Freeze-thaw Injury Research Articles

Cross‐disciplinary insights into the mechanisms of plant cold hardiness: From molecules to ecosystems

Cross‐disciplinary insights into the mechanisms of plant cold hardiness: From molecules to ecosystems

Transcriptome analysis of Populus × canadensis ‘Zhongliao1’ in response to low temperature stress

BackgroundLow temperatures are known to limit the growth and geographical distribution of poplars. Although some transcriptomic studies have been conducted to explore the response of poplar leaves to cold stress, only a few have comprehensively analyzed the effects of low temperature on the transcriptome of poplars and identified genes related to cold stress response and repair of freeze–thaw injury.ResultsWe exposed the Euramerican poplar Zhongliao1 to low temperatures; after stems were exposed to − 40℃, 4℃, and 20℃, the mixture of phloem and cambium was collected for transcriptome sequencing and bioinformatics analysis. A total of 29,060 genes were detected, including 28,739 known genes and 321 novel genes. Several differentially expressed genes (n = 36) were found to be involved in the Ca2+ signaling pathway, starch–sucrose metabolism pathway, abscisic acid signaling pathway, and DNA repair. They were functionally annotated; glucan endo-1,3-beta-glucosidase and UDP-glucuronosyltransferase genes, for instance, showed a close relationship with cold resistance. The expression of 11 differentially expressed genes was verified by qRT-PCR; RNA-Seq and qRT-PCR data were found to be consistent, which validated the robustness of our RNA-Seq findings. Finally, multiple sequence alignment and evolutionary analysis were performed, the results of which suggested a close association between several novel genes and cold resistance in Zhongliao1.ConclusionWe believe that the cold resistance and freeze–thaw injury repair genes identified in this study are of great significance for cold tolerance breeding.

An ice-binding protein from an Arctic population of American dunegrass, Leymus mollis.

Several cold-hardy grasses have been shown to have ice-binding proteins (IBPs) that protect against freeze-thaw injury. Here, we looked for IBP activity in an Alaskan coastal grass, Leymus mollis (Pooidae), that had not previously been examined. Rhizome tissue had strong ice-structuring and ice recrystallization inhibiting (IRI) activities, indicating the probable presence of IBPs. The gene sequence of an IBP was obtained. The sequence encoded a 118-amino acid IRI domain composed of eight repeats and that was 80% identical to the IRI domain of the IBP of perennial ryegrass Lolium perenne. The predicted 3D structure of the IRI domain had eight beta-roll coils like those in L. perenne IBP.

An ice-binding protein from an Arctic grass, Leymus mollis.

Several cold-hardy grasses have been shown to have ice-binding proteins (IBPs) that protect against freeze-thaw injury. Here, we looked for IBP activity in an Alaskan coastal grass that had not previously been examined, Leymus mollis (Pooidae). Rhizome tissue had strong ice-structuring and ice recrystallization inhibiting (IRI) activities, indicating the probable presence of IBPs. The gene sequence of an IBP was obtained. The sequence encoded a 118-amino acid IRI domain that contained eight repeats. A 3D structure of the IRI domain was predicted from the structure of the IRI domain of the perennial ryegrass Lolium perenne. The predicted structure appeared to have the same eight beta-roll coils found in the L. perenne IBP.

Repair of sub-lethal freezing damage in leaves of Arabidopsis thaliana

BackgroundThe detrimental effects of global climate change direct more attention to the survival and productivity of plants during periods of highly fluctuating temperatures. In particular in temperate climates in spring, temperatures can vary between above-zero and freezing temperatures, even during a single day. Freeze-thaw cycles cause cell membrane lesions that can lead to tissue damage and plant death. Whereas the processes of cold acclimation and freeze-thaw injury are well documented, not much is known about the recovery of plants after a freezing event. We therefore addressed the following questions: i. how does the severity of freezing damage influence repair; ii. how are respiration and content of selected metabolites influenced during the repair process; and iii. how do transcript levels of selected genes respond during repair?ResultsWe have investigated the recovery from freezing to sub-lethal temperatures in leaves of non-acclimated and cold acclimated Arabidopsis thaliana plants over a period of 6 days. Fast membrane repair and recovery of photosynthesis were observed 1 day after recovery (1D-REC) and continued until 6D-REC. A substantial increase in respiration accompanied the repair process. In parallel, concentrations of sugars and proline, acting as compatible solutes during freezing, remained unchanged or declined, implicating these compounds as carbon and nitrogen sources during recovery. Similarly, cold-responsive genes were mainly down regulated during recovery of cold acclimated leaves. In contrast, genes involved in cell wall remodeling and ROS scavenging were induced during recovery. Interestingly, also the expression of genes encoding regulatory proteins, such as 14–3-3 proteins, was increased suggesting their role as regulators of repair processes.ConclusionsRecovery from sub-lethal freezing comprised membrane repair, restored photosynthesis and increased respiration rates. The process was accompanied by transcriptional changes including genes encoding regulatory proteins redirecting the previous cold response to repair processes, e.g. to cell wall remodeling, maintenance of the cellular proteome and to ROS scavenging. Understanding of processes involved in repair of freeze-thaw injury increases our knowledge on plant survival in changing climates with highly fluctuating temperatures.

