Five lncRNAs associated with the survival of hepatocellular carcinoma: a comprehensive study based on WGCNA and competing endogenous RNA network.

OBJECTIVE: The competing endogenous RNA (ceRNA) presents a comprehensive regulatory network among lncRNAs, miRNAs and mRNA. The ceRNA provides significant information in understanding the pathology of cancer. This study aimed to explore a lncRNA-associated ceRNA network for predicting the overall survival of patients with hepatocellular carcinoma (HCC). MATERIALS AND METHODS: In this study, RNA-sequencing data of HCC were downloaded from The Cancer Genomes Atlas (TCGA) database. The module-trait relationship was analyzed with Weighted gene co-expression network analysis (WGCNA). The key module associated with tumor was identified, as well as the involved lncRNAs, mRNAs and miRNAs. The preliminary ceRNA network was constructed with Cytoscape. The survival analysis was further performed to screen survival-relevant lncRNAs, mRNAs and miRNAs, and then the survival-associated ceRNA network was reconstructed. RESULTS: Eventually, 5 lncRNAs, 10 miRNAs, and 25 mRNAs were included in the reconstructed ceRNA network. CONCLUSIONS: The identified lncRNAs were promising candidate biomarkers in HCC diagnosis and therapeutics. This analysis process was effective to construct ceRNA network. The result will be conductive to explore the significant lncRNAs and regulatory mechanism.

[The influence factors of pathologic complete response of the ipsilateral supraclavicular lymph node of breast cancer after neoadjuvant chemotherapy].

Objective: To investigate the factors affecting the pathologic complete response (PCR) of the ipsilateral supraclavicular lymph node (ISLN) of breast cancer after neoadjuvant chemotherapy (NAC). Methods: A total of 178 patients with breast cancer who had primary ipsilateral supraclavicular lymph node metastasis (ISLNM), receiving NAC and subsequent ISLN dissection, were retrospectively reviewed. The single factor and multi factor analysis were carried out by the chi square test and the Logistic regression model. Results: The enrolled patients were all female, 28 to 74 years old. The rate of PCR on the ISLN was 52.2%. Single factor analysis showed that KI67 expression level (χ(2)=7.717,P=0.005), breast PCR (bPCR) (χ(2)=33.564,P<0.001), and axillary PCR (aPCR) (χ(2)=31.750, P<0.001) were associated with the ISLN PCR. Multifactor analysis showed that KI67 expression level (OR=4.096, 95%CI: 1.176-14.263, P=0.027), bPCR (OR=4.452, 95%CI: 1.894-10.461, P<0.001) and aPCR (OR=5.183, 95%CI: 1.974-13.605, P<0.001) were independent predictors of ISLN PCR. The rate of PCR on the ISLN was 90.9% in the patients with KI67>30% and simultaneous breast and axilla PCR. Conclusions: The PCR rate of the ISLN after neoadjuvant chemotherapy is higher than that of the breast and axillary PCR. The expression level of KI67, the bPCR and the aPCR are independent predictors of the PCR on the ISLN.

[Analysis of factors influencing the axillary lymph node metastasis and breast cancer-specific survival in patients with T1 breast cancer].

Objective: To investigate the predictors of axillary lymph node metastasis and the breast cancer-specific survival (BCSS) in patients with T1 breast cancer. Methods: A retrospective analysis of clinical and pathological data of 840 T1 invasive breast cancer cases between January 2009 and January 2014 in Henan Cancer Hospital was conducted.Chi square test and Logistic regression analysis were carried out to identify relevant factors of lymph node metastasis. Analysis of prognostic factors were analyzed by Log-rank test and Cox regression. Results: Among the 840 T1 breast cancer cases, positive axillary lymph nodes were found in 150 (17.9%) cases. Univariate analysis showed that tumor size, histological grade, tumor location, and HER2 status were associated with axillary lymph node status (P<0.05). Multivariate analysis showed that tumor size, histological grade, tumor location, and HER2 status were independent predictive factors of axillary lymph node metastasis (P<0.05). Log-rank test showed that tumor size, histological grade, HER2 status, partial response (PR) status and number of positive lymph nodes were important factors influencing BCSS of the patients with positive axillary lymph nodes (P<0.05). Cox analysis showed that the size of the primary tumors and the number of positive lymph nodes were independent factors affecting the BCSS of the patients(P<0.05). Conclusions: Tumor size, histological grade, tumor location and HER2 status correlated with axillary lymph nodes status of T1 breast cancer. For T1 breast cancer patients with positive axillary lymph node, more positive lymph nodes involved and smaller primary tumor correlated with worse prognosis.

