Mouse Model Of Pseudoxanthoma Elasticum Research Articles

Novel Treatments for PXE: Targeting the Systemic and Local Drivers of Ectopic Calcification.

Pseudoxanthoma elasticum (PXE) is a heritable multisystem ectopic calcification disorder. The gene responsible for PXE, ABCC6, encodes ABCC6, a hepatic efflux transporter regulating extracellular inorganic pyrophosphate (PPi), a potent endogenous calcification inhibitor. Recent studies demonstrated that in addition to the deficiency of plasma PPi, the activated DDR/PARP signaling in calcified tissues provides an additional possible mechanism of ectopic calcification in PXE. This study examined the effects of etidronate (ETD), a stable PPi analog, and its combination with minocycline (Mino), a potent inhibitor of DDR/PARP, on ectopic calcification in an Abcc6-/- mouse model of PXE. Abcc6-/- mice, at 4 weeks of age, before the development of ectopic calcification, were treated with ETD, Mino, or both for 18 weeks. Micro-computed tomography, histopathologic examination, and quantification of the calcium content in Abcc6-/- mice treated with both ETD and Mino revealed further reduced calcification than either treatment alone. The effects were associated with reduced serum alkaline phosphatase activity without changes in plasma PPi concentrations. These results suggest that ETD and Mino combination therapy might provide an effective therapeutic approach for PXE, a currently intractable disease.

INZ-701, a recombinant ENPP1 enzyme, prevents ectopic calcification in an Abcc6-/- mouse model of pseudoxanthoma elasticum.

Pseudoxanthoma elasticum (PXE), a heritable multisystem ectopic calcification disorder, is predominantly caused by inactivating mutations in ABCC6. The encoded protein, ABCC6, is a hepatic efflux transporter and a key regulator of extracellular inorganic pyrophosphate (PPi). Recent studies demonstrated that deficiency of plasma PPi, a potent endogenous calcification inhibitor, is the underlying cause of PXE. This study examined whether restoring plasma PPi levels by INZ-701, a recombinant human ENPP1 protein, the principal PPi-generating enzyme, prevents ectopic calcification in an Abcc6-/- mouse model of PXE. Abcc6-/- mice, at 6 weeks of age, the time of earliest stages of ectopic calcification, were injected subcutaneously with INZ-701 at 2 or 10mg/kg for 2 or 8 weeks. INZ-701 at both doses increased steady-state plasma ENPP1 activity and PPi levels. In the 8-week treatment study, histopathologic examination and quantification of the calcium content in INZ-701-treated Abcc6-/- mice revealed significantly reduced calcification in the muzzle skin containing vibrissae, a biomarker of the calcification process in these mice. The extent of calcification corresponds to the local expression of two calcification inhibitors, osteopontin and fetuin-A. These results suggest that INZ-701 might provide a therapeutic approach for PXE, a disease with high unmet needs and no approved treatment.

Abcc6 Null Mice-a Model for Mineralization Disorder PXE Shows Vertebral Osteopenia Without Enhanced Intervertebral Disc Calcification With Aging.

Chronic low back pain is a highly prevalent health condition intricately linked to intervertebral disc degeneration. One of the prominent features of disc degeneration that is commonly observed with aging is dystrophic calcification. ATP-binding cassette sub-family C member 6 (ABCC6), a presumed ATP efflux transporter, is a key regulator of systemic levels of the mineralization inhibitor pyrophosphate (PPi). Mutations in ABCC6 result in pseudoxanthoma elasticum (PXE), a progressive human metabolic disorder characterized by mineralization of the skin and elastic tissues. The implications of ABCC6 loss-of-function on pathological mineralization of structures in the spine, however, are unknown. Using the Abcc6 −/− mouse model of PXE, we investigated age-dependent changes in the vertebral bone and intervertebral disc. Abcc6 −/− mice exhibited diminished trabecular bone quality parameters at 7 months, which remained significantly lower than the wild-type mice at 18 months of age. Abcc6 −/− vertebrae showed increased TRAP staining along with decreased TNAP staining, suggesting an enhanced bone resorption as well as decreased bone formation. Surprisingly, however, loss of ABCC6 resulted only in a mild, aging disc phenotype without evidence of dystrophic mineralization. Finally, we tested the utility of oral K3Citrate to treat the vertebral phenotype since it is shown to regulate hydroxyapatite mechanical behavior. The treatment resulted in inhibition of the osteoclastic response and an early improvement in mechanical properties of the bone underscoring the promise of potassium citrate as a therapeutic agent. Our data suggest that although ectopic mineralization is tightly regulated in the disc, loss of ABCC6 compromises vertebral bone quality and dysregulates osteoblast-osteoclast coupling.

Functional Assessment of Missense Variants in the ABCC6 Gene Implicated in Pseudoxanthoma Elasticum, a Heritable Ectopic Mineralization Disorder

Pseudoxanthoma elasticum, a heritable multisystem ectopic mineralization disorder, is caused by inactivating mutations in the ABCC6 gene. The encoded protein, ABCC6, a transmembrane transporter, has a specialized efflux function in hepatocytes by contributing to plasma levels of inorganic pyrophosphate, a potent inhibitor of mineralization in soft connective tissues. Reduced plasma inorganic pyrophosphate levels underlie the ectopic mineralization in pseudoxanthoma elasticum. In this study, we characterized the pathogenicity of three human ABCC6 missense variants using an adenovirus-mediated liver-specific ABCC6 transgene expression system in an Abcc6-/- mouse model of pseudoxanthoma elasticum. Variants p.L420V and p.R1064W were found benign because they had abundance and plasma membrane localization in hepatocytes similar to the wild-type human ABCC6 transgene, normalized plasma inorganic pyrophosphate levels, and prevented mineralization in the dermal sheath of vibrissae in muzzle skin, a phenotypic hallmark in the Abcc6-/- mice. In contrast, p.S400F was shown to be pathogenic because it failed to normalize plasma inorganic pyrophosphate levels and had no effect on ectopic mineralization despite its normal expression and proper localization in hepatocytes. These results showed that adenovirus-mediated hepatic ABCC6 expression in Abcc6-/- mice can provide a model system to effectively elucidate the multifaceted functional consequences of human ABCC6 missense variants identified in patients with pseudoxanthoma elasticum.

A phytic acid analogue INS-3001 prevents ectopic calcification in an Abcc6-/- mouse model of pseudoxanthoma elasticum.

Pseudoxanthoma elasticum (PXE), a prototype of heritable ectopic calcification disorders, affects the skin, eyes and the cardiovascular system due to inactivating mutations in the ABCC6 gene. There is no effective treatment for the systemic manifestations of PXE. In this study, the efficacy of INS-3001, an analogue of phytic acid, was tested for inhibition of ectopic calcification in an Abcc6-/- mouse model of PXE. In prevention study, Abcc6-/- mice, at 6weeks of age, the time of onset of ectopic calcification, were treated with INS-3001 with 0.16, 0.8, 4, 20 or 100mg/kg/day administered by subcutaneous implantation of osmotic pumps, as well as 4mg/kg/day by subcutaneous injection thrice weekly or 14, 4 and 0.8mg/kg/day once weekly subcutaneous injection. Mice were necropsied at 12weeks of age. Histologic examination and quantitative calcium assay revealed that mice receiving 6weeks of continuous INS-3001 administration via osmotic pumps showed dose-dependent inhibition of muzzle skin calcification with complete response at 4mg/kg/day and a minimum effective dose at 0.8mg/kg/day. INS-3001 plasma concentrations were dose-dependent and largely consistent during treatment for each dose. thrice weekly and once weekly subcutaneous injections of INS-3001 also prevented calcification. In established disease study, 12-week-old Abcc6-/- mice with extensive calcification were continuously administered INS-3001 at 4mg/kg/day for a follow-up of 12weeks. INS-3001 treatment was found to stabilize existing calcification that had developed at start of treatment. These results suggest that INS-3001 may provide a promising preventive treatment strategy for PXE, a currently intractable ectopic calcification disorder.

405 Adenovirus-mediated ABCC6 gene therapy prevents ectopic mineralization in an Abcc6-/- mouse model of pseudoxanthoma elasticum

Biallelic loss-of-function mutations in the ABCC6 gene cause pseudoxanthoma elasticum (PXE) and type 2 generalized arterial calcification of infancy (GACI), heritable ectopic mineralization disorders without effective treatment. ABCC6 encodes the putative efflux transporter ABCC6 which is predominantly expressed in the liver. While the substrate of ABCC6 protein remains unknown, recent studies demonstrated that PXE is a metabolic disorder caused by reduced circulating levels of inorganic pyrophosphate (PPi), a potent inhibitor of mineralization. Here, we hypothesized that reconstitution of ABCC6 might counteract ectopic mineralization in an Abcc6-/- mouse model of PXE. Intravenous administration of a recombinant adenovirus expressing wild type human ABCC6 in Abcc6-/- mice showed sustained high-level expression of human ABCC6 in the liver for up to 4 weeks, increasing PPi levels in plasma. In addition, adenovirus injection every 4 weeks restored plasma PPi levels, and consequently, significantly reduced ectopic mineralization in the skin of young mice. By contrast, the same treatment in old mice, 43 weeks of age, with already established mineral deposits failed to reduce mineralization. These results suggest that adenovirus-mediated ABCC6 gene delivery, when initiated early, is a promising prevention therapy for PXE and GACI, diseases that currently lack preventive or therapeutic options to counteract ABCC6 deficiency.

Adenovirus-Mediated ABCC6 Gene Therapy for Heritable Ectopic Mineralization Disorders

Loss-of-function mutations in the ABCC6 gene cause pseudoxanthoma elasticum and type 2 generalized arterial calcification of infancy, heritable ectopic mineralization disorders without effective treatment. ABCC6 encodes the putative efflux transporter ABCC6, which is predominantly expressed in the liver. Although the substrate of ABCC6 remains unknown, recent studies showed that pseudoxanthoma elasticum is a metabolic disorder caused by reduced circulating levels of pyrophosphate, a potent mineralization inhibitor. We hypothesized that reconstitution of ABCC6 might counteract ectopic mineralization in an Abcc6-/- mouse model of pseudoxanthoma elasticum. Intravenous administration of a recombinant adenovirus expressing wild-type human ABCC6 in Abcc6-/- mice showed sustained high-level expression of human ABCC6 in the liver for up to 4 weeks, increasing pyrophosphate levels in plasma. In addition, adenovirus injection every 4 weeks restored plasma pyrophosphate levels and, consequently, significantly reduced ectopic mineralization in the skin of young mice. By contrast, the same treatment in old mice with already established mineral deposits failed to reduce mineralization. These results suggest that adenovirus-mediated ABCC6 gene delivery, when initiated early, is a promising prevention therapy for pseudoxanthoma elasticum and generalized arterial calcification of infancy, diseases that currently lack preventive or therapeutic options.

Dietary Pyrophosphate Modulates Calcification in a Mouse Model of Pseudoxanthoma Elasticum: Implication for Treatment of Patients

Pseudoxanthoma elasticum is a heritable disease caused by ABCC6 deficiency. Patients develop ectopic calcification in skin, eyes, and vascular tissues. ABCC6, primarily found in liver and kidneys, mediates the cellular efflux of ATP, which is rapidly converted into inorganic pyrophosphate (PPi), a potent inhibitor of calcification. Pseudoxanthoma elasticum patients and Abcc6-/- mice display reduced PPi levels in plasma and peripheral tissues. Pseudoxanthoma elasticum is currently incurable, although some palliative treatments exist. In recent years, we have successfully developed therapeutic methodologies to compensate the PPi deficit in animal models and humans. Here, we inadvertently discovered that modulating dietary PPi can also be an effective approach to reducing calcification in Abcc6-/- mice. Our findings were prompted by a change in institutional rodent diet. The new chow was enriched in PPi, which increased plasma PPi, and significantly reduced mineralization in Abcc6-/- mice. We also found that dietary PPi is readily absorbed in humans. Our results suggest that the consumption of food naturally or artificially enriched in PPi represents a possible intervention to mitigate calcification progression in pseudoxanthoma elasticum, that dietary preferences of patients may explain pseudoxanthoma elasticum heterogeneous manifestations, and that animal chow has the potential to influence data reproducibility.