Extracellular mitochondria released from traumatized brains induced platelet procoagulant activity.

Coagulopathy often develops soon after acute traumatic brain injury and its cause remains poorly understood. We have shown that injured brains release cellular microvesicles that disrupt the endothelial barrier and induce consumptive coagulopathy. Morphologically intact extracellular mitochondria accounted for 55.2% of these microvesicles, leading to the hypothesis that these extracellular mitochondria are metabolically active and serve as a source of oxidative stress that activates platelets and renders them procoagulant. In testing this hypothesis experimentally, we found that the extracellular mitochondria purified from brain trauma mice and those released from brains subjected to freeze-thaw injury remained metabolically active and produced reactive oxygen species. These extracellular mitochondria bound platelets through the phospholipid-CD36 interaction and induced α-granule secretion, microvesiculation, and procoagulant activity in an oxidant-dependent manner, but failed to induce aggregation. These results define an extracellular mitochondria-induced and redox-dependent intermediate phenotype of platelets that contribute to the pathogenesis of traumatic brain injury-induced coagulopathy and inflammation.

Effect of short-term versus prolonged freezing on freeze–thaw injury and post-thaw recovery in spinach: Importance in laboratory freeze–thaw protocols

‘Duration of freezing’ at a given treatment temperature can significantly influence the laboratory evaluation of plant freeze–thaw stress tolerance (FTST) by potentially affecting the extent of freeze–thaw injury and, therefore, also the ability of post-thaw recovery. The objective of this study was to compare the extent of freeze–thaw injury, estimated by various physiological indicators [electrolyte leakage, photosystem II efficiency (Fv/Fm), accumulation of reactive oxygen species (O2− and H2O2), and malondialdehyde (MDA) accumulation], in the leaves of spinach (Spinacia oleracea L.) when exposed for various durations (0.5, 3.0, 5.5, and 10.5h) of freezing. Leaf LT50 was first determined (approximately −5.5°C) and two sub-lethal temperatures, −4.0 and −4.5°C, were selected as the freezing stress. The ability of post-thaw recovery was compared in leaves stressed for shorter versus longer durations at −4.5°C. All measurements indicated that injury progressively increased with longer freezing durations. While no injury was observed (based on the electrolyte leakage) at −4°C for 0.5h, tissues were ∼50% injured by the same temperature when frozen for 10.5h. Moreover, −4.5°C stress appears as sub-lethal at 3h of freezing (∼30% injury), but, injury doubled (and exceeded the LT50 threshold) after additional 2.5h at this temperature. Finally, leaves were identically injured to a recoverable point (∼30% injury) when frozen for 3h at either −4°C or −4.5°C while 5.5h of freezing at −4.5°C caused irreversible injury but no change for −4.0°C. Our results and corresponding discussion highlight the importance of considering ‘duration of freezing’ as one of the factors for objective interpretation of FTST/LT50 data derived from artificial freeze–thaw tests.

Understanding the cellular mechanism of recovery from freeze–thaw injury in spinach: possible role of aquaporins, heat shock proteins, dehydrin and antioxidant system