[The influence of lumpectomy on the axillary lymph node status of breast cancer patients].

Objective: To investigate the influence of lumpectomy on axillary lymph node status of breast cancer patients. Methods: The clinical data of 738 invasive breast cancer patients with non-palpable axillary lymph node and sentinel lymph node (SLN) biopsy from November 2011 to August 2013 in Henan Provincial Cancer Hospital were collected and retrospectively analyzed. Among them, 136 patients underwent preoperative lumpectomy (lumpectomy group) and 602 patients underwent puncture biopsy only (biopsy group). The difference of axillary lymph node status and positive ratio of SLN detected by color Doppler ultrasound were compared between these two groups. Results: Among the 738 breast cancer patients, the axillary lymph nodes of 444 (60.2%) cases could be detected by ultrasound. Among them, 92 cases belonged to lumpectomy group, significantly less than 352 cases of biopsy group (P=0.048). Among the patients with ultrasound-visible lymph nodes, the proportion of the biggest diameter of axillary lymph node >1 cm of lumpectomy group or biopsy group was 58.7% (54/92) or 52.8% (186/352), respectively, without significant difference (P=0.316). The proportion of patients with the ratio of long diameter to short diameter <2 of lumpectomy group or biopsy group was 37.0% (34/92) or 38.6% (136/352), respectively, with marginal difference (P=0.768). The positive rate of SLN of lumpectomy group or biopsy group was 23.5% (32/136) or 26.9% (162/602), respectively, without significant difference (P=0.419). The incidence rate of the ultrasound visible axillary lymph nodes of patients whose postoperative time ≤ 7 days or > 7days was 71.1% (64/90) or 60.9% (8/46), respectively, without significant difference (P=0.227). However, the positive rate of SLN of these two groups was 28.9% (26/90) and 13.0% (6/46), respectively, with significant difference (P=0.039). The number of ultrasound visible axillary lymph nodes, the biggest diameter of axillary lymph nodes and the ratio of the long diameter to short diameter <2 were substantially correlated with the positive rate of SLN (P<0.05). Conclusions: The incidence rate of ultrasound visible axillary lymph node in the patients with lumpectomy is higher than that of patients with puncture biopsy only. The positive rate of SLN of the patients with a long postoperative time is lower than that of patients with a short postoperative time, even though the axillary lymph nodes are ultrasound visible.

[Analysis of predictive effect of Androgen receptor on the response to neoadjuvant chemotherapy in breast cancer patients].

Objective: To explore the expression of androgen receptor (AR) in the tissues as well as its association with the clinicopathological factors of primary breast cancer patients treated with neoadjuvant chemotherapy (NAC), and analyze the effect of AR in the prediction of pathologic complete response (PCR) rate. Method: A total of 668 breast cancer patients treated with NAC in Henan Cancer Hospital between March 2014 and June 2017 were retrospectively reviewed. The relationship of AR expression and clinicopathological characteristics was calculated using chi square test. Multivariate analysis using binary Logistic regression was used to analyze correlations of different factors with PCR. Result: All patients were female, with the age of 20-76 years old. AR was detected in 74.6% of tumors, and significantly correlated with hormone receptor (HR), human epidermalgrowth factor receptor-2 (HER-2), Ki-67, CK5/6, epidermal growth factor receptor (EGFR) and molecular subtypes (all P<0.05). Multivariate analysis showed that AR, HR and HER-2 were independent predictors for PCR (all P<0.05). Conclusions: The expressions of AR were more frequently in HR positive breast cancer tissues (86.7%), and lowest in triple-negative breast cancer (TNBC) group (23.2%). AR was independent predictor for PCR.

Expression of CDCA8 correlates closely with FOXM1 in breast cancer: public microarray data analysis and immunohistochemical study.

Forkhead Box M1 (FOXM1) is an oncogenic transcription factor implicated in breast cancer progression and metastasis. However, the clinical significance of FOXM1 and its associated signaling genes in human breast cancer still needed to be clarified. In this study, we first analyzed the co-expression gene pattern of FOXM1 in three breast cancer gene expression microarray datasets from the Oncomine database. Cell division cycle associated 8 (CDCA8) gene was identified to correlate closely with FOXM1. In silico analysis further indicated that CDCA8 overexpressed in breast cancer tissues compared with the normal controls is significantly associated with the triple-negative phenotype. Experimentally, we performed a immunohistochemical study to detect the expression of CDCA8 in 112 breast cancer samples, and evaluated its clinicopathological and prognostic significance. We found that CDCA8 was frequently over-expressed in breast cancer tissues, and increased expression of CDCA8 was positively associated with FOXM1 expression, triple-negative phenotype and shorter overall survival. Moreover, we also found that combination of CDCA8 and FOXM1 showed a higher hazard ratio than the individual markers. Our results suggest that FOXM1-CDCA8 signature might be involved in breast cancer progression, and serves as a potential prognostic factor and a promising therapeutical target.

New methods for time-resolved fluorescence spectroscopy data analysis based on the Laguerre expansion technique--applications in tissue diagnosis.

OBJECTIVES: A new deconvolution method for the analysis of time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) data is introduced and applied for tissue diagnosis. METHOD: The intrinsic TR-LIFS decays are expanded on a Laguerre basis, and the computed Laguerre expansion coefficients (LEC) are used to characterize the sample fluorescence emission. The method was applied for the diagnosis of atherosclerotic vulnerable plaques. RESULTS: At a first stage, using a rabbit atherosclerotic model, 73 TR-LIFS in-vivo measurements from the normal and atherosclerotic aorta segments of eight rabbits were taken. The Laguerre deconvolution technique was able to accurately deconvolve the TR-LIFS measurements. More interesting, the LEC reflected the changes in the arterial biochemical composition and provided discrimination of lesions rich in macrophages/foam-cells with high sensitivity (> 85%) and specificity (> 95%). At a second stage, 348 TR-LIFS measurements were obtained from the explanted carotid arteries of 30 patients. Lesions with significant inflammatory cells (macrophages/foam-cells and lymphocytes) were detected with high sensitivity (> 80%) and specificity (> 90%), using LEC-based classifiers. CONCLUSION: This study has demonstrated the potential of using TR-LIFS information by means of LEC for in vivo tissue diagnosis, and specifically for detecting inflammation in atherosclerotic lesions, a key marker of plaque vulnerability.

Diagnosis of vulnerable atherosclerotic plaques by time-resolved fluorescence spectroscopy and ultrasound imaging.

In this study, time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) and ultrasonography were applied to detect vulnerable (high-risk) atherosclerotic plaque. A total of 813 TR-LIFS measurements were taken from carotid plaques of 65 patients, and subsequently analyzed using the Laguerre deconvolution technique. The investigated spots were classified by histopathology as thin, fibrotic, calcified, low-inflamed, inflamed and necrotic lesions. Spectral and time-resolved parameters (normalized intensity values and Laguerre expansion coefficients) were extracted from the TR-LIFS data. Feature selection for classification was performed by either analysis of variance (ANOVA) or principal component analysis (PCA). A stepwise linear discriminant analysis algorithm was developed for detecting inflamed and necrotic lesion, representing the most vulnerable plaques. These vulnerable plaques were detected with high sensitivity (>80%) and specificity (>90%). Ultrasound (US) imaging was obtained in 4 carotid plaques in addition to TR-LIFS examination. Preliminary results indicate that US provides important structural information of the plaques that could be combined with the compositional information obtained by TR-LIFS, to obtain a more accurate diagnosis of vulnerable atherosclerotic plaque.