Inhibition of Tissue-Nonspecific Alkaline Phosphatase Attenuates Ectopic Mineralization in the Abcc6–/– Mouse Model of PXE but Not in the Enpp1 Mutant Mouse Models of GACI

Pseudoxanthoma elasticum (PXE), a prototype of heritable ectopic mineralization disorders, is caused by mutations in the ABCC6 gene encoding a putative efflux transporter ABCC6. It was recently shown that the absence of ABCC6-mediated adenosine triphosphate release from the liver and, consequently, reduced inorganic pyrophosphate levels underlie the pathogenesis of PXE. Given that tissue-nonspecific alkaline phosphatase (TNAP), encoded by ALPL, is the enzyme responsible for degrading inorganic pyrophosphate, we hypothesized that reducing TNAP levels either by genetic or pharmacological means would lead to amelioration of the ectopic mineralization phenotype in the Abcc6-/- mouse model of PXE. Thus, we bred Abcc6-/- mice to heterozygous Alpl+/- mice that display approximately 50% plasma TNAP activity. The Abcc6-/-Alpl+/- double-mutant mice showed 52% reduction of mineralization in the muzzle skin compared with the Abcc6-/-Alpl+/+ mice. Subsequently, oral administration of SBI-425, a small molecule inhibitor of TNAP, resulted in 61% reduction of plasma TNAP activity and 58% reduction of mineralization in the muzzle skin of Abcc6-/- mice. By contrast, SBI-425 treatment of Enpp1 mutant mice, another model of ectopic mineralization associated with reduced inorganic pyrophosphate, failed to reduce muzzle skin mineralization. These results suggest that inhibition of TNAP might provide a promising treatment strategy for PXE, a currently intractable disease.

Etidronate prevents, but does not reverse, ectopic mineralization in a mouse model of pseudoxanthoma elasticum (Abcc6-/- ).

Pseudoxanthoma elasticum (PXE) and generalized arterial calcification of infancy (GACI) are heritable disorders manifesting with ectopic tissue mineralization. Most cases of PXE and some cases of GACI are caused by mutations in the ABCC6 gene, resulting in reduced plasma pyrophosphate (PPi) levels. There is no effective treatment for these disorders. It has been suggested that administration of bisphosphonates, stable and non-hydrolyzable PPi analogs, could counteract ectopic mineralization in these disorders. In this study we tested the potential efficacy of etidronate, a first generation bisphosphonate, on ectopic mineralization in the muzzle skin of Abcc6−/− mice, a model of PXE. The Abcc6−/− mice received subcutaneous injections of etidronate, 0.283 and 3.40 mg/kg per injection (0.01× and 0.12×), twice a week, in both prevention and reversal studies. Ectopic mineralization in the dermal sheath of vibrissae in muzzle skin was determined by histopathologic analysis and by direct chemical assay for calcium content. Subcutaneous injection of etidronate prevented ectopic mineralization but did not reverse existing mineralization. The effect of etidronate was accompanied by alterations in the trabecular bone microarchitecture, determined by micro-computed tomography. The results suggest that etidronate may offer a potential treatment modality for PXE and GACI caused by ABCC6 mutations. Etidronate therapy should be initiated in PXE patients as soon as the diagnosis is made, with careful monitoring of potential side effects.

Plasma PPi Deficiency Is the Major, but Not the Exclusive, Cause of Ectopic Mineralization in an Abcc6–/– Mouse Model of PXE

Pseudoxanthoma elasticum (PXE), a prototype of heritable ectopic mineralization disorders, is caused in most cases by inactivating mutations in the ABCC6 gene. It was recently discovered that absence of ABCC6-mediated adenosine triphosphate release from the liver and consequently reduced plasma inorganic pyrophosphate (PPi) levels underlie PXE. This study examined whether reduced levels of circulating PPi, an antimineralization factor, is the sole mechanism of PXE. The Abcc6-/- and Enpp1asj mice were crossed with transgenic mice expressing human ENPP1, an ectonucleotidase that generates PPi from adenosine triphosphate. We generated Abcc6-/- and Enpp1asj mice, either wild-type or hemizygous for human ENPP1. Plasma levels of PPi and the degree of ectopic mineralization were determined. Overexpression of human ENPP1 in Enpp1asj mice normalized plasma PPi levels to that of wild-type mice and, consequently, completely prevented ectopic mineralization. These changes were accompanied by restoration of their bone microarchitecture. In contrast, although significantly reduced mineralization was noted in Abcc6-/- mice expressing human ENPP1, small mineralization foci were still evident despite increased plasma PPi levels. These results suggest that PPi is the major mediator of ectopic mineralization in PXE, but there might be an alternative, as yet unknown mechanism, independent of PPi, by which ABCC6 prevents ectopic mineralization under physiologic conditions.

Functional Rescue of ABCC6 Deficiency by 4-Phenylbutyrate Therapy Reduces Dystrophic Calcification in Abcc6–/– Mice

Soft-tissue calcification is associated with aging, common conditions such as diabetes or hypercholesterolemia, and with certain genetic disorders. ABCC6 is an efflux transporter primarily expressed in liver facilitating the release of adenosine triphosphate from hepatocytes. Within the liver vasculature, adenosine triphosphate is converted into pyrophosphate, a major inhibitor of ectopic calcification. ABCC6 mutations thus lead to reduced plasma pyrophosphate levels, resulting in the calcification disorder pseudoxanthoma elasticum and some cases of generalized arterial calcification of infancy. Most mutations in ABCC6 are missense, and many preservetransport activity but are retained intracellularly. We have previously shown that the chemical chaperone 4-phenylbutyrate (4-PBA) promotes the maturation of ABCC6 mutants to the plasma membrane. In a humanized mouse model of pseudoxanthoma elasticum, we investigated whether 4-PBA treatments could rescue the calcification inhibition potential of selected ABCC6 mutants. We used the dystrophic cardiac calcification phenotype of Abcc6-/- mice as an indicator of ABCC6 function to quantify the effect of 4-PBA on human ABCC6 mutants transiently expressed in the liver. We showed that 4-PBA administrations restored the physiological function of ABCC6 mutants, resulting in enhanced calcification inhibition. This study identifies 4-PBA treatment as a promising strategy for allele-specific therapy of ABCC6-associated calcification disorders.

0356 : Disseminated arterial calcification and enhanced myogenic response are associated with Abcc6 deficiency in a mouse model of pseudoxanthoma elasticum

Beside calcification, the impact of ABCC6 deficiency on the vasculature remains unclear. We investigated arterial structure and function in Abcc6-/- mice, a model of the human Pseudoxanthoma Elasticum (PXE). Arterial calcium accumulation determined by atomic absorption spectrometry was 1.5 – to 2-fold higher in Abcc6-/- than in wild-type mice. Calcium also accumulated locally leading to a specific punctuated pattern. Abcc6-/- mesenteric arteries mounted on a wire myograph displayed slight increase in arterial vasoconstrictor tone in response to phenylephrine and thromboxane A2. Interestingly, myogenic tone (Bayliss effect) determined using a pressure myograph was significantly elevated in Abcc6-/- compared to wild type arteries. Arterial blood pressure was not significantly modified in Abcc6-/- animals, despite higher variability. These changes were accompanied with deregulated gene expression (RTqPCR) in both liver and resistance arteries. Old Abcc6-/- mouse mesenteric arteries expressed markers of both osteogenic (Runx2, opn) and chondrogenic lineage (Sox9, col2a1). Surprisingly Enpp1 and Alpl genes encoding ectonucleotide pyrophosphatase/phosphodiesterase 1 and alkaline phosphatase were deregulated within Abcc6-/- liver and this was corroborated with reduced alkaline phosphatase circulating levels in PXE patients. As a conclusion, scattered calcium depositions result from osteochondrogenic transdifferentiation of vascular cells. The lower elasticity and increased myogenic tone evidenced in aged Abcc6-/- mice suggest a reduced control of local blood flow, which in turn may alter vascular homeostasis. Our findings argue in favor of a deregulated arterial function and may help to decipher consequences of ABCC6 deficiency since PXE is a significant risk factor for small vessel disease and particularly ischemic stroke.

Development of atherosclerotic plaques in a mouse model of pseudoxanthoma elasticum

Objective Pseudoxanthoma elasticum (PXE) is an autosomal recessive disorder, characterized by extensive mineralization of connective tissues and fragmentation of elastin fi bres. PXE patients may sporadically suff er from severe cardiovascular complications caused by accelerated atherosclerosis. Consistent with this fi nding, recent evidence suggests that elastin fragmentation in arteries of atherosclerotic mice leads to unstable plaques and humanlike complications such as myocardial infarction, stroke and sudden death. Because Abcc6–/– mice manifest the human features of PXE including the fragmentation of elastin fi bres, Abcc6–/– mice were crossbred with ApoE–/– mice to investigate the level of plaque formation and potential complications.Methods and results ApoE–/– and ApoE–/– Abcc6–/– mice were fed a Western-type diet (WD) for 25 weeks to induce plaque formation. WD-fed animals showed neither signs of neurological dysfunction nor sudden death. Cardiac function of ApoE–/– Abcc6–/– mice, as assessed by echocardiography, was not diff erent from ApoE–/– control mice. Histochemical analysis did not reveal elastin fragmentation or pronounced mineral deposition in the vessel wall. Plaques from the proximal ascending aorta and brachiocephalic artery of ApoE–/– Abcc6–/– mice were similar in size and composition as compared to ApoE–/– mice. Moreover, en faceoil red O stainings of the aortic arch and descending thoracic aorta did not reveal enhanced plaque formation in ApoE–/– Abcc6–/– mice as compared to ApoE–/– controls.Conclusion ApoE–/–Abcc6–/– mice do not represent an adequate model of accelerated atherosclerosis and therefore are not useful to study atherosclerosis-related complications as observed in PXE patients.

Disseminated Arterial Calcification and Enhanced Myogenic Response Are Associated With Abcc6 Deficiency in a Mouse Model of Pseudoxanthoma Elasticum

Pseudoxanthoma elasticum is an inherited metabolic disorder resulting from ABCC6 gene mutations. It is characterized by progressive calcification and fragmentation of elastic fibers in the skin, retina, and the arterial wall. Despite calcium accumulation in the arteries of patients with pseudoxanthoma elasticum, functional consequences remain unknown. In the present study, we investigated arterial structure and function in Abcc6(-/-) mice, a model of the human disease. Arterial calcium accumulation was evaluated using alizarin red stain and atomic absorption spectrometry. Expression of genes involved in osteochondrogenic differentiation was measured by polymerase chain reaction. Elastic arterial properties were evaluated by carotid echotracking. Vascular reactivity was evaluated using wire and pressure myography and remodeling using histomorphometry. Arterial calcium accumulation was 1.5- to 2-fold higher in Abcc6(-/-) than in wild-type mice. Calcium accumulated locally leading to punctuate pattern. Old Abcc6(-/-) arteries expressed markers of both osteogenic (Runx2, osteopontin) and chondrogenic lineage (Sox9, type II collagen). Abcc6(-/-) arteries displayed slight increase in arterial stiffness and vasoconstrictor tone in vitro tended to be higher in response to phenylephrine and thromboxane A2. Pressure-induced (myogenic) tone was significantly higher in Abcc6(-/-) arteries than in wild type. Arterial blood pressure was not significantly changed in Abcc6(-/-), despite higher variability. Scattered arterial calcium depositions are probably a result of osteochondrogenic transdifferentiation of vascular cells. Lower elasticity and increased myogenic tone without major changes in agonist-dependent contraction evidenced in aged Abcc6(-/-) mice suggest a reduced control of local blood flow, which in turn may alter vascular homeostasis in the long term.

Atorvastatin counteracts aberrant soft tissue mineralization in a mouse model of pseudoxanthoma elasticum (Abcc6 −/− )

Pseudoxanthoma elasticum (PXE), a multisystem heritable disorder with aberrant mineralization of arterial blood vessels, is caused by mutations in the ABCC6 gene. Previous studies have suggested that carriers of the ABCC6 mutations, particularly of p.R1141X, are at increased risk for coronary artery disease. In this study, we used Abcc6 (tm1Jfk) knock-out mice to determine the serum lipid profiles and examine the effects of atorvastatin on the aberrant mineralization in this model of PXE. First, serum lipid profiles at 12 weeks of age, after overnight fasting, revealed a statistically significant increase in total cholesterol and triglyceride levels in Abcc6 (tm1Jfk) mice compared to their wild-type littermates. Placing these mice at 4 weeks of age for 20 weeks on atorvastatin, either 0.01 % or 0.04 % of the diet (low statin and high statin groups, respectively), reduced the total triglyceride and cholesterol levels, which was accompanied with significantly reduced mineralization of the dermal sheath of vibrissae, a biomarker of the aberrant mineralization process in these mice. However, if the mice were placed on atorvastatin for 12 weeks at 12 weeks of age, at which time point significant mineralization had already taken place, no difference in the amount of mineralization was noted. These observations suggest that statins, particularly atorvastatin, can prevent, but not reverse, aberrant mineralization in this mouse model of PXE. For a clinical perspective, a survey of 1,747 patients with PXE was conducted regarding their present or past use of statins. The results indicated that about one third of all PXE patients are currently or have previously been on cholesterol-lowering drugs. Thus, a sizable number of patients with PXE could be subject to modulation of their mineralization processes by concomitant statin treatment. The Abcc6 (-/-) mice serve as a model system for PXE, an ectopic mineralization disorder Abcc6 (-/-) mice were shown to have elevated serum cholesterol and triglyceride levels Feeding of the Abcc6 (-/-) mice with atorvastatin prevented connective tissue mineralization A third of patients with PXE was found to be on cholesterol-lowering therapy Atorvastatin may potentially be beneficial for patients with PXE.

Restricting dietary magnesium accelerates ectopic connective tissue mineralization in a mouse model of pseudoxanthoma elasticum (Abcc6−/−)

Ectopic mineralization, linked to a number of diseases, is a major cause of morbidity and mortality in humans. Pseudoxanthoma elasticum (PXE) is a heritable multisystem disorder characterized by calcium phosphate deposition in various tissues. The mineral content of diet has been suggested to modify the disease severity in PXE. The aim of this study is to explore the role of diet with reduced magnesium in modifying tissue mineralization in a mouse model of PXE. Abcc6(-/-) mice were placed on either standard rodent diet (control) or an experimental diet low in magnesium at weaning (4weeks) and examined for mineralization in the skin and internal organs at the ages of 1.5, 2 or 6months by computerized morphometric analysis of histopathological sections and by chemical assay of calcium and phosphate. Abcc6(-/-) mice on experimental diet demonstrated an accelerated, early-onset mineralization of connective tissues, as compared to control mice. Wild-type or heterozygous mice on experimental diet did not show evidence of mineralization up to 6months of age. All mice on experimental diet showed decreased urinary calcium, increased urinary phosphate and elevated parathyroid serum levels. However, no difference in bone density at 6months of age was noted. Our findings indicate that the mineral content, particularly magnesium, can modify the extent and the onset of mineralization in Abcc6(-/-) mice and suggest that dietary magnesium levels may contribute to the phenotypic variability of PXE. The control of mineralization by dietary magnesium may have broader implications in general population in the context of vascular mineralization.

Magnesium reduces carotid intima-media thickness in a mouse model of pseudoxanthoma elasticum: a novel treatment biomarker.

Pseudoxanthoma elasticum (PXE), which demonstrates progressive build-up of calcium phosphate and proteoglycan deposits in skin, eye, and arteries, has been associated with myocardial infarctions, stroke, and blindness. In a mouse model of PXE, a magnesium-enriched diet prevents mineralization of the vibrissae capsule, an early biomarker for PXE. However, biomarkers for therapeutic responses in PXE have not been identified in humans. Because PXE patients have an increased carotid intima-media thickness (CIMT), a risk factor for cardiovascular disease and stroke, we analyzed the feasibility of CIMT as a treatment endpoint before and after magnesium supplementation in a mouse model of PXE (Abcc6(-/-) ). CIMT was measured in 1-year-old Abcc6(-/-) and Abcc6(+/+) mice fed either standard rodent diet with or without magnesium oxide supplementation for 2 months. Baseline CIMT in Abcc6(-/-) versus Abcc6(+/+) mice was increased (p value = 0.009), whereas CIMT in magnesium-treated versus untreated Abcc6(-/-) mice was reduced (p value = 0.024). CIMT is a novel treatment endpoint in this mouse model and may serve as a predictive biomarker of therapeutic response in PXE patients. In that context, magnesium oxide significantly reduced CIMT in PXE mice, and may be useful for disease prevention in PXE patients.

Vitamin K does not prevent soft tissue mineralization in a mouse model of pseudoxanthoma elasticum

Pseudoxanthoma elasticum (PXE) is a heritable disease characterized by calcified elastic fibers in cutaneous, ocular, and vascular tissues. PXE is caused by mutations in ABCC6, which encodes a protein of the ATP-driven organic anion transporter family. The inability of this transporter to secrete its substrate into the circulation is the likely cause of PXE. Vitamin K plays a role in the regulation of mineralization processes as a co-factor in the carboxylation of calcification inhibitors such as Matrix Gla Protein (MGP). Vitamin K precursor or a conjugated form has been proposed as potential substrate(s) for ABCC6. We investigated whether an enriched diet of vitamin K1 or vitamin K2 (MK4) could stop or slow the disease progression in Abcc6-/- mice. Abcc6-/- mice were placed on a diet of either vitamin K1 or MK4 at 5 or 100 mg/kg at prenatal, 3 weeks or 3 months of age. Disease progression was quantified by measuring the calcium content of one side of the mouse muzzle skin and histological staining for calcium of the opposing side. Raising the vitamin K1 or MK4 content of the diet increased the concentration of circulating MK4 in the serum. However, this increase did not significantly affect the MGP carboxylation status or reduce its abnormal abundance, the total calcium content or the pathologic calcification in the whiskers of the 3 treatment groups compared to controls. Our findings showed that raising the dietary intake of vitamin K1 or MK4 was not beneficial in the treatment of PXE and suggested that the availability of vitamin K may not be a limiting factor in this pathology.

Expression of the Abca-subfamily of genes in Abcc6−/− mice - upregulation of Abca4

Pseudoxanthoma elasticum (PXE), a heritable multi-system disorder, is caused by mutations in the ABCC6 gene primarily expressed in the liver. Recent analysis of cultured fibroblasts from patients with PXE has suggested compensatory alterations in the expression of the ABCA-subfamily of genes. We have now determined by quantitative RT-PCR the level of expression of Abca-family of genes in a mouse model of PXE developed by targeted ablation of Abcc6. The results indicated variable levels of mRNA for different Abca genes in the liver; however, only one of them, Abca4, was significantly, ∼6.5-fold, upregulated in the Abcc6(-/-) mice in comparison with wild-type mice. In the same mice, Abca4 was not upregulated in the eyes or the kidney, suggesting that the upregulation of Abca4 in the liver is a tissue-specific compensatory consequence of the 'knock-out' of Abcc6.