Recovery from reversible freeze-thaw injury in plants is a critical component of ultimate frost survival. However, little is known about this aspect at the cellular level. To explore possible cellular mechanism(s) for post-thaw recovery (REC), we used Spinacia oleracea L. cv. Bloomsdale leaves to first determine the reversible freeze-thaw injury point. Freeze (-4.5°C)-thaw-injured tissues (32% injury vs <3% in unfrozen control) fully recovered during post-thaw, as assessed by an ion leakage-based method. Our data indicate that photosystem II efficiency (Fv/Fm) was compromised in injured tissues but recovered during post-thaw. Similarly, the reactive oxygen species (O2 (•-) and H2 O2 ) accumulated in injured tissues but dissipated during recovery, paralleled by the repression and restoration, respectively, of activities of antioxidant enzymes, superoxide dismutase (SOD) (EC. 1.14.1.1), and catalase (CAT) (EC.1.11.1.6) and ascorbate peroxidase (APX) (EC.1.11.1.11). Restoration of CAT and APX activities during recovery was slower than SOD, concomitant with a slower depletion of H2 O2 compared to O2 (•-) . A hypothesis was also tested that the REC is accompanied by changes in the expression of water channels [aquaporines (AQPs)] likely needed for re-absorption of thawed extracellular water. Indeed, the expression of two spinach AQPs, SoPIP2;1 and SoδTIP, was downregulated in injured tissues and restored during recovery. Additionally, a notion that molecular chaperones [heat shock protein of 70 kDa (HSP70s)] and putative membrane stabilizers [dehydrins (DHNs)] are recruited during recovery to restore cellular homeostasis was also tested. We noted that, after an initial repression in injured tissues, the expression of three HSP70s (cytosolic, endoplasmic reticulum and mitochondrial) and a spinach DHN (CAP85) was significantly restored during the REC.

Protective Role of (RS)-glucoraphanin Bioactivated with Myrosinase in an Experimental Model of Multiple Sclerosis

The discovery of new natural compounds with pharmacological properties is a field of interest widely growing. Recent literature shows that Brassica vegetables (Cruciferae) possess therapeutic effects particularly ascribed due to their content in glucosinolates, which upon myrosinase hydrolysis release the corresponding isothiocyanates. This study examines the potential neuroprotective and immunomodulatory effects of (RS )-glucoraphanin from Tuscan black kale (Brassica oleracea L. var. acephala sabellica) bioactivated with myrosinase (bioactive RS -GRA) (10 mg/kg/day intraperitoneally), in an experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. EAE was induced by immunization with myelin oligodendroglial glycoprotein peptide (MOG35-55 ) in mice. After immunization, mice were observed daily for signs of EAE and weight loss. Clinical score was evaluated using a standardized scoring system. By Western blot analysis of spinal cord tissues, we have demonstrated that treatment with bioactive RS -GRA significantly decreased nuclear factor (NF)-kB translocation, pro-inflammatory cytokine production such as interleukin-1β (IL-1β), and apoptosis (Bax and caspase 3 expression). Our results clearly demonstrate that bioactive RS -GRA treatment may represent a useful therapeutic perspective in the treatment of this disease.

Proteomic changes associated with freeze‐thaw injury and post‐thaw recovery in onion (Allium cepa L.) scales

The ability of plants to recover from freeze-thaw injury is a critical component of freeze-thaw stress tolerance. To investigate the molecular basis of freeze-thaw recovery, here we compared the proteomes of onion scales from unfrozen control (UFC), freeze-thaw injured (INJ), and post-thaw recovered (REC) treatments. Injury-related proteins (IRPs) and recovery-related proteins (RRPs) were differentiated according to their accumulation patterns. Many IRPs decreased right after thaw without any significant re-accumulation during post-thaw recovery, while others were exclusively induced in INJ tissues. Most IRPs are antioxidants, stress proteins, molecular chaperones, those induced by physical injury or proteins involved in energy metabolism. Taken together, these observations suggest that while freeze-thaw compromises the constitutive stress protection and energy supply in onion scales, it might also recruit 'first-responders' (IRPs that were induced) to mitigate such injury. RRPs, on the other hand, are involved in the injury-repair program during post-thaw environment conducive for recovery. Some RRPs were restored in REC tissues after their first reduction right after thaw, while others exhibit higher abundance than their 'constitutive' levels. RRPs might facilitate new cellular homeostasis, potentially by re-establishing ion homeostasis and proteostasis, cell-wall remodelling, reactive oxygen species (ROS) scavenging, defence against possible post-thaw infection, and regulating the energy budget to sustain these processes.