Application of the laguerre deconvolution method for time-resolved fluorescence spectroscopy to the characterization of atherosclerotic plaques.

This study investigates the ability of time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) to detect inflammation in atherosclerotic lesion, a key feature of plaque vulnerability. A total of 348 TR-LIFS measurements were taken from carotid plaques of 30 patients, and subsequently analyzed using the Laguerre deconvolution technique. The investigated spots were classified as Early, Fibrotic/Calcified or Inflamed lesions. A stepwise linear discriminant analysis algorithm was developed using spectral and TR features (normalized intensity values and Laguerre expansion coefficients at discrete emission wavelengths, respectively). Features from only three emission wavelengths (390, 450 and 500 nm) were used in the classifier. The Inflamed lesions were discriminated with sensitivity > 80% and specificity > 90 %, when the Laguerre expansion coefficients were included in the feature space. These results indicate that TR-LIFS information derived from the Laguerre expansion coefficients at few selected emission wavelengths can discriminate inflammation in atherosclerotic plaques. We believe that TR-LIFS derived Laguerre expansion coefficients can provide a valuable additional dimension for the detection of vulnerable plaques.

Novel methods of time-resolved fluorescence data analysis for in-vivo tissue characterization: application to atherosclerosis.

This study investigates the ability of new analytical methods of time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) data to characterize tissue in-vivo, such as the composition of atherosclerotic vulnerable plaques. A total of 73 TR-LIFS measurements were taken in-vivo from the aorta of 8 rabbits, and subsequently analyzed using the Laguerre deconvolution technique. The investigated spots were classified as normal aorta, thin or thick lesions, and lesions rich in either collagen or macrophages/foam-cells. Different linear and nonlinear classification algorithms (linear discriminant analysis, stepwise linear discriminant analysis, principal component analysis, and feedforward neural networks) were developed using spectral and TR features (ratios of intensity values and Laguerre expansion coefficients, respectively). Normal intima and thin lesions were discriminated from thick lesions (sensitivity >90%, specificity 100%) using only spectral features. However, both spectral and time-resolved features were necessary to discriminate thick lesions rich in collagen from thick lesions rich in foam cells (sensitivity >85%, specificity >93%), and thin lesions rich in foam cells from normal aorta and thin lesions rich in collagen (sensitivity >85%, specificity >94%). Based on these findings, we believe that TR-LIFS information derived from the Laguerre expansion coefficients can provide a valuable additional dimension for in-vivo tissue characterization.

Role of group II secretory phospholipase A2 in atherosclerosis: 1. Increased atherogenesis and altered lipoproteins in transgenic mice expressing group IIa phospholipase A2.

Some observations have suggested that the extracellular group IIa phospholipase A2 (sPLA2), previously implicated in chronic inflammatory conditions such as arthritis, may contribute to atherosclerosis. We have examined this hypothesis by studying transgenic mice expressing the human enzyme. Compared with nontransgenic littermates, the transgenic mice exhibited dramatically increased atherosclerotic lesions when maintained on a high-fat, high-cholesterol diet. Surprisingly, the transgenic mice also exhibited significant atherosclerotic lesions when maintained on a low-fat chow diet. Immunohistochemical staining indicated that sPLA2 was present in the atherosclerotic lesions of the transgenic mice. On both chow and atherogenic diets, the transgenic mice exhibited decreased levels of HDLs and slightly increased levels of LDLs compared with nontransgenic littermates. These data indicate that group IIa sPLA2 may promote atherogenesis, in part, through its effects on lipoprotein levels. These data also provide a possible mechanism for the observation that there is an increased incidence of coronary artery disease in many chronic inflammatory diseases.

