Myeloid CD34+CD13+ precursor cells transdifferentiate into chondrocyte-like cells in atherosclerotic intimal calcification.

Chondrogenic differentiation is pivotal in the active regulation of artery calcification. We investigated the cellular origin of chondrocyte-like cells in atherosclerotic intimal calcification of C57BL/6 LDLr(-/-) mice using bone marrow transplantation to trace ROSA26-LacZ-labeled cells. Immunohistochemical costaining of collagen type II with LacZ and leukocyte defining surface antigens was performed and analyzed by high-resolution confocal microscopy. Chondrocyte-like cells were detected in medium and advanced atherosclerotic plaques accounting for 7.1 +/- 1.6% and 14.1 +/- 1.7% of the total plaque cellularity, respectively. Chimera analysis exhibited a mean of 89.8% LacZ(+) cells in peripheral blood and collagen type II costaining with LcZ revealed an average 88.8 +/- 7.6% cytoplasmatic LacZ(+) evidence within the chondrocyte-like cells. To examine whether hematopoietic stem cells contribute to the phenotype, stem cell marker CD34 and myeloid progenitor-associated antigen CD13 were analyzed. CD34(+) was detectable in 86.9 +/- 8.1% and CD13(+) evidence in 54.2 +/- 7.6% of chondrocyte-like cells, attributable most likely because of loss of surface markers during transdifferentiation. Chondrocyte differentiation factor Sox-9 was detected in association with chondrocyte-like cells, whereas Sm22alpha, a marker for smooth muscle cells, could not be demonstrated. The results show that the majority of chondrocyte-like cells were of bone marrow origin, whereas CD34(+)/CD13(+) myeloid precursors appeared to infiltrate the plaque actively and transdifferentiated into chondrocytes-like cells in the progression of atherosclerosis.

An alternative splice variant in Abcc6, the gene causing dystrophic calcification, leads to protein deficiency in C3H/He mice.

Dystrophic cardiac calcification (DCC) is an autosomal recessive trait characterized by calcium phosphate deposits in myocardial tissue. The Abcc6 gene locus was recently found to mediate DCC; however, at the molecular level the causative variants remain to be determined. Examining the sequences of Abcc6 cDNA in DCC-resistant C57BL/6 and DCC-susceptible C3H/He mice, we identified a missense mutation (Cys to Thr at codon 619, rs32756904) at the 3'-border of exon 14 that creates an additional donor splice site (GT). Accordingly, an alternative transcript variant was detected, lacking the last 5 bp of exon 14 (-AGG(C/T)GCTgtga-) in DCC-susceptible C3H/He mice that carry the Thr allele. The 5-bp deletion was found to result in premature termination at codon 684, in turn leading to protein deficiency in DCC-susceptible mouse tissue as well as in cells transfected with Abcc6 cDNA lacking the last 5 bp of exon 14. All mouse strains that were found to carry the Thr allele, including C3H/He, DBA/2J, and 129S1/SvJ, were also found to be positive for DCC. In summary, we identified a splice variant leading to a 5-bp deletion in the Abcc6 transcript that gives rise to protein deficiency both in vivo and in vitro. The fact that all mouse strains that carry the deletion also develop dystrophic calcifications further suggests that the underlying splice variant affects the biological function of MRP6 protein and is a cause of DCC in mice.

Ultrafine mapping of Dyscalc1 to an 80-kb chromosomal segment on chromosome 7 in mice susceptible for dystrophic calcification.

In mice, dystrophic cardiovascular calcification (DCC) is controlled by a major locus on proximal mouse chromosome 7 named Dyscalc1. Here we present a strategy that combines in silico analysis, expression analysis, and extensive sequencing for ultrafine mapping of the Dyscalc1 locus. We subjected 15 laboratory mouse strains to freeze-thaw injury of the heart, and association with respective genotypes allowed condensation of the Dyscalc1 locus to 1 Mb. Within this region, 51 known and predicted genes were studied in DCC-susceptible C3H/He and DCC-resistant C57BL/6 mice with respect to mRNA expression in response to injury. Five genes displayed differential expression. Genotyping of seven novel single nucleotide polymorphisms (SNPs) within these genes revealed an 80-Kb region in NZB mice that were found positive for calcification though carrying otherwise alleles from DCC-resistant mice. This microheterogeneity in NZB mice was evolutionary conserved in all DCC-susceptible mouse strains and contains the genes EMP-3, BC013491, and Abcc6 (partially). The flanking SNPs are rs3703247 and NT_039420.5_2757991. mRNA levels of EMP-3 were found to be upregulated in response to injury in both C57BL/6 and C3H/He mice. Sequencing of EMP-3 revealed an SNP leading to an amino acid substitution (p.T153I) that was found in all mouse strains susceptible for DCC but not in resistant strains such as C57BL/6 mice. Thus, the p.T153I changes might affect the biological function of EMP-3 gene product after injury. Using this combined approach, we ultrafine-mapped the Dyscalc1 locus to an 80-Kb region and identified EMP-3 as a new candidate gene for DCC.

The effects of thermochemotherapy using cyclophosphamide plus hyperthermia on the malignant pleural mesothelioma in vivo.

The human malignant pleural mesothelioma is related to the use of asbestos in the majority of cases. Though the use of asbestos has been prohibited since the 1990s, the incidence of pleural mesothelioma is still increasing because of a latency period of at least 20 years. This study investigated the benefit of single therapy with cyclophosphamide or hyperthermia or the combination of both on cells of a human pleural mesothelioma cell line, xenotransplanted subcutaneously in the paw of mice. A CONTROL group received the same volume of physiological saline. The oxygenation of tumours was measured, tumour growth was followed over 3 weeks, immunohistochemical studies and a light and electron microscopic evaluation were performed. Chemotherapy or hyperthermia alone was only temporarily effective. The greatest benefit was achieved using combined thermochemotherapy consisting of cyclophosphamide plus hyperthermia: 50% of this group had partial remissions, and 67% responded to this therapy. After 3 weeks tumours grew again. Superior effects could be achieved by performing additional cycles of chemotherapy or adding another drug or radiation for instance. This study shows promising results in the treatment of malignant pleural mesothelioma.

Periosteum stimulates subchondral bone densification in autologous chondrocyte transplantation in a sheep model.

In this sheep study, we have tested the hypothesis that an osteogenic response is triggered in the subchondral bone by periosteum implanted in full thickness cartilage defects and can be prevented by replacing the periosteum by a cell-free collagen type I/III membrane. Two 7-mm diameter osteochondral defects were made in the trochlea groove and in the medial femoral condyle of one of the knees in each of 15 adult sheep. The animals were divided into three groups (n=5): a control group with untreated cartilage defects, a group treated with autologous chondrocyte transplantation (ACT) and periosteum, and a group treated with ACT in combination with a collagen I/III membrane cover. Histological examination was performed 1 year later. The optical density of the subchondral bone in the histological sections was measured with digital imaging software. There was a dramatic, statistically significant (P<0.0001; power=1) increase in bone density of 45%-70% under defects that were treated with the periosteal cover, compared with the collagen membrane and control groups, which displayed the same bone density. There was no difference in the cartilaginous reparative tissue in the defects in the three groups. Periosteum thus stimulates the remodelling process in subchondral bone. Stiffening of the subchondral bone can lead to degeneration of the overlying reparative cartilaginous tissue because of an increase in the mechanical stress in the tissue. These findings warrant evaluation of subchondral bone changes in patients treated by ACT and the correlation of these changes with clinical outcome.

Analyzing the migration of labeled T cells in vivo: an essential approach with challenging features.

T cells are involved in the pathogenesis of many diseases. To exert a pathological effect, T cells enter the tissues. We show that the determination of their entry site requires isolation of the respective T cell population, injection into genetically un-manipulated animals, and identification of the cells in vivo at various time points after injection. We indicate variables influencing in vivo migration experiments artificially, and outline how resulting problems can be either avoided or taken into account. Reviewing experiments performed according to the outlined criteria reveals two types of migration patterns for T cell subsets in vivo: 1). Naïve and memory T cells enter lymphoid and non-lymphoid organs in comparable numbers, but selectively accumulate in lymphoid tissues over time, 2). Effector T cells, too, enter lymphoid and non-lymphoid organs in comparable numbers. However, most of them die within 24 hours. Depending on the presence of cytokines, chemokines and extracellular matrix compounds they are able to survive, thereby preferentially accumulating in their target tissues. This information might help to understand the role of migration in the pathogenesis of T cell mediated diseases.