Comparative observation of freeze–thaw‐induced damage in pig, rabbit, and human corneal stroma

Although variations exist between species with respect to outcomes after cryopreservation, little is known about the differences in the susceptibility of the corneal stroma to cryoinjury. We performed this study to investigate freeze-thaw-induced damage in keratocytes and collagen in rabbit, pig, and human corneas. Rabbit, pig, and human. We prepared 250-microm-thick anterior stroma from rabbit, pig, and human corneas after scraping off the epithelium and endothelium. Each 250-microm-thick corneal stroma without epithelium was placed in a 50-mL tube, frozen with liquid N2 for 15 min and taken out to thaw rapidly at 37 degrees C. This procedure of rapid freezing and thawing was repeated three times. Differences between the species with respect to cells and collagen structures were examined using hematoxylin-eosin (H&E) staining, terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay, and transmission electron microscopy (TEM). We orthotopically transplanted the pig and rabbit corneal transplants after the triple freeze-thaw cycle into rabbit eyes and evaluated graft survival. On gross examination, rabbit corneas became opaque after the triple freeze-thaw procedure, while pig and human corneas remained transparent. Histologically, keratocytes were apoptotic on TUNEL assay and TEM in rabbit, pig, and human corneas. Collagen fibrils were fragmented and the arrangement of collagen fibrils was severely disturbed in rabbit corneas on H&E staining and TEM; collagen was well preserved in pig and human corneas. Rabbit corneal stroma underwent autolysis after transplantation, whereas the pig corneal stroma remained clear for 1 month. Our study showed that rabbit corneal stroma was more susceptible to freeze-thaw injury than pig and human corneas.

Insufficiency of Copper Ion Homeostasis Causes Freeze-Thaw Injury of Yeast Cells as Revealed by Indirect Gene Expression Analysis

Saccharomyces cerevisiae is exposed to freeze-thaw stress in commercial processes, including frozen dough baking. Cell viability and fermentation activity after a freeze-thaw cycle were dramatically decreased due to freeze-thaw injury. Because this type of injury involves complex phenomena, the injury mechanisms are not fully understood. We examined freeze-thaw injury by indirect gene expression analysis during postthaw incubation after freeze-thaw treatment using DNA microarray profiling. The results showed that genes involved in the homeostasis of metal ions were frequently contained in genes that were upregulated, depending on the freezing period. We assessed the phenotype of deletion mutants of the metal ion homeostasis genes that exhibited freezing period-dependent upregulation and found that the strains with deletion of the MAC1 and CTR1 genes involved in copper ion homeostasis exhibited freeze-thaw sensitivity, suggesting that copper ion homeostasis is required for freeze-thaw tolerance. We found that supplementation with copper ions during postthaw incubation increased intracellular superoxide dismutase activity and intracellular levels of reactive oxygen species were decreased. Moreover, cell viability was increased by supplementation with copper ions. These results suggest that insufficiency of copper ion homeostasis may be one of the causes of freeze-thaw injury.

Adjuvant Approaches to Enhance Cryosurgery

Molecular adjuvants can be used to enhance the natural destructive mechanisms of freezing within tissue. This review discusses their use in the growing field of combinatorial or adjuvant enhanced cryosurgery for a variety of disease conditions. Two important motivations for adjuvant use are: (1) increased control of the local disease in the area of freezing (i.e., reduced local recurrence of disease) and (2) reduced complications due to over-freezing into adjacent tissues (i.e., reduced normal functional tissue destruction near the treatment site). This review starts with a brief overview of cryosurgical technology including probes and cryogens and major mechanisms of cellular, vascular injury and possible immunological effects due to freeze-thaw treatment in vivo. The review then focuses on adjuvants to each of these mechanisms that make the tissue more sensitive to freeze-thaw injury. Four broad classes of adjuvants are discussed including: thermophysical agents (eutectic forming salts and amino acids), chemotherapuetics, vascular agents and immunomodulators. The key issues of selection, timing, dose and delivery of these adjuvants are then elaborated. Finally, work with a particularly promising vascular adjuvant, TNF-alpha, that shows the ability to destroy all cancer within a cryosurgical iceball is highlighted.