Role of group II secretory phospholipase A2 in atherosclerosis: 2. Potential involvement of biologically active oxidized phospholipids.

Secretory nonpancreatic phospholipase A2 (group II sPLA2) is induced in inflammation and present in atherosclerotic lesions. In an accompanying publication we demonstrate that transgenic mice expressing group II sPLA2 developed severe atherosclerosis. The current study was undertaken to determine whether 1 mechanism by which group II sPLA2 might contribute to the progression of inflammation and atherosclerosis is by increasing the formation of biologically active oxidized phospholipids. In vivo measurements of bioactive lipids were performed, and in vitro studies tested the hypothesis that sPLA2 can increase the accumulation of bioactive phospholipids. We have shown previously that 3 oxidized phospholipids derived from the oxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC) stimulated endothelial cells to bind monocytes, a process that is known to be an important step in atherogenesis. We now show that these 3 biologically active phospholipids are significantly increased in livers of sPLA2 transgenic mice fed a high-fat diet as compared with nontransgenic littermates. We present in vitro evidence for several mechanisms by which these phospholipids may be increased in sPLA2 transgenics. These studies demonstrated that polyunsaturated free fatty acids, which are liberated by sPLA2, increased the formation of bioactive phospholipids in LDL, resulting in increased ability to stimulate monocyte-endothelial interactions. Moreover, sPLA2-treated LDL was oxidized by cocultures of human aortic endothelial cells and smooth muscle cells more efficiently than untreated LDL. Analysis by electrospray ionization-mass spectrometry revealed that the bioactive phospholipids, compared with unoxidized PAPC, were less susceptible to hydrolysis by human recombinant group II sPLA2. In addition, HDL from the transgenic mice and human HDL treated with recombinant sPLA2 in vitro failed, in the coculture system, to protect against the formation of biologically active phospholipids in LDL. This lack of protection may in part relate to the decreased levels of paraoxonase seen in the HDL isolated from the transgenic animals. Taken together, these studies show that levels of biologically active oxidized phospholipids are increased in sPLA2 transgenic mice; they also suggest that this increase may be mediated by effects of sPLA2 on both LDL and HDL.

Ultrastructure of early lipid accumulation in ApoE-deficient mice.

Apolipoprotein (apo) E-deficient mice develop severe hypercholesterolemia and have lesions that progress from fatty streaks to fibrous plaques distributed in lesion-prone areas throughout the aorta. Lesions develop in apoE-deficient mice on a regular chow diet and will occur faster on a diet higher in cholesterol. Examination of the aortas from these mice on a chow diet by high-resolution, freeze-etch electron microscopy demonstrated lipid retention in the intima by 3 weeks of age. Lipid was retained in the matrix as individual particles between 33 and 48 nm in diameter, aligned along the collagen fibrils and in aggregates consisting of lipid particles with average diameters of 33 and 68 nm. Larger particles seemed to have formed from fusion of smaller particles. Lipid retention was more widespread in 5- and 9-week-old mice. Monocyte attachment to endothelial cells was observed by electron microscopy at 5 weeks of age. The appearance of the intimal lipid was similar to that previously described in rabbit models and suggests that lipid interaction with matrix filaments and subsequent aggregation of lipid particles are critical first steps in the process of foam cell formation.

Genetic determinants of autoimmune disease and coronary vasculitis in the MRL-lpr/lpr mouse model of systemic lupus erythematosus.

MRL-lpr/lpr (MRL/lpr) mice are a model of human autoimmune disease. They exhibit a number of characteristics of systemic lupus erythematosus, including anti-DNA Abs, anti-cardiolipin Abs, immune complex-mediated vasculitis, lymphadenopathy, and severe glomerulonephritis. Although the autoimmune disorder is mediated primarily by mutation of the Fas gene (lpr), which interferes with lymphocyte apoptosis, MRL/lpr mice also have other predisposing genetic factors. In an effort to identify these additional factors, we have applied quantitative trait locus (QTL) mapping using an intercross between MRL/lpr mice and the nonautoimmune inbred strain BALB/cJ. A complete linkage map spanning the entire genome was constructed for 189 intercross progeny, and genetic loci contributing to features of the autoimmunity were identified using statistical analytic procedures. As expected, the primary genetic determinant of autoimmune disease in this cross was the Fas gene on mouse chromosome 19, exhibiting a lod score of 60. In addition, two novel loci, one on chromosome 2 (lod score, 4.3) and one on chromosome 11 (lod score, 3.1), were found to contribute to levels of anti-DNA Abs. Interestingly, the chromosome 19 and chromosome 11 QTLs, but not the chromosome 2 QTL, also exhibited associations with anti-cardiolipin Abs (lod scores, 38.4 and 2.6). We further examined the effects of these QTLs on the development of coronary vasculitis in the F2 mice. Our results indicate that the QTLs on chromosomes 11 and 19 also control the development of vasculitis, demonstrating common genetic determinants of autoantibody levels and vasculitis.

Heterozygous osteopetrotic (op) mutation reduces atherosclerosis in LDL receptor- deficient mice.

Previous studies of osteopetrotic (op) mice lacking macrophage colony-stimulating factor (M-CSF) have revealed an inhibition of atherosclerosis development in the apolipoprotein E (apo E)-deficient model and in a diet-induced model. Using LDL receptor-deficient mice, we now show that atheroma development depends on M-CSF concentration, as not only did homozygous osteopetrotic (op/op) mice have dramatically reduced lesions (approximately 0.3% of control lesion size) but heterozygous (op/+) mice had lesions < 1% of controls. Mice heterozygous for the op mutation (op/+) had plasma levels of M-CSF about half those in controls (+/+). The finding that an approximately 2-fold reduction in M-CSF expression reduced lesion size approximately 100-fold suggests the requirement for a threshold level of M-CSF. The effect of M-CSF on atherosclerosis did not appear to be mediated either by changes in plasma lipoprotein levels or alterations in the number of circulating monocytes, since both op/op and op/+ mice exhibited higher levels of atherogenic lipoprotein particles and (op/+) mice showed a near normal number of circulating monocytes. LDL receptor-null littermates of genotypes from op/op, op/+, to +/+ showed monocyte differentials of approximately 4.5, 8, and 10%, respectively. Taken together, these results suggest that the effects of M-CSF on atherogenesis may not be mediated by expression of M-CSF systemically or by modulation of the number of circulating monocytes. These studies support the conclusion that M-CSF participates critically in fatty streak formation and progression to a complex fibrous lesion.

Role of macrophage colony-stimulating factor in atherosclerosis: studies of osteopetrotic mice.

Previous in vitro and in vivo studies have suggested that macrophage colony-stimulating factor (M-CSF) plays a role in atherogenesis. To examine this hypothesis, we have studied atherogenesis in osteopetrotic (op/op) mice, which lack M-CSF due to a structural gene mutation. Atherogenesis was induced either by feeding the mice a high fat, high cholesterol diet or by crossing op mice with apolipoprotein E (apo E) knockout mice to generate mice lacking both M-CSF and apo E. In both the dietary and apo E knockout models, M-CSF deficiency resulted in significantly reduced atherogenesis. For example, in the apo E knockout model, homozygosity for the op mutation totally abolished aortic atherogenesis in male mice and reduced the size of the lesions approximately 97% in female mice. Mice heterozygous for the op mutation also exhibited a significant decrease in lesion size. Among apo E knockout mice, the frequency of atherosclerosis in aortic arch was 0/6 (op/op), 1/15 (op/+), and 12/16 (+/+). The effect of the M-CSF on atherosclerosis did not appear to be mediated by changes in plasma lipoproteins, as the op mice exhibited higher levels of atherogenic lipoprotein particles. The effects of the op mutation on atherogenesis may have resulted from decreased circulating monocytes, reduced tissue macrophages, or diminished arterial M-CSF.

