albumin present in 0.37 % serum (p < 0.05). Serum depletion also caused marked membrane translocation as evidenced through a specific apoptosis dye uptake by cells. The proteomic analysis of calcifying vesicles, which can be released by serum depletion, revealed several calcification-related proteins. CONCLUSION: The aortic smooth muscle cell culture model suggests that serum depletion may play a role in the initiation of aortic calcification. The serum exhibits remarkable ability to inhibit cell-mediated calcification.

Mechanisms of focal calcification in rabbit aortas: role of calcifying vesicles.

BACKGROUND: We previously demonstrated that calcification in rabbit thoracic aortas is initiated in the lower zone of the extensively thickened plaques (LZP) adjacent to the media. Whether osteogenesis or a local increase in calcifying vesicles underlies the focal calcification remains to be established. METHODS: To determine that focal calcification is related to osteogenesis, an obligatory osteogenic biomarker, alkaline phosphatase (ALP) activity, was evaluated in the unfixed thoracic aorta sections of rabbits fed a supplemental cholesterol diet and of humans with advanced atherosclerosis. To determine whether blood shortage to the smooth muscle cells (SMCs) imposed by intima thickening may increase calcifying vesicles, the effect of serum deprivation on the biogenesis of calcifying vesicles in cultured SMCs was investigated. RESULTS: (1) In contrast to positive rabbit kidney cross sections and consistent with the activity in various isolated subcellular fractions of aortas, ALP activity was absent in the media, adventitia, lesions, and LZP of rabbit aortas or in the fibrointima of human aortas. (2) Histologic assessments of the lesions indicate the absence of bone cells or osteoid. (3) Depletion of the serum from culture media caused a twofold increase in the levels of ALP-deficient and adenosine triphosphatase-rich calcifying vesicles, which were released from the cells by treatment with 0.05% trypsin-0.02% ethylenediaminetetraacetic acid for 15 minutes. CONCLUSIONS: (1) Focal calcification in rabbit aortas and diffuse mineralization in human fibrointima are not associated with osteogenesis, and (2) blockade of the blood supply to SMCs may trigger the cells to produce more calcifying vesicles, thereby leading to site-specific calcification in rabbit aortas.

Accumulation of low density lipoprotein associated cholesterol in calcifying vesicle fractions correlates with intimal thickening in thoracic aortas of juvenile rabbits fed a supplemental cholesterol diet.

BACKGROUND: It has been shown that calcifying vesicles play an important role in aortic calcification and that cholesterol content in the isolated vesicle fraction is increased when rabbits are fed supplemental cholesterol diets. Whether lipoprotein-associated cholesterols and other lipids are also increased in the vesicle fraction and whether the increase correlates with atherosclerosis remain unknown. RESULTS: Fourteen juvenile male rabbits fed an atherogenic diet containing 0.5% cholesterol and 2% peanut oil for 3 months developed varying degrees of hypercholesterolemia and intimal thickening in the ascending thoracic aorta. The correlation between these two parameters was insignificant, and likely attributable to the use of small numbers of rabbits in this study. Despite this lack of correlation, we demonstrate that the accumulation of cholesterol in calcifying vesicle fractions obtained from the collagenase-digested aorta fragments correlates well with intimal thickening (r2 = 0.98, p < 0.0001). To a smaller degree, the correlation was also significant between intimal thickening and the cholesterol accumulation in the microsomal and post-vesicle fractions. The cholesterol supplemental diet increased the low density lipoprotein-cholesterol (LDL-C) content in calcifying vesicle fractions by 3-fold but did not affect the triglyceride content. High density lipoprotein-cholesterol (HDL-C) and very low-density lipoprotein cholesterol (VLD-C) were absent in calcifying vesicle fractions. CONCLUSION: When limited numbers of rabbits are used, LDL-C accumulation in calcifying vesicle fractions is a better biomarker for atherosclerosis than LDL-C levels in the serum. The close association of LDL-C with calcifying vesicles may play an important role in atherosclerosis and calcification.

Effects of dietary calcium on atherosclerosis, aortic calcification, and icterus in rabbits fed a supplemental cholesterol diet.

BACKGROUND: Vascular calcification is implicated in myocardial infarction, instability and rigidity of the aortic wall, and bioprosthetic failures. Although an increase in the calcium (Ca) content in atherogenic diets has been shown to decrease atherosclerosis in rabbits, whether Ca supplementation and deficiency can affect atherosclerosis-related aortic calcification remains unknown. RESULTS: New Zealand White male rabbit littermates were fed an atherogenic diet containing 0.5% cholesterol and 2% peanut oil. The Ca content of the diet, which normally contains 1%, was adjusted to 0.5 or 3%. Segments of thoracic aortas were dissected from rabbits for histological evaluations and Ca and Pi determinations. Rabbits with calcium supplementation were maintained for 4 months, whereas those with calcium deficiency were maintained for 2 1/2 months due to severe icterus beyond this stage. The ratios of intimal to medial areas and calcified to intimal areas were used to semi-quantify lesion accumulation and calcification, respectively. Icterus was estimated from the extent of yellowing of the skin, sclera, and mucous membranes along with gross evidence of hepatic lipidosis and/or biliary obstructions. Statistical analysis of 16 matched littermates shows that Ca supplementation significantly decreased the lesions by 41% (p < 0.05) and markedly inhibited calcification by 62% (p < 0.05). Statistical analysis of 11 matched littermates shows that Ca deficiency significantly increased the lesions by 2.7-fold (p < 0.05) and that the diet caused a small but significant calcification not seen in the sibling groups with normal dietary Ca. Ca supplementation caused a significant 30% decrease in serum cholesterol (p < 0.05). Calcium deficiency increased serum cholesterol by 57% (p < 0.001). Serum cholesterol and LDL-cholesterol levels in Ca deficient rabbits were 2-fold higher than those with high Ca diets. Ca supplementation decreased soluble Ca and Pi content in aortas, suggesting that this effect may underlie the effects of Ca supplementation on calcification. Calcium deficiency increased icterus by 33% (p < 0.05), which may affect hepatic clearance of cholesterol, while calcium supplementation decreased it by 43% (p < 0.001). CONCLUSION: Ca supplementation to an atherogenic diet inhibits atherosclerosis, aortic calcification, and icterus, whereas a Ca deficient-diet promotes them.

