Arterial Calcification as a Pseudoxanthoma Elasticum-like Manifestation in Beta-Thalassemia: Molecular Mechanisms and Significance

Abstract

Thalassemia, once associated with limited survival, now sees extended life expectancy due to treatment advancements, but new complications such as pseudoxanthoma elasticum (PXE)-like syndrome are emerging. In fact, thalassemia patients develop PXE-like features more frequently than the general population. These features include skin lesions, ocular changes, and vascular issues like arterial calcifications, all linked to oxidative damage from iron overload. PXE-like syndrome in thalassemia mimics inherited PXE but is acquired. The underlying cause is thought to be oxidative stress due to iron overload, which induces free radicals and damages elastic tissues. Unlike inherited PXE, this form does not involve mutations in the ABCC6 gene, suggesting different pathogenic mechanisms, including abnormal fibroblast metabolism and oxidative processes. The vascular calcification seen in this syndrome often follows elastic fiber degeneration, with proteoglycans and glycoproteins acting as nucleation sites for mineralization. The condition can lead to severe cardiovascular and gastrointestinal complications. Studies have shown a significant incidence of PXE-like skin lesions in thalassemia patients, with some dying from cardiovascular complications. Research on ABCC6, a transporter protein involved in ectopic mineralization, has highlighted its role in various conditions, including PXE, beta-thalassemia, and generalized arterial calcification of infancy. ABCC6 mutations or reduced expression led to ectopic mineralization, affecting cardiovascular, ocular, and dermal tissues. The exact molecular mechanisms linking ABCC6 deficiency to ectopic mineralization remain unclear, though it is known to influence calcification-modulating proteins. This review focuses on the role of ABCC6 in the pathogenesis of calcifications, especially intracranial vascular calcifications in PXE and beta-thalassemia.

1. Introduction

In the past, individuals with thalassemia faced significantly shortened life spans, rarely surviving beyond their teens. Advances in medical care have drastically improved survival rates, but this has also led to the emergence of new complications, such as pseudoxanthoma elasticum (PXE)-like syndrome [1]. The first documented connection between PXE and sickle-cell anemia appeared in 1964 [2], while Aessopos et al. established its association with thalassemia in 1989 [3]. Subsequent research has confirmed that PXE-like manifestations are more prevalent among thalassemia patients compared to the general population [4,5,6,7]. For example, a 1998 study investigating thalassemia intermedia patients over 30 years old revealed that 55% exhibited arterial calcifications, 20% had skin lesions, and 52% showed ocular alterations, with 85% having at least one of these features [8].

PXE is characterized by elastorrhexia, involving mineralization and fragmentation of elastic fibers in the skin, eyes, and vasculature [9,10]. While inherited PXE results from genetic mutations, the PXE-like syndrome seen in thalassemia is acquired. It presents similarly with hallmark features such as yellowish papular skin lesions (often coalescing into plaques on the neck, axillae, and periumbilical regions) [11], ocular changes (including retinal hyperpigmentation, “peau d’orange” appearance, and angioid streaks that may lead to vision loss) [9,10,12,13], and vascular issues (early arterial calcifications, degeneration of the elastic laminae, and peripheral occlusive disease) [14].

The PXE-like syndrome associated with thalassemia is likely driven by oxidative stress resulting from iron overload. Free iron and hemoglobin-derived microparticles generate reactive oxygen species that overwhelm antioxidant defenses [8,15,16,17,18]. Elevated neutrophil elastase levels and calcium deposits exacerbate tissue damage and calcification [12]. Unlike inherited PXE, this acquired form is not linked to mutations in the ABCC6 gene, a transporter implicated in PXE pathogenesis [13]. Instead, fibroblast metabolism and oxidative processes play central roles [14,15].

Elastic fiber degeneration in PXE-like syndrome often leads to extensive vascular calcification. Calcium ions bind to proteoglycans and glycoproteins, which act as nucleation sites for mineral deposition [7,14]. Studies like Samarkos et al. [12] have shown that thalassemia-associated PXE-like features do not involve ABCC6 mutations, indicating an alternative pathophysiology. The resulting elastic tissue damage can lead to severe complications, including cardiovascular events (e.g., stroke, myocardial infarction, valvular calcification, and hypertension) [8,9,11,19,20] and gastrointestinal or urologic bleeding [21]. A 12-year longitudinal study by Cianciulli et al. [11] followed 80 Italian beta-thalassemia patients, finding that 17% developed PXE-like skin lesions. Histological findings mirrored those of inherited PXE, and five patients succumbed to cardiovascular complications within 8–132 months of diagnosis.