Reduced aortic lesions and elevated high density lipoprotein levels in transgenic mice overexpressing mouse apolipoprotein A-IV.

Transgenic mouse lines carrying several copies of the mouse apo A-IV gene were produced. Lipoprotein composition and function, and aortic lesion development were examined. Apo A-IV levels in the plasma of transgenic mice were elevated threefold compared with nontransgenic littermates on a chow diet, and sixfold in mice fed an atherogenic diet. Plasma concentrations of total cholesterol, HDL cholesterol, triglycerides, and free fatty acids were similar in transgenic and control mice fed a chow diet. However, with the atherogenic diet, male transgenic mice exhibited significantly higher levels of plasma triglycerides (P < 0.05), total cholesterol (P < 0.01), HDL cholesterol (P < 0.0001), and free fatty acids (P < 0.05), and lower levels of unesterified cholesterol (P < 0.05), than nontransgenic littermates. Expression of the apo A-IV transgene had a protective effect against the formation of diet-induced aortic lesions, with transgenics exhibiting lesion scores of approximately 30% those seen in control mice. HDL-sized lipoproteins isolated from transgenic mice fed the atherogenic diet promoted cholesterol efflux from cholesterol-loaded human monocytes more efficiently than comparable lipoproteins from nontransgenic counterparts. Plasma from transgenics also exhibited higher endogenous cholesterol esterification rates. Taken together, these results suggest that apo A-IV levels influence the metabolism and antiatherogenic properties of HDL.

A locus on chromosome 7 determines myocardial cell necrosis and calcification (dystrophic cardiac calcinosis) in mice.

Dystrophic cardiac calcinosis, an age-related cardiomyopathy that occurs among certain inbred strains of mice, involves myocardial injury, necrosis, and calcification. Using a complete linkage map approach and quantitative trait locus analysis, we sought to identify genetic loci determining dystrophic cardiac calcinosis in an F2 intercross of resistant C57BL/6J and susceptible C3H/HeJ inbred strains. We identified a single major locus, designated Dyscalc, located on proximal chromosome 7 in a region syntenic with human chromosomes 19q13 and 11p15. The statistical significance of Dyscalc (logarithm of odds score 14.6) was tested by analysis of permuted trait data. Analysis of BxH recombinant inbred strains confirmed the mapping position. The inheritance pattern indicated that this locus influences susceptibility of cells both to enter necrosis and to subsequently undergo calcification.

Genetic determination of cartilaginous metaplasia in mouse aorta.

Calcification frequently occurs in atherosclerotic plaques in humans, but the cellular and genetic factors contributing to this pathological trait are unknown. We previously reported that the arterial calcification among inbred strains is genetically determined, and we now report that cartilaginous metaplasia, associated with the presence of arterial chondrocytes that express type II collagen, may underlie this calcification. Both uncalcified and calcified cartilaginous metaplasia were often colocalized with aortic atheromatous lesions and calcification, and clear genetic differences were observed in the occurrence of aortic cartilaginous metaplasia among inbred strains. Analysis of a genetic cross between strains C57BL/6J (exhibiting aortic cartilaginous metaplasia) and C3H/HeJ (no aortic cartilaginous metaplasia) revealed a recessive inheritance pattern; thus, F1 mice were entirely devoid of cartilaginous metaplasia, in common with the C3H/HeJ parental strain. Analyses of an F2 cross and a set of recombinant inbred strains derived from parental strains C57BL/6J and C3H/HeJ were consistent with a major gene effect exhibiting incomplete penetrance. The occurrence of aortic calcification was correlated with the occurrence of cartilaginous metaplasia in these genetic crosses, suggesting a link between the traits. Finally, we observed widespread calcified cartilaginous metaplasia within spontaneous atherosclerotic lesions in mice targeted for a null mutation in the apoE gene, suggesting that cartilaginous metaplasia is a potential pathway for artery wall calcification associated with the atherosclerotic plaque.