The salting-in hypothesis of post-hypertonic lysis

The phenomenon of slow cooling cryoinjury has remained one of the primary areas of research in cryobiology since the early 1950s when it was first investigated thoroughly. Lovelock demonstrated that cell death from freezing and thawing was mainly due to exposure to hypertonic solutions and the subsequent dilution back to isotonic conditions. He suggested that the cell became permeable to sodium in hypertonic conditions leading to a loading of sodium during the hypertonic exposure, which caused the cell to swell past its elastic limit during resuspension in isotonic media (post-hypertonic lysis). This idea was pursued by Zade-Oppen, Farrant, and others who were able to show that the membrane became leaky to cations in hypertonic media but they could not provide any mechanism that would cause the cell to load up with sodium (other than an exchange of extracellular sodium for intracellular potassium, leaving the cell with the same cation concentration that it started out with). In the absence of such a mechanism, predicting post-hypertonic lysis from osmotic simulations cannot be done. A simplified model is proposed in which the intracellular milieu is composed of both KCl and a proteinaceous component that normally forms many salt bridges between amino acids with fixed charges. When the intracellular salt concentration increases, the proteins are “salted in” to solution (salt bridges are replaced with ionic interactions) thereby decreasing the intracellular cation concentration. Cation channels in the plasma membrane are opened by exposure to a high salt concentration (either inside or outside the membrane) allowing extracellular sodium to take the place of the intracellular potassium that is interacting with anionic groups on the proteins. Dilution of the external medium (which also occurs during melting) causes water to move into the cells, diluting the cytoplasm. The proteins are then “salted out” of solution and release the salt back to free ions in solution. The cell has an excess of intracellular ions and may swell past its elastic limit due to water influx. A simulation engine is developed based on the model and compared to results in the literature for freeze–thaw injury in human red blood cells.

Suicide through stress: A bacterial response to sub-lethal injury in the food environment

The response of bacteria to sub-lethal injury is an important aspect of food microbiology as many inimical processes to which bacteria are subjected during processing are non-lethal. For pathogens like Salmonella and Escherichia coli, the difference in injury levels of exponential phase cells compared to their stationary phase counterparts in this regard is well recognised and evident for a variety of inimical processes. The expression of a range of stress resistance genes under the control of the sigma factor RpoS provides some explanation for the greater resistance of stationary phase cells. However in 1997 the suicide response hypothesis was put forward as an explanation for the observed response of Salmonella and E. coli to sub-lethal stresses. This hypothesis arose as an explanation for the observed protection of Salmonella and E. coli strains to heat and freeze–thaw injury by the presence of a high level of competitor organisms, a protection that had been shown to be RpoS independent. The central tenet of this theory was that under sub-lethal stress bacteria produce a burst of intracellular free radicals and it is these that lead to sub-lethal injury and/or death. Exponential phase cells because of their more active metabolism are more susceptible to this effect and suffer greater damage. This paper reviews the origins of this theory, the evidence for a free radical response and explores the potential mechanisms by which competitor cells produce a protective effect.

Identification and classification of genes required for tolerance to freeze–thaw stress revealed by genome-wide screening ofSaccharomyces cerevisiaedeletion strains

Yeasts used in bread making are exposed to freeze-thaw stress during frozen-dough baking. To clarify the genes required for freeze-thaw tolerance, genome-wide screening was performed using the complete deletion strain collection of diploid Saccharomyces cerevisiae. The screening identified 58 gene deletions that conferred freeze-thaw sensitivity. These genes were then classified based on their cellular function and on the localization of their products. The results showed that the genes required for freeze-thaw tolerance were frequently involved in vacuole functions and cell wall biogenesis. The highest numbers of gene products were components of vacuolar H(+)-ATPase. Next, the cross-sensitivity of the freeze-thaw-sensitive mutants to oxidative stress and to cell wall stress was studied; both of these are environmental stresses closely related to freeze-thaw stress. The results showed that defects in the functions of vacuolar H(+)-ATPase conferred sensitivity to oxidative stress and to cell wall stress. In contrast, defects in gene products involved in cell wall assembly conferred sensitivity to cell wall stress but not to oxidative stress. Our results suggest the presence of at least two different mechanisms of freeze-thaw injury: oxidative stress generated during the freeze-thaw process, and defects in cell wall assembly.

Calcification and cellularity in human aortic heart valve tissue determine the differentiation of bone-marrow-derived cells

