Augustine Blood Group System and Equilibrative Nucleoside Transporter 1

Augustine (AUG) is a blood group system comprising four antigens: AUG1, AUG2 (At^a), and AUG4 are of very high frequency; AUG3 is of very low frequency. These antigens are located on ENT1, an equilibrative nucleoside transporter encoded by <i>SLC19A1</i>. AUG antibodies are of clinical relevance in blood transfusion and pregnancy: anti-AUG2 have caused haemolytic transfusion reactions; the only anti-AUG3 was associated with severe haemolytic disease of the fetus and newborn. ENT1 is present in almost all human tissues. It facilitates the transfer of purine and pyrimidine nucleosides and is responsible for the majority of adenosine transport across plasma membranes. Adenosine transport appears to be an important factor in the regulation of bone metabolism. The AUG_null phenotype (AUG:–1,–2,–3,–4) has been found in three siblings, who are homozygous for an inactivating splice-site mutation in <i>SLC29A1</i>. Although ENT1 is very likely to be absent from all cells in these three individuals, they were apparently healthy with normal lifestyles. However, they suffered frequent attacks of pseudogout, a form of arthritis, in various joints with multiple calcifications around their hand joints. Ectopic calcification in the hips, pubic symphysis, and lumbar discs was present in the propositus. The three AUG_null individuals had misshapen red cells with deregulated protein phosphorylation, but no anaemia or shortening of red cell lifespan. Defective in vitro erythropoiesis in the absence of ENT1 was confirmed by shRNA-mediated knockdown of ENT1 during in vitro erythropoiesis of CD34⁺ progenitor cells from individuals with normal ENT1. Nucleoside transporters, such as ENT1, are vital in the uptake of synthetic nucleoside analogue drugs, used in cancer and viral chemotherapy. It is feasible that the efficacy of these drugs would be compromised in patients with the extremely rare AUG_null phenotype.

Abstract 16747: Rapidly Lethal Dilated Cardiomyopathy Secondary To Generalized Arterial Calcification Of Infancy

Case Presentation: A previously healthy 3-month-old infant presented with cardiorespiratory arrest, from which she was successfully resuscitated. A dilated cardiomyopathy with severely depressed systolic function was diagnosed. ECG showed high voltage QRS complexes and generalized alteration of repolarization. In the following 12 hours, she suffered 2 other cardiac arrests, recuperated with defibrillation. Hemodinamic stability was achieved but brain death was diagnosed 36 hours after the onset of symptoms. Autopsy showed striking calcification of the right and left coronary arteries, with narrowing of the arterial lumen, causing extensive subendocardial infarction. Calcification also affected the aorta, pulmonary arteries, thyroid, kidney and other splanchnic arteries. Generalized arterial calcification of infancy (GACI) was diagnosed and a genetic study found two biallelic variants in ABCC6 gene: p.Arg1114Cys and p.Trp38Ser, both previously described in elastic pseudoxanthoma (PXE), but not in GACI. Genotyping of the healthy parents confirmed genetic segregation with biallelic variants. Discussion: GACI is an extremely rare genetic disease characterized by widespread arterial calcification and narrowing of large and medium-sized vessels. The usual clinical presentation is heart failure in fetal life or in the first months of infancy. In most cases it is lethal, with death occurring within a few hours or days after the onset of symptoms, although clinical involvement is highly variable and cases with long survival have been described. GACI is an autosomal recessive disease secondary to biallelic variants in the ENPP1 gene (67% of cases) and in the ABCC6 gene (9%). The variants found in our patient had not been previously described in GACI, just in PXE, a much milder disease with usually normal lifespan. This case confirms that both entities reflect two extremes of a clinical spectrum of ectopic calcification instead of two different disorders. The aim of presenting this case is to remind clinicians of this rare etiology in neonates or infants with dilated cardiomyopathy. In case of death, autopsy should always be requested. When this condition is diagnosed, genetic study will be positive in 75% of cases, allowing prenatal counseling.

Engineered osteoclasts as living treatment materials for heterotopic ossification therapy

Osteoclasts (OCs), the only cells capable of remodeling bone, can demineralize calcium minerals biologically. Naive OCs have limitations for the removal of ectopic calcification, such as in heterotopic ossification (HO), due to their restricted activity, migration and poor adhesion to sites of ectopic calcification. HO is the formation of pathological mature bone within extraskeletal soft tissues, and there are currently no reliable methods for removing these unexpected calcified plaques. In the present study, we develop a chemical approach to modify OCs with tetracycline (TC) to produce engineered OCs (TC-OCs) with an enhanced capacity for targeting and adhering to ectopic calcified tissue due to a broad affinity for calcium minerals. Unlike naive OCs, TC-OCs are able to effectively remove HO both in vitro and in vivo. This achievement indicates that HO can be reversed using modified OCs and holds promise for engineering cells as “living treatment agents” for cell therapy.