Recently, research has focused on the molecular mechanisms underlying abnormal soft tissue and vascular calcifications, identifying ABCC6 as a central player. Ectopic mineralization occurs in various conditions, including aging, diabetes, hypercholesterolemia, chronic renal failure, and genetic disorders. Although these processes differ mechanistically, ABCC6 has emerged as a common pathway. This ATP-dependent transporter, primarily expressed in hepatocytes, facilitates the export of unknown substrates into systemic circulation. ABCC6 deficiencies are strongly implicated in diseases such as PXE [22,23], beta-thalassemia [5,11], generalized arterial calcification of infancy (GACI) [24,25], and dystrophic cardiac calcification (DCC) in mice [26]. These disorders share the pathological hallmark of calcification in the cardiovascular, ocular, and dermal tissues.

Inherited PXE and GACI result from inactivating ABCC6 mutations, whereas reduced hepatic ABCC6 expression underlies the ectopic mineralization in beta-thalassemia. Conversely, DCC, an acquired phenotype linked to cardiovascular damage (e.g., ischemia or hyperlipidemia), is also associated with ABCC6 insufficiency. Studies on ABCC6-deficient mice have provided insight into ectopic calcifications, as these models replicate the PXE and DCC phenotypes. Despite significant progress, the exact mechanisms linking hepatic ABCC6 deficiency to distal tissue mineralization remain elusive. Current evidence suggests ABCC6 modulates other calcification regulators, hinting at a broader physiological role than previously understood [27].

This review aims to consolidate current knowledge on ABCC6′s role in calcification processes, focusing particularly on its involvement in PXE and beta-thalassemia-related vascular calcifications, with an emphasis on intracranial manifestations.

2. Molecular Mechanisms Underlying the Role of ABCC6 in Ectopic Calcifications

Calcification of soft tissues in the absence of systemic mineral imbalance is categorized as ectopic or dystrophic calcification. The term “dystrophic” is specifically applied to calcification occurring in injured, damaged, or necrotic tissues. In contrast, elevated calcium or phosphate levels due to imbalanced absorption or secretion result in metastatic calcification. Both forms typically involve calcium phosphate salts, such as hydroxyapatite, and can affect various soft tissues, with the skin, kidneys, tendons, and cardiovascular system being particularly vulnerable.

Vascular calcification, a hallmark of aging, is frequently associated with conditions like hyperlipidemia (atherosclerosis), chronic renal insufficiency, and diabetes, as well as rare genetic disorders. Historically considered a passive process involving calcium and phosphate precipitation, calcification is now understood as a tightly regulated phenomenon. It involves the osteogenic transformation of resident cells, including smooth muscle cells (SMCs), pericytes, and adventitial myofibroblasts. Once thought harmless, calcification is now recognized as a key driver of cardiovascular events and chronic tissue damage, independent of the underlying disease [28]. Despite advances in understanding, the interplay between normal osteogenic signaling and pathological ectopic mineralization remains poorly defined.

The ATP-binding cassette (ABC) transporter ABCC6 has emerged as a critical regulator of ectopic calcification. Reduced ABCC6 levels or loss of its function in the liver are implicated in four distinct mineralization disorders in humans and mice. Pseudoxanthoma elasticum (PXE, MIM 264800) is an autosomal recessive disorder characterized by progressive calcification of elastic fibers in the skin, eyes, and blood vessels, caused by inactivating mutations in the ABCC6 gene [22,23]. In β-thalassemia, calcification phenotypes—especially among individuals of Mediterranean descent—are associated with reduced hepatic ABCC6 expression rather than direct mutations [5,11,29]. Generalized arterial calcification of infancy (GACI), primarily linked to ENPP1 mutations, is also associated with ABCC6 mutations in some cases, highlighting overlapping genetic contributions [24,25]. Dystrophic cardiac calcification (DCC), observed in mice with ABCC6 deficiency, involves acute myocardial and large artery calcification in response to injury, particularly in strains like C3H/HeJ and DBA/2J [30,31].

