ABCC6, Pyrophosphate and Ectopic Calcification: Therapeutic Solutions.

Pathological (ectopic) mineralization of soft tissues occurs during aging, in several common conditions such as diabetes, hypercholesterolemia, and renal failure and in certain genetic disorders. Pseudoxanthoma elasticum (PXE), a multi-organ disease affecting dermal, ocular, and cardiovascular tissues, is a model for ectopic mineralization disorders. ABCC6 dysfunction is the primary cause of PXE, but also some cases of generalized arterial calcification of infancy (GACI). ABCC6 deficiency in mice underlies an inducible dystrophic cardiac calcification phenotype (DCC). These calcification diseases are part of a spectrum of mineralization disorders that also includes Calcification of Joints and Arteries (CALJA). Since the identification of ABCC6 as the "PXE gene" and the development of several animal models (mice, rat, and zebrafish), there has been significant progress in our understanding of the molecular genetics, the clinical phenotypes, and pathogenesis of these diseases, which share similarities with more common conditions with abnormal calcification. ABCC6 facilitates the cellular efflux of ATP, which is rapidly converted into inorganic pyrophosphate (PPi) and adenosine by the ectonucleotidases NPP1 and CD73 (NT5E). PPi is a potent endogenous inhibitor of calcification, whereas adenosine indirectly contributes to calcification inhibition by suppressing the synthesis of tissue non-specific alkaline phosphatase (TNAP). At present, therapies only exist to alleviate symptoms for both PXE and GACI; however, extensive studies have resulted in several novel approaches to treating PXE and GACI. This review seeks to summarize the role of ABCC6 in ectopic calcification in PXE and other calcification disorders, and discuss therapeutic strategies targeting various proteins in the pathway (ABCC6, NPP1, and TNAP) and direct inhibition of calcification via supplementation by various compounds.

Adaptive Optics Imaging in Patients Affected by Pseudoxanthoma Elasticum.

PURPOSE: To describe the retinal findings of patients affected by pseudoxanthoma elasticum (PXE) using a multimodal imaging approach including flood-illumination adaptive optics ophthalmoscopy (AO). DESIGN: Retrospective case series. MATERIALS AND METHODS: Patients affected by PXE were retrospectively studied. Clinical data, color, infrared and autofluorescence fundus imaging, optical coherence tomographic scans, and AO examinations were collected. Furthermore, the photoreceptor count was assessed. PXE diagnosis was confirmed by a positive skin biopsy and/or genetic testing. RESULTS: Twenty-one eyes of 18 patients (11 females and 7 males) were included in the study. In 3 patients, both eyes were studied. The mean age at examination was 37.7 ± 16.4 years (range 14-66) and the mean best-corrected visual acuity (BCVA) was 0.1 ± 0.2 logMAR (range 0-1). We identified 3 types of angioid streaks (AS) using AO: "crack," "band," and "hypopigmented." The first 2 were very similar and they differed in size; the third type showed specific clinical features. Comet lesions appeared as hyper-reflective round lesions on AO imaging. In all eyes, the cone mosaic appeared reduced inside the streaks compared to the neighboring areas (13,532.8 ± 1,366.5 cones/mm2 vs 16,817.1 ± 1,263.0 cones/mm2 respectively). CONCLUSION: Using AO imaging in PXE-related retinopathy, we were able to observe the presence of the photoreceptors within the angioid streaks, differentiate 3 types of angioid streaks, based on size and reflective features, and identify the very small crystalline bodies not identifiable using other retinal imaging techniques.

PSEUDOXANTHOMA ELASTICUM: SUCCESSFUL LONG-TERM MANAGEMENT OF CHOROIDAL NEOVASCULARIZATION SECONDARY TO ANGIOID STREAKS WITH PRO RE NATA INTRAVITREAL BEVACIZUMAB INJECTIONS.