Human bone-marrow-derived mesenchymal stem cells (MSC) are responsible the remodeling of human tissue. However, damaged aortic valves are lack the ability to regenerate which is an active cell-mediated process. Diseased aortic valve remodeling has similarities even to bone formation. In this study, the prerequisites for cultured MSCs to undergo osteoblastic differentiation on aortic valves were explored. An ex vivo model using a human aortic valve microenvironment was developed. The expression of type I procollagen, alkaline phosphatase activity, osteocalcin secretion and osteocalcin immunostaining were studied to evaluate the induction of osteogenesis of the MSCs on noncalcified and calcified human aortic valves. Aortic valves were exposed to freeze–thaw injury to devitalize valves in order to separately study the role of valve matrix vs. endothelial cells in the explants. Thus, valves were assigned to 1 of 4 treatment groups: noncalcified uninjured valves, calcified uninjured valves, noncalcified injured and calcified injured. Finally, valves were decalcified to separately explore the effect of a calcified matrix on the osteogenesis. In this co-culture system, the noncalcified uninjured valves inhibited osteogenesis of MSCs, whereas the calcified valves promoted differentiation towards osteoblastic lineage. Devitalization of the valve matrix inflicted a significant increase in the osteogenesis of co-cultured MSCs. Calcified matrix in the valves seemed to have a role in the spontaneous osteogenesis of the MSCs. This spontaneous matrix induced differentiation of MSCs into osteoblast lineage could not be inhibited by pravastatin, indomethacin or tetracycline. In conclusion, these results suggest that interactions between MSCs and aortic valve matrix components and cells modulate MSC phenotype in this environment. Further studies are required to characterize this interesting phenomenon in greater detail.

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.

Fine mapping of Dyscalc1, the major genetic determinant of dystrophic cardiac calcification in mice

Calcification of severely dystrophic muscle is occasionally observed in targeted mouse models of muscular dystrophy and cardiomyopathy. Intracellular calcium deposition occurs in necrotic myocytes in the absence of plasma calcium and phosphate imbalances. In the heart, this recessive trait is referred to as dystrophic cardiac calcinosis (DCC). We identified previously Dyscalc1, a major genetic determinant of DCC, in a 15.2-Mbp region on proximal chromosome 7. We report now further steps toward the identification of the Dyscalc1 gene by reverse genetics. Transferring the Dyscalc1 locus from susceptible mouse strain C3H/He onto a resistant C57BL/6 strain background, we generated congenic inbred strains B6.C3-(D7Mit56-D7Mit230) and B6.C3-(D7Nds5-D7Mit230). Three days after myocardial freeze-thaw injury, both strains exhibited calcification of necrotic lesions, confirming the pathogenetic relevance of Dyscalc1. Analysis of two (129S1 x C57BL/6) x 129S1 backcrosses allowed mapping of Dyscalc1 more precisely to a region spanning 0.76 Mbp between genes Fgf21 (39.70 Mbp) and Myod1 (40.46 Mbp). This interval contains 31 known and putative genes in three large, ancestral haplotypes shared by susceptible strains C3H/He, 129S1, and DBA/2. Thus we were able to exclude previously proposed candidate genes Bax and Hrc. Instead, a potential candidate may be the gene encoding the ATP-binding cassette C6. Mutations in the orthologous human ABCC6 gene cause pseudoxanthoma elasticum, or Gronblad-Strandberg syndrome, an elastic tissue disorder with cardiovascular calcifications.

Arterial calcification in mice after freeze-thaw injury

Vascular calcification is highly correlated with atherosclerosis and cardiovascular disease and is a significant predictor of cardiovascular morbidity and mortality. Studies in mice indicate a genetic contribution to this dystrophic extra osseous calcification. We sought to elaborate a method to induce dystrophic arterial calcification in mice and further examine the pathogenetical mechanisms involved in the phenotype. We established a method of freeze-thaw injury of the infrarenal aorta producing a limited tissue necrosis and histologically investigated the occurrence of dystrophic calcification within the aortic wall 1, 3 and 7 days after injury in C57BL/6 (a mouse strain shown to be resistant to dystrophic cardiac calcification after injury) and C3H/He (susceptible to dystrophic cardiac calcification). C57BL/6 mice exhibited no dystrophic calcification at all within the vessel wall upon injury of the infrarenal aorta (0/5 mice 1 day after injury and 0/10 animals 7 days after injury). By contrast C3H/He mice displayed a remarkable extent of calcification mainly present within the media of the infrarenal aorta which was evident as early as 24 h (three out of five animals 1 day after injury) and reached its maximum extent 7 days after injury (10 out of 10 animals at the seventh postoperative day, p<0.001 compared to C57BL/6 mice). Upon immuno-histological analysis calcification was accompanied by the occurrence of certain bone-matrix associated proteins. Osteopontin and Bone Morphogenetic Protein 2/4 expression was detected co-localized with the calcified lesions. Our results demonstrate that freeze-thaw injury of the infrarenal aorta is a sufficient method to induce dystrophic arterial calcification in mice. We present evidence that the occurrence of arterial calcification in C3H/He mice seems to be actively regulated by certain bone-matrix associated proteins.