Role of bicarbonate/CO2 buffer in the initiation of vesicle-mediated calcification: mechanisms of aortic calcification related to atherosclerosis.

Calcifying vesicles play an important role in the mechanism of aortic calcification induced by dietary cholesterol interventions. This study was initiated to test the hypothesis that alterations in the ratio of bicarbonate/CO2, which is a main physiological buffer, could affect vesicle-mediated calcification. Using rabbits as a model, in vitro calcification of vesicles isolated from aortas was performed to study the effect of the bicarbonate buffer on the mineralization process. When Tris buffer was initially used to maintain pH of the media, ATP-dependent vesicle calcification increased with pH of calcifying media. By replacing Tris with physiological bicarbonate/CO2 buffer, ATP-dependent vesicle calcification increased rapidly with increased ratios of bicarbonate/CO2. The increase appears to be a result of elevated levels of pH through the alteration in the ratios of bicarbonate/CO2. The effect of the physiological concentration of bicarbonate (30 mM) on pH of calcifying media was remarkable since 50 mM of Tris buffer at pH 7.6 failed to prevent a rapid rise in pH under atmospheric CO2. The effect of bicarbonate and CO2 on vesicle calcification was dependent on the ratio of the surface area to the volume of calcifying media, since the ratio profoundly affects the exchange rate between the gas and liquid phases of CO2. Although the pathological conditions that alter the pH remain unknown, it is conceivable that blockage in the supply of blood CO2 to the media by intimal thickening in the lesions could contribute to focal calcification. We conclude that bicarbonate buffer could provide a dynamic and rapid transitional increase in pH of extracellular fluids, thereby creating a favorable condition for the initiation of vesicle-mediated calcification under pathological conditions.

Mechanism of dystrophic calcification in rabbit aortas: temporal and spatial distributions of calcifying vesicles and calcification-related structural proteins.

INTRODUCTION: We have previously demonstrated the accumulation of calcifying vesicles in the thoracic aorta undergoing dystrophic calcification. Whether the distributions of other structural proteins related to calcification such as collagen and elastin fibers undergo coordinated modifications has not been established. METHODS: Young rabbits with various degrees of aortic calcification induced by cholesterol dietary interventions were used as an animal model to study the correlations. RESULTS: Rabbits fed a diet enriched in cholesterol for 3 months accumulated calcifying vesicles in the ascending thoracic aortas but did not develop histologically identifiable calcification. There were concomitant marked thickenings of the intima with focal deposition of collagen and disruption of the internal elastic fibers at this stage. By the 6th month, calcification was predominantly present in the intimal area adjacent to the media. At this calcified stage, calcifying activity of vesicles was higher than earlier stages. Concomitantly, collagen deposition in the lesions was intensified and the internal elastic fibers were completely disintegrated. These changes were found to be more profound in the proximal than in the distal portion of the aortas. CONCLUSION: The coordinated changes in the accumulation of collagen, disintegration of internal elastic fibers, and the appearance of calcifying vesicles in the lesions before calcification may set the stage for aortic calcification.

In vitro effect of cholesterol on calcifying activity of vesicles isolated from rabbit aortas.

It has been shown that vesicles play a key role in the onset mechanism of aortic calcification related to cholesterol-induced atherosclerosis. This study using a rabbit model was conducted to determine whether cholesterol exerts a direct effect on vesicle's calcifiability. Inclusion of cholesterol in calcifying media stimulated ATP-initiated deposition of calcium in a dose-dependent manner by vesicles isolated from normal aortas using crude collagenase digestion. By contrast, cholesterol did not significantly affect ATP-promoted calcification if vesicles were isolated from atherosclerotic aortas. To determine whether high cholesterol levels in atherosclerotic vesicle preparations may have already maximized calcifying activity and therefore account for lack of the vesicle's response to the sterol, Fourier transform infrared spectroscopy (FT-IR) was used to compare the cholesterol contents in control and atherosclerotic vesicles. The spectral patterns revealed higher levels of cholesterol in vesicle preparations from atherosclerotic aortas than those from normal aortas. Removal of extra-vesicular cholesterol micelles from atherosclerotic vesicles by a relatively low centrifugal force sensitized the vesicles to cholesterol stimulation causing a 2-fold increase in calcifying activity. Of various oxidized forms of cholesterol tested, 7-keto and 6-keto cholesterol enhanced the activity by 2-fold. Altogether, these observations suggest that cholesterol and especially its oxidized forms may induce aortic calcification by directly enhancing the vesicle's ability to calcify.