Defining matrix Gla protein expression in the Dunkin-Hartley guinea pig model of spontaneous osteoarthritis

Background Matrix Gla (γ-carboxyglutamate) protein (MGP) is considered a strong inhibitor of ectopic calcification, and it has been associated with OA severity, although not conclusively. We utilized male Dunkin-Hartley (DH) guinea pigs to investigate the expression of MGP throughout aging and disease pathogenesis in a spontaneous model. Method Twenty-five male DH guinea pigs were obtained and nurtured to several timepoints, and then randomly and equally divided by age into five subgroups (1-, 3-, 6-, 9-, and 12-months, with the 1-month group as the reference group). DH guinea pigs in each group were euthanized at the designated month-age and the left or right medial tibial plateaus cartilages were randomly excised. OA severity was described by modified Mankin Score (MMS) at microscopy (Safranin O/Fast Green stain). Proteomic evaluation using isobaric tags for relative and absolute quantification (iTRAQ) was performed to validate the age-related changes in the MGP profiles, and immunohistochemistry (IHC) methods were applied for semi-quantitative determination of MGP expression in articular cartilage. Results The histopathologic findings validated the increasing severity of cartilage degeneration with age in the DH guinea pigs. The MMS showed significant, stepwise (every adjacent comparison P < 0.05) disease progression with month-age. The iTRAQ indicated that MGP levels increased significantly with advancing age (P < 0.05), as supported by the IHC result (P < 0.05). Conclusion Increased expression of MGP in male DH guinea pigs was present throughout aging and disease progression and may be link to increased OA severity. Further studies are needed to investigate and confirm the association between MGP levels and OA severity.

Biomolecules Orchestrating Cardiovascular Calcification

Vascular calcification, once considered a degenerative, end-stage, and inevitable condition, is now recognized as a complex process regulated in a manner similar to skeletal bone at the molecular and cellular levels. Since the initial discovery of bone morphogenetic protein in calcified human atherosclerotic lesions, decades of research have now led to the recognition that the regulatory mechanisms and the biomolecules that control cardiovascular calcification overlap with those controlling skeletal mineralization. In this review, we focus on key biomolecules driving the ectopic calcification in the circulation and their regulation by metabolic, hormonal, and inflammatory stimuli. Although calcium deposits in the vessel wall introduce rupture stress at their edges facing applied tensile stress, they simultaneously reduce rupture stress at the orthogonal edges, leaving the net risk of plaque rupture and consequent cardiac events depending on local material strength. A clinically important consequence of the shared mechanisms between the vascular and bone tissues is that therapeutic agents designed to inhibit vascular calcification may adversely affect skeletal mineralization and vice versa. Thus, it is essential to consider both systems when developing therapeutic strategies.

Morphological and Crystal-Chemical Features of Macro- and Microcalcifications of Human Aorta

Ectopic calcification or pathological biomineralization correlates with morbidity and mortality from cardiovascular diseases. Aortas with atherosclerotic lesions and biomineralization were selected for the study. Thirty samples of mineralized abdominal aortas (group M) were examined by histology. Depending on the calcifications size, samples were separated into group M1 (macroscopic calcifications) and M2 (microscopic calcifications). Each group consists of 15 samples. Calcification 2 mm or less were considered as microscopic, >2 mm—macroscopic. Thirty samples of aortic tissue without biomineralization (group C) were used as a control group. Aortic tissue was examined by macroscopic description, histology, histochemistry, immunohistochemistry (IHC), scanning electron microscopy (SEM) with microanalysis, and transmission electron microscopy (TEM). The results of IHC showed the involvement of OPN in the formation and development of pathological biomineralization, but the obvious role of OPN in the differentiation of macro- and microcalcifications of atherosclerotic aorta was not revealed. SEM with X-ray microanalysis confirmed that the biomineral part of the aortic samples of the M1 group consisted mainly of apatites, which correspond to previous studies. The Ca/P ratio was less in the M2 group than in the M1 group. It means that microcalcifications can be formed by more defective (immature) hydroxyapatite.

Vascular Effects of Pseudoxanthoma Elasticum

Background: Pseudoxanthoma elasticum (PXE) is a rare hereditary disease caused by mutations in the ABCC6 gene, characterized by ectopic calcification of connective tissue throughout the body. Vascular conditions associated with PXE have been well-documented in the literature, but to our knowledge, analysis of the myriad of PXE case reports with associated vascular diseases in addition to larger cohort studies, has not been undertaken. Objective: To review existing literature reporting peripheral vascular disease (PVD), cardiovascular disease (CVD), cerebrovascular disease (CeVD), hypertension, and carotid rete mirabile (CRM) in PXE patients as of June 2021. Methods: A search of the PubMed database using the key words “pseudoxanthoma elasticum” and “vascular” was performed. Results: A total of 345 cases of PVD, 97 cases of CVD, and 123 case of CeVD were reported. Additionally, 88 cases of hypertension and 5 cases of CRM were reported. Conclusions: PXE patients are at risk of developing serious vascular conditions, particularly peripheral vascular disease. This condition also appears to have some connection to carotid rete mirabile, which is extremely rare in humans. Further research should be conducted to analyze the connection between PXE and CRM in order to better understand and treat both conditions.