PURPOSE: To demonstrate how a patient with recurrent episodes of choroidal neovascularization (CNV), secondary to angioid streaks, can be managed successfully with a pro re nata regime of intravitreal bevacizumab injection over an eight-year period. METHOD: A 32-year-old white woman with pseudoxanthoma elasticum has been followed up over an eight-year period for management of recurrent episodes of CNV in both eyes. She was educated to recognize the early signs and symptoms of CNV. Physical examination including visual acuity and slit-lamp examination as well as investigations such as macula optical coherence tomography and optical coherence tomography angiography were performed. Bevacizumab injections were given to her when she was diagnosed with CNV. RESULTS: Multiple episodes of CNV were successfully treated with pro re nata regimes of intravitreal bevacizumab injections. The patient was able to maintain excellent visual acuity of 0 logarithm of the minimum angle of resolution even after suffering recurrent episodes of CNV. CONCLUSION: This case report supports that a pro re nata regime of intravitreal bevacizumab injection therapy can be used successfully to treat recurrent episodes of CNV in a patient with pseudoxanthoma elasticum over an eight-year period. Early diagnosis through patient education and the use of appropriate diagnostic tools such as optical coherence tomography angiography have enabled us to deliver early treatment, resulting in an excellent outcome for this patient.

Generalized Arterial Calcification of Infancy: New Insights, Controversies, and Approach to Management.

PURPOSE OF REVIEW: This review summarizes current understanding of generalized arterial calcification of infancy (GACI), emphasizing pathophysiology, clinical presentation, and approaches and controversies in management. RECENT FINDINGS: Identification of causative ENPP1 mutations revealed that GACI arises from deficiencies in inorganic pyrophosphate (leading to calcifications) and adenosine monophosphate (leading to intimal proliferation). Identification of genotypic and phenotypic overlap with pseudoxanthoma elasticum and autosomal recessive hypophosphatemic rickets further advanced understanding of GACI as a complex, multisystemic disease. Clinical data is limited to small, retrospective samples; it is therefore unknown whether commonly used medications, such as bisphosphonates and hypophosphatemia treatment, are therapeutic or potentially harmful. ENPP1-Fc replacement represents a promising approach warranting further study. Knowledge gaps in natural history place clinicians at high risk of assigning causality to interventions that are correlated with changes in clinical status. There is thus a critical need for improved natural history studies to develop and test targeted therapies.

ABCC6 knockdown in HepG2 cells induces a senescent-like cell phenotype.

BACKGROUND: Pseudoxanthoma elasticum (PXE) is characterized by progressive ectopic mineralization of elastic fibers in dermal, ocular and vascular tissues. No effective treatment exists. It is caused by inactivating mutations in the gene encoding for the ATP-binding cassette, sub-family C member 6 transporter (ABCC6), which is mainly expressed in the liver. The ABCC6 substrate (s) and the PXE pathomechanism remain unknown. Recent studies have shown that overexpression of ABCC6 in HEK293 cells results in efflux of ATP, which is rapidly converted into nucleoside monophosphates and pyrophosphate (PPi). Since the latter inhibits mineralization, it was proposed that the absence of circulating PPi in PXE patients results in the characteristic ectopic mineralization. These studies also demonstrated that the presence of ABCC6 modifies cell secretory activity and suggested that ABCC6 can change the cell phenotype. METHODS: Stable ABCC6 knockdown HepG2 clones were generated using small hairpin RNA (shRNA) technology. The intracellular glutathione and ROS levels were determined. Experiments using cell cycle analysis, real-time PCR and western blot were performed on genes involved in the senescence phenotype. RESULTS: To shed light on the physiological role of ABCC6, we focused on the phenotype of HepG2 cells that lack ABCC6 activity. Interestingly, we found that ABCC6 knockdown HepG2 cells show: 1) intracellular reductive stress; 2) cell cycle arrest in G1 phase; 3) upregulation of p21Cip p53 independent; and 4) downregulation of lamin A/C. CONCLUSIONS: These findings show that the absence of ABCC6 profoundly changes the HepG2 phenotype, suggesting that the PXE syndrome is a complex metabolic disease that is not exclusively related to the absence of pyrophosphate in the bloodstream.

Pseudoxanthoma elasticum.

Pseudoxanthoma elasticum (PXE) is characterized by elastic tissue fragmentation and calcification. The deterioration of elastic fibers leads to characteristic yellowish papules and plaques (pseudoxanthomas) and retinal angioid streaks. Although these findings may begin in childhood, the diagnosis is typically not made until the second or third decade after the skin and retinal findings become more prominent. Cerebrovascular complications include brain infarction due to narrowing and occlusion of cerebral arteries and aneurysm formation. Intracranial hemorrhage can occur in the absence of aneurysm, and gastrointestinal hemorrhage is common. Peripheral arterial vascular disease can lead to intermittent leg claudication. A skin biopsy often demonstrates calcified elastic fibers, even in a mildly affected area of skin. The inheritance is autosomal recessive, although heterozygotes may exhibit some features of the disease. PXE is due to mutation of the ABCC6 gene on chromosome 16. There is no treatment, but certain lifestyle modifications may limit the complications. The potential for retinal hemorrhage has led to recommendations for limitations of contact sports or other activities that might facilitate eye trauma. Other recommendations include maintaining a normal lipid profile, avoidance of aspirin and nonsteroidal anti-inflammatory agents, and limiting dietary calcium intake.

Disseminated arterial calcification and enhanced myogenic response are associated with abcc6 deficiency in a mouse model of pseudoxanthoma elasticum.

OBJECTIVE: 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. APPROACH AND RESULTS: 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. CONCLUSIONS: 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.

Relationship between ankle brachial index and arterial remodeling in pseudoxanthoma elasticum.

OBJECTIVES: Pseudoxanthoma elasticum (PXE) is an inherited metabolic disease characterized by elastic fiber fragmentation and calcification in the cutaneous, ophthalmologic, and vascular tissues. Cardiovascular manifestations such as peripheral arterial disease (PAD) are frequent in PXE. Because of the changes in the elastic properties and medial calcification of the arterial wall in PXE, the impact of the arterial remodeling on the ankle brachial index (ABI), a well-established diagnostic method for the detection and follow-up of PAD, remains to be determined in this disease. METHODS: This was a cross-sectional, comparative, open study, which took place at the PXE Consultation Center, University Hospital of Angers. The subjects were 53 patients (mean age, 49 ± 14 years; 35 females) with PXE clinically proven on the basis of established criteria (skin changes, angioid streaks, and skin biopsy). The ABI at rest, symptoms of intermittent claudication (IC), carotid intima-media thickness (IMT), carotid-femoral pulse wave velocity (c-f PWV), compliance (CC), and ß stiffness index were measured in a single-center cohort. RESULTS: Forty-five percent of the PXE patients had an ABI ≤0.90, but only one patient had an ABI >1.40. IC was found in 23% of the patients with an ABI ≤0.90. There were no significant differences between the patients with a low and normal ABI in terms of IMT (P = .566) or ß stiffness index (P = .194), but differences were significant for c-f PWV (P = .010) and CC (P = .011). Adjusted multivariate linear regression for the Framingham-Laurier score showed that patients with a low ABI had less compliant carotid arteries (B = 0.318, P = .039). CONCLUSIONS: PAD detected by a low ABI is very frequent in PXE, although with limited prevalence of symptomatic claudication. Unexpectedly, ABI was low in such calcifying PAD and associated with lower CC, independently of atherosclerosis risk factors. These findings demonstrate that PXE represents a unique monogenic model of PAD in which the specific arterial wall remodeling could change the diagnostic value of the ABI to detect PAD.

Long-term effectiveness of intravitreal bevacizumab for choroidal neovascularization secondary to angioid streaks in pseudoxanthoma elasticum.

PURPOSE: To investigate the long-term effectiveness of intravitreal bevacizumab for treating active choroidal neovascularizations in pseudoxanthoma elasticum (PXE). METHODS: Fourteen patients (16 eyes) received intravitreal bevacizumab (1.5 mg) and were investigated monthly. Further treatments were administered depending on disease activity. Examinations included best-corrected visual acuity, biomicroscopy, optical coherence tomography, fluorescein angiography and indocyanine green angiography, fundus autofluorescence, and digital fundus photography. Areas of atrophy of the retinal pigment epithelium and retinal fibrosis were quantified using semiautomated detection on fundus autofluorescence images. RESULTS: Mean age of the cohort was 55 ± 13 years, and mean best-corrected visual acuity at baseline was 20/80 (logarithm of the minimum angle of resolution, 0.56, SD, 0.51). At last follow-up, after an average of 6.5 ± 5.7 injections over 28 months, best-corrected visual acuity was 20/40 (logarithm of the minimum angle of resolution, 0.31, SD, 0.32; P = 0.04). Central retinal thickness was reduced from 254 ± 45 µm to 214 ± 40 µm (P = 0.035). The size of retinal pigment epithelial atrophy and retinal fibrosis measured on fundus autofluorescence images increased in both the treated eye and the fellow eye (P < 0.05). Best-corrected visual acuity of patients with early disease compared with that of those with advanced disease improved significantly more over the treatment course (20/25 vs. 20/63; P = 0.008). CONCLUSION: Intravitreal bevacizumab therapy demonstrates long-term effectiveness by preserving function in advanced disease and improving function in early disease. Best results of treating active choroidal neovascularizations in PXE are achieved when treatment starts the earliest possible.

Calcium may preferentially deposit in areas of elastic tissue damage.

BACKGROUND: Cutaneous calcification is an acquired disorder whereby insoluble, amorphous calcium salts deposit in the skin. Classically, cutaneous calcification is categorized as metastatic, dystrophic, idiopathic, or iatrogenic. OBJECTIVE: The purpose of this study was to further elucidate the underlying pathogenic mechanism for cutaneous calcification. METHODS: Three cases of cutaneous calcification, including clinical characteristics and associated histopathology, were reviewed. Previous reports of cutaneous calcification were searched for in the published literature and included. RESULTS: Calcium is distributed within areas of underlying tissue damage (ie, locus minoris resistentiae), and in our cases, occurred specifically at sites of chronic actinic damage and intravenous extravasation tissue injury. LIMITATIONS: A small number of clinical cases and previously published reports were reviewed. CONCLUSION: We hypothesize that cutaneous calcification may preferentially occur at anatomic sites where tissue integrity has been compromised (ie, locus minoris resistentiae). We suggest one potential mechanism: that cutaneous calcification occurs within dermis that contains damaged elastic fibers. Pseudoxanthoma elasticum may serve as a possible genetic disease model for this process.

Pseudoxanthoma elasticum-like lesions in association with thalassaemia major.

A 33-year-old woman with a background of thalassaemia major presented with a 3-month history of yellowish plaques on the back of her neck bilaterally and alterations in the texture of the skin in both axillae. Examination of these lesions showed yellowish cobblestone plaques and coalescing papules in both axillae. Pseudoxanthoma elasticum (PXE)-like lesions were confirmed histopathologically. PXE-like lesions in association with thalassaemia major are an uncommon occurrence. Due to the increase in the survival rate of thalassaemia major patients on treatment, the development of these lesions is likely to increase. The histopathological manifestation of these lesions mimics that of hereditary PXE and is associated with severe vascular complications. This case highlights the importance of recognising this rare disorder and the role dermatologists have in establishing the diagnosis and advising on appropriate screening for complications.

Oxidative stress in fibroblasts from patients with pseudoxanthoma elasticum: possible role in the pathogenesis of clinical manifestations.

Pseudoxanthoma elasticum (PXE) is a genetic disease characterized by calcification and fragmentation of elastic fibres of the skin, cardiovascular system and eye, caused by mutations of the ABCC6 gene, which encodes the membrane transporter MRP6. The pathogenesis of the lesions is unknown. Based on studies of similar clinical and histopathological damage present in haemolytic disorders, our working hypothesis is that PXE lesions may result from chronic oxidative stress occurring in PXE cells as a consequence of MRP6 deficiency. Our results show that PXE fibroblasts suffer from mild chronic oxidative stress due to the imbalance between production and degradation of oxidant species. The findings also show that this imbalance results, at least in part, from the loss of mitochondrial membrane potential (DeltaPsi(m)) with overproduction of H2O2. Whether mitochondrial dysfunction is the main factor responsible for the oxidative stress in PXE cells remains to be elucidated. However, mild chronic generalized oxidative stress could explain the great majority of structural and biochemical alterations already reported in PXE.

Oral phosphate binders in the treatment of pseudoxanthoma elasticum.

BACKGROUND: Pseudoxanthoma elasticum (PXE) is a systemic connective tissue disorder involving elastic fiber calcification and fragmentation with major clinical manifestations occurring in the cutaneous, ocular, and cardiovascular systems. Normalization of the serum calcium-phosphate product through hemodialysis in a previous patient with perforating periumbilical PXE and elevated serum phosphate resulted in regression of skin lesions. OBJECTIVE: We sought to study the effect of pharmacologically limiting the intestinal absorption of phosphate in patients with PXE. METHODS: Patients received baseline skin examinations, target skin lesion evaluation, and photography; renal function tests and serum calcium and phosphate levels; urine calcium, phosphate, and creatinine levels; skin biopsy; and eye examinations and indocyanine-green angiography. Patients were treated with aluminium hydroxide tablets or liquid and returned every 2 to 4 months for skin photography and lesion evaluation. Repeated skin biopsies were performed on clinically improved target sites. Ophthalmologic evaluation was obtained at yearly intervals. RESULTS: Of 6 patients, 3 showed significant clinical improvement of skin lesions and all 3 of these patients showed histopathologic regression of disease in their target lesions. No deterioration of eye disease was seen in any of the 6 patients at 1-year follow-up. CONCLUSION: Our results demonstrate that the calcification seen in PXE may be reversible in some patients. This could hold true for eye and vascular lesions and for skin. Further studies supporting these results could reveal the first real treatment option for PXE.

[Mendelian arterial diseases. Pseudoxanthoma elasticum, Ehlers-Danlos vascular syndrome, Rendu-Osler disease]. / Maladies artérielles mendéliennes. Pseudoxanthome élastique, syndrome d'Ehlers-Danlos vasculaire, maladie de Rendu-Osler.

Understanding the features of the various hereditary vascular pathologies allows consideration and confirmation of the diagnosis, and a search for treatable hidden disorders, avoiding harmful investigations, initiating follow up, performing family investigations and providing genetic counselling. Pseudoxanthoma elasticum must be considered in the presence of calcified distal arteriopathy of the lower limbs in a young subject without any other aetiological aspects. Cutaneous or mucosal lesions confirmed on histological examination, angioid streaks at the back of the eye and a family history support the diagnosis, which is confirmed by showing pathogenic mutations of the ABCC6 gene. It is then important to search for a peripheral disorder in other arterial territory, a low grade coronaropathy, hypertension and an endocardial disorder. Prescription of antithrombotics must be made carefully because of the risk of gastro-intestinal haemorrhage. Vascular Ehlers-Danlos syndrome is suspected in a subject less than 30 years old with diffuse aneurysmal disease, spontaneous arterial rupture or dissection, a carotido-cavernous fistula or early onset varices. Demonstrating an ecchymotic tendency or an acrogeric morphology, especially in a familial context, warrants cutaneous biopsy for anatomopathological examination and fibroblast culture for a study of the C0L3A1 gene. When the diagnosis is suggested, it is advisable to prohibit any arterial puncture, cold surgery or gastro-intestinal endoscopy. The search for aneurysmal lesions must be performed by non-invasive imaging. The therapeutic management requires specialised teams. The combination of repeated epistaxis, muco-cutaneous telangectasia and similar characteristics in a family suggests the diagnosis of Osler-Weber-Rendu disease. The search for iron deficiency, gastro-intestinal bleeds and pulmonary, hepatic or cerebral arterio-venous malformations is then necessary. Besides skilled endovascular management when indicated, it is important to advise every patient with a pulmonary arterio-venous malformation to take antibiotic prophylaxis against cerebral abscess in situations at risk of bacteraemia.

Pseudoxanthoma elasticum with abnormal nailfold microcirculatory findings.

Pseudoxanthoma elasticum (PXE) is an inherited disorder of elastic tissue. Here we report a 34-year-old male patient who developed multiple symptomless yellowish papules over his neck for several months. He visited our dermatologic out-patient-clinic because his sister had similar skin lesions and mild visual impairment. The pathologic features of the skin biopsy showed fragmented calcified elastic fibers in the mid-to-lower dermis under the H&E, Verhoeff-van Gieson and Von Kossa stains. Under electron microscopy, calcified degenerated elastic fibers were noted. No other internal organ involvement was found except angioid streaks on the fundus. In addition, morphological changes of the nailfold capillaries, including increased tortuosity, dilated venous limbs of capillary loops, and decreased red-blood-cell velocity, were observed under the capillaroscopy. Though former reports have indicated that cardiovascular manifestations are caused by degeneration of elastic fibers of blood vessels, this study is the first to emphasize the microcirculatory disturbance of nailfold capillary, including morphology and blood-cell velocity, in PXE.

[Grönblad-Strandberg syndrome from the angiological viewpoint]. / Das Grönblad-Strandberg-Syndrom aus angiologischer Sicht.

HISTORY AND CLINICAL FINDINGS: A 42-year-old man was admitted for treatment of peripheral vascular disease in the left leg (stage III of Fontaine). A year before he had undergone a right aortofemoral bypass operation. On admission there was stenosis of the left pelvic axis and bilateral femoral artery occlusion. In addition there were changes in the skin with abnormal folds, loss of elasticity and yellowish spots over the sides of the neck and the flexor surfaces of all large joints. In addition vision in the left eye was impaired. These findings suggested connective tissue disease involving the skin, eye and arterial system. INVESTIGATIONS: Routine haematological tests were normal as were clotting parameters. Serum concentration of GOT, GPT, gamma-GT were slightly increased. There was a dysproteinaemia with raised HDL and LDL levels. Resting electrocardiogram was normal, showing sinus rhythm and left axis deviation. The crurobrachial pressure index was clearly abnormal: 0.6 on the right and 0.5 on the left. Angiography of the pelvic and left arteries revealed long-segment femoral and partial lower-leg occlusions bilaterally. Abdominal sonography indicated diffuse parenchymal calcifications in both kidneys and angioid streaks on bilateral fundoscopy. Skin biopsy showed defects of elastic fibres and perivascular inflammatory infiltration, while capillary microscopy revealed twisting of the capillaries, most of them with normal lumen. These findings taken together indicated pseudoxanthoma elasticum (PXE) or Grönblad-Strandberg syndrome. TREATMENT AND COURSE: A thrombendarterectomy was performed on the left superficial femoral artery, after which the left popliteal artery became palpable, the pressure indices for the left leg were slightly better, and the patient was discharged home without further complications and improved leg perfusion. CONCLUSION: Possible cardiovascular involvement had to be taken into account in patients with PXE, and long-term angiological monitoring is indicated.

Pseudoxanthoma elasticum and pregnancy.

Reports of complications of pseudoxanthoma elasticum occurring during pregnancy have dissuaded some women with the disorder from attempting to conceive for fear of exacerbating the disease. The actual risks of serious complications during pregnancy, however, may be overstated. We report a 40-year-old woman with pseudoxanthoma elasticum who delivered a healthy male infant after an uneventful pregnancy. At the time of delivery, the placenta was noted to be heavily calcified. The effects of pregnancy on pseudoxanthoma elasticum and the effects of maternal pseudoxanthoma elasticum on a developing fetus are reviewed, with a review of the relevant medical literature.