ABCC6 belongs to the ABC transporter subfamily C, encompassing active transporters and ion-channel-related proteins. This group includes ABCC1–5 (classical transporters) and channel regulators such as ABCC7 (CFTR), ABCC8, and ABCC9 (SUR1 and SUR2). ABCC6 functions as an ATP-driven efflux pump for glutathione conjugates, including N-ethylmaleimide-glutathione (NEM-GS) and leukotriene C4 (LTC4), though its affinity for LTC4 is significantly lower than for NEM-GS [32,33]. Unlike other glutathione-conjugate transporters, ABCC6 does not efficiently transport 17-β-estradiol-17-β-D-glucuronide [34]. Benzbromarone and indomethacin are potent inhibitors of ABCC6, suggesting a narrow substrate specificity. Despite extensive research, the endogenous substrates of ABCC6 and their roles in disorders like PXE, β-thalassemia, GACI, and DCC remain elusive.

Structural insights into ABCC6 have been advanced using homology models based on the prokaryotic Sav1866 export pump. These models have pinpointed critical regions, such as the transmission interface and domain interactions, as sites where mutations disrupt function [35,36]. ABCC6 is localized to the basolateral membrane of hepatocytes in humans, rats, and mice [37,38,39], with additional expression in kidney proximal tubules [40,41]. PXE-causing ABCC6 mutations result in defective localization, ATP utilization, or protein folding, leading to functional impairments and variability in disease presentation [2,23,42].

Vitamin K metabolism has been explored as a potential link to PXE. While early studies suggested vitamin K-dependent pathways might involve ABCC6 substrates, subsequent research has found no evidence that ABCC6 transports vitamin K or its derivatives effectively [43,44,45,46,47,48]. Another proposed substrate, adenosine, linked to arterial calcification in CD73-deficient ACDC patients, has also been ruled out as a direct ABCC6 substrate [49,50,51].

A summary of the molecular mechanisms of ABCC6-related ectopic calcifications is proposed in

Table 1. Summary of the molecular mechanisms of ectopic calcifications related to ABCC6.

Molecular Mechanism	Involved Molecules/Proteins	Involved Cells
ABCC6 Deficiency	ABCC6 (transport protein)	Fibroblasts, endothelial cells
Alteration of PPi Metabolism	Adenosine pyrophosphate (PPi)	Smooth muscle cells, fibroblasts
Accumulation of Phosphates	Phosphates, extracellular matrix proteins	Smooth muscle cells, osteoblasts
Inhibition of Calcification	Inhibitory proteins: MGP (Matrix Gla Protein), OPN (Osteopontin)	Endothelial cells, fibroblasts
Activation of Pro-calcific Pathways	BMP (Bone Morphogenetic Proteins), TGF-β (Transforming Growth Factor-beta)	Mesenchymal cells, osteoblasts
Ectopic Mineralization	Hydroxyapatite crystals, calcium phosphate	Connective tissue, vascular tissue

.

The following steps are involved:

• ABCC6 Deficiency: Mutations in the ABCC6 gene lead to reduced expression of the protein, which is essential for the transport of metabolites and regulation of PPi.
• Alteration of PPi Metabolism: A decrease in PPi, an inhibitor of mineralization, results in increased calcification in non-bone tissues.
• Accumulation of Phosphates: The rise in phosphates due to impaired mineral metabolism promotes mineral deposition.
• Inhibition of Calcification: Proteins like MGP and OPN are crucial for inhibiting calcification; ABCC6 deficiency compromises their functionality.
• Activation of Pro-calcific Pathways: Proteins such as BMP and TGF-β promote the differentiation of mesenchymal cells into osteoblasts, increasing calcification.
• Ectopic Mineralization: An abnormal deposition of hydroxyapatite crystals and calcium phosphate occurs in tissues, contributing to ectopic calcifications.

Despite progress, ABCC6’s exact physiological role remains fragmented, hinting at a signaling or hormonal function affecting connective tissues. While calcification is the most visible consequence of PXE, GACI, and DCC, it may reflect only part of a broader, underlying pathological process. Further research is essential to elucidate the molecular and systemic pathways modulated by ABCC6.

3. PXE and PXE-like Diseases and the Role of ABCC6 as a Genetic Modifier

ABCC6 is a member of the ATP-binding cassette (ABC) transporter subfamily C, which includes multi-drug resistance proteins. It encodes a transmembrane protein that utilizes ATP hydrolysis to transport organic anions across cellular membranes. Although its specific physiological role remains incompletely understood, ABCC6 expression is predominantly observed in the liver and kidneys, with lesser expression in the intestine, retina, lung, skin, and vasculature [22,39,40]. The exact function of ABCC6 in these tissues remains unclear, but its deficiency is strongly associated with ectopic calcification in conditions such as pseudoxanthoma elasticum (PXE), β-thalassemia, and generalized arterial calcification of infancy (GACI) in humans, as well as dystrophic cardiac calcification (DCC) in mice. Notably, studies suggest that the liver plays a critical role in the calcification phenotype [46,52].

PXE (MIM 264800) is an autosomal recessive disorder affecting connective tissues, characterized by the calcification and fragmentation of elastic fibers [42]. Clinically, PXE predominantly affects the skin, Bruch’s membrane in the eyes, and the cardiovascular system. Symptoms include skin sagging, visual impairment due to retinal hemorrhages, and peripheral arterial disease (PAD) with associated gastrointestinal bleeding and intermittent claudication. ABCC6 is localized to the basolateral membrane of hepatocytes and proximal kidney tubule cells, where its inability to secrete a yet-unknown substrate into circulation likely drives ectopic calcification. This positions PXE as a metabolic disorder with connective tissue manifestations.

β-thalassemia (MIM 141900) is a monogenic disorder caused by mutations in the β-globin gene, leading to insufficient β-globin production. This results in unstable α-globin chain precipitation, red blood cell precursor destruction, and ineffective erythropoiesis. Clinical complications include bone deformities, iron overload, and transfusion-related issues [53]. Over the past decade, many Mediterranean β-thalassemia and sickle cell anemia patients have exhibited PXE-like manifestations [54]. While β-thalassemia and PXE are distinct genetic disorders, the ectopic calcification in β-thalassemia patients is clinically and structurally indistinguishable from PXE [6,7,14,55].

PXE-like mineralization in β-thalassemia patients occurs independently of ABCC6 mutations [29]. However, it is hypothesized that ABCC6 expression or function in the liver and kidneys is secondarily affected by the hemoglobinopathy. In a β-thalassemia mouse model (Hbbth3/+), ABCC6 expression progressively declined in the liver, stabilizing at 25% of wild-type levels by advanced age. This downregulation was attributed to the absence of the transcription factor NF-E2, a key regulator of hemoglobin-related genes [56]. While these mice did not develop spontaneous calcification—likely due to the incomplete downregulation of ABCC6—this molecular pathway may parallel PXE-like mineralization in human β-thalassemia patients [52].

GACI is a rare autosomal recessive disorder characterized by severe arterial media calcification, vascular occlusion, hypertension, myocardial ischemia, and congestive heart failure. Most patients succumb within the first six months of life. Biallelic ENPP1 mutations are the primary cause, but a subset of GACI patients without ENPP1 mutations have ABCC6 mutations. These findings highlight a shared mechanism between GACI and PXE. PXE-specific features, such as angioid streaks and characteristic skin lesions, are occasionally observed in GACI patients with ENPP1 mutations [25]. This overlap suggests that PXE and GACI represent a spectrum of ectopic calcification disorders, with both ABCC6 and ENPP1 playing critical roles in mineralization inhibition.

A PXE-like syndrome associated with gamma-glutamyl carboxylase (GGCX) mutations shares phenotypic similarities with PXE. GGCX deficiency affects the activation of Matrix Gla Protein (MGP), a key calcification inhibitor [44]. Unlike the PXE-GACI connection, the similarities between PXE and GGCX-related syndromes reflect converging phenotypes rather than shared molecular pathways. These syndromes differ structurally, with GGCX-related conditions exhibiting greater skin laxity compared to PXE [43,57].

ABCC6 may function as a phenotype modifier in related diseases. Mungrue et al. [58] found that myocardial necrosis and calcification in Abcc6-null mice occurred only after prolonged ischemic injury, indicating that ABCC6 dysfunction exacerbates damage under specific conditions. While some studies suggest that heterozygous carriers of ABCC6 mutations may be at increased risk for cardiovascular complications [59,60], larger studies involving 66,831 individuals found no significant association between the common ABCC6 p.R1141X mutation and ischemic heart disease or stroke [61]. Thus, the role of ABCC6 mutations in vascular-related conditions remains contentious, warranting further investigation.

4. Calcifications and Vascular Remodeling

Arterial remodeling is driven by complex and interrelated pathophysiological mechanisms that affect both the cellular and non-cellular components of the vascular wall. These mechanisms include fibrosis, hyperplasia of the arterial intima and media, changes in vascular collagen and elastin composition, endothelial dysfunction, and arterial calcification. The migration and proliferation of vascular smooth muscle cells (VSMCs) significantly contribute to intimal thickening. Furthermore, VSMC phenotypic switching—from a contractile to a secretory or osteogenic state—can increase vascular tone and promote extracellular matrix (ECM) calcification. Alterations in the activity of vitamin K-dependent proteins also play a role in vascular remodeling, particularly in the induction of calcification. Due to the complexity and interplay of these processes, isolating the contribution of a single mechanism to arterial remodeling remains a challenge [62,63].

Monogenic diseases, such as PXE, PXE-like syndrome, Marfan syndrome, and Kuehtel syndrome, provide valuable insights into arterial remodeling. These conditions, though rare, share clinical features with common arterial remodeling processes and are caused by specific genetic defects that impact one or more underlying mechanisms. Studying these diseases offers a unique opportunity to unravel the pathophysiology of more prevalent cardiovascular conditions, including hypertension, diabetes mellitus, chronic kidney disease, and vascular aging.

Arterial remodeling refers to structural and functional changes in the vascular wall in response to mechanical, hemodynamic, or pathological stimuli. Broadly, it is categorized into (I) atherosclerosis (a localized inflammatory process characterized by lipid plaque accumulation) and (II) arteriosclerosis (generalized changes in the arterial media, often associated with aging, cardiovascular diseases, or metabolic disorders) [64]. Arterial remodeling can manifest in various structural alterations, including (I) hypertrophic remodeling (vascular wall thickening); (II) eutrophic remodeling (maintenance of constant wall thickness); and (III) hypotrophic remodeling (vascular wall thinning) [65].

The type of remodeling observed often depends on the size and type of the affected artery. For example, larger elastic arteries commonly undergo outward hypertrophic remodeling, whereas smaller muscular arteries exhibit inward eutrophic or hypertrophic remodeling, often in response to sustained vasoconstriction [66,67]. These remodeling types and their characteristics are summarized in

Table 2. Subtypes of vascular remodeling.

Type of Vascular Remodeling	Description	Wall-Lumen Ratio	Vessel Changes	Common in
Hypotrophic Remodeling	Thinning of the vascular wall	Lower wall-to-lumen ratio	Vessel wall becomes thinner	Rare, can be seen in certain cardiovascular conditions
Eutrophic Remodeling	Constant wall thickness, but diameter may change	No change in wall-to-lumen ratio	Diameter of the vessel changes, but wall thickness stays the same	May occur with certain cardiovascular diseases
Hypertrophic Remodeling	Thickening of the vascular wall due to cellular changes or ECM deposition	Higher wall-to-lumen ratio	Vessel diameter may or may not increase, but the wall becomes thicker	Common in arteriosclerosis, hypertension, and atherosclerosis

.

The arterial remodeling process involves thickening of the arterial wall through mechanisms such as intimal hyperplasia, smooth muscle cell (SMC) hypertrophy, and ECM deposition, including mineralization [68,69]. Under normal conditions, ECM fibers in the arterial media, primarily elastin, provide elasticity and maintain vessel stability. During remodeling, these fibers fragment, leading to increased vessel stiffness as SMCs predominantly synthesize non-elastic collagen instead of elastin [70]. This stiffness is further exacerbated by calcium deposition within the ECM [71].

Endothelial cells play a pivotal role in regulating arterial remodeling by producing nitric oxide (NO), which modulates SMC contraction and relaxation. However, endothelial dysfunction—common in aging and cardiovascular diseases—can lead to altered blood flow and the release of pro-remodeling cytokines and growth factors, such as transforming growth factor-beta (TGF-β) and bone morphogenetic proteins (BMPs). These signaling molecules stimulate SMC and pericyte proliferation, as well as ECM deposition, contributing to arterial stiffening and structural remodeling [72,73].

Arterial remodeling is governed by several interconnected processes, including (I) SMC proliferation (expansion of SMC populations in response to hemodynamic stress or biochemical signals); (II) elastin degradation (breakdown of elastic fibers, reducing vascular compliance and stability); and (III) ECM calcification (deposition of calcium in the ECM, further enhancing stiffness and dysfunction).

Genetic diseases, such as PXE and Marfan syndrome, have provided key insights into the mechanisms underlying vascular remodeling. For instance, mutations affecting ECM composition or maintenance pathways in these diseases highlight the critical role of structural and regulatory proteins in vascular homeostasis.

Arterial remodeling is a major contributor to vascular diseases, which remain leading causes of morbidity and mortality worldwide. Conditions such as myocardial infarction, stroke, and ischemia are directly linked to pathological remodeling processes. Factors like hypertension, diabetes, smoking, and genetic abnormalities accelerate arterial remodeling, increasing arterial stiffness. This stiffening impairs organ function, contributes to end-organ damage, and elevates mortality risk.

The interplay between genetic and acquired conditions in the pathophysiology of arterial remodeling is summarized in

Table 3. Pathophysiological pathways leading to arterial remodeling in genetic and cardiovascular disease. Abbreviations: MMP, matrix metalloproteinases; VSMC, vascular smooth muscle cell; GGCX, gamma glutamyl transferase.

Pathophysiological Pathways	Associated Mechanisms
Arterial Remodeling in Genetic Diseases	Genetic mutations leading to altered vascular function, extracellular matrix (ECM) changes
Matrix Metalloproteinase (MMP) Activity	MMPs contribute to ECM degradation, affecting arterial structure and leading to remodeling
Vascular Smooth Muscle Cell (VSMC) Changes	VSMC proliferation, migration, and phenotypic switching contributing to vascular remodeling
Gamma Glutamyl Transferase (GGCX) Activity	GGCX is involved in the post-translational modification of matrix proteins, influencing vessel tone

, providing a comprehensive overview of the cellular and molecular contributors to vascular disease.

Structural changes in arterial walls during remodeling include modifications to extracellular matrix (ECM) components, especially elastic fibers, which are essential for vascular elasticity. As remodeling advances, these fibers lose integrity, leading to increased vessel stiffness and conditions like aortic stiffening [74]. Vascular smooth muscle cells (VSMCs) play a crucial role in this process, switching between contractile and synthetic phenotypes in response to stimuli. This switching is vital for remodeling and is linked to diseases like Marfan’s syndrome, where defective elastic fiber synthesis worsens vascular dysfunction [75]. Elevated phosphate levels drive VSMCs toward an osteogenic phenotype, contributing to vascular calcification and arterial stiffening [76,77]. Under certain conditions, including high extracellular calcium and phosphate or lack of calcification inhibitors, VSMCs can differentiate into osteogenic-like cells, characterized by downregulation of mineralization inhibitors, increased alkaline phosphatase activity, and release of Matrix Vesicles (MVs) for calcium phosphate deposition [78]. In phosphate-rich environments, VSMCs upregulate osteogenic markers while downregulating VSMC-specific markers [78]. Bone morphogenetic protein-2 (BMP-2), which promotes osteogenic differentiation, is also upregulated in atherosclerotic lesions [79]. The transition of VSMCs to osteogenic or chondrocytic-like cells induced by BMP-2 is inhibited by calcification regulators like matrix Gla-protein (MGP), which helps maintain VSMC contractility by binding to BMP-2 [80,81]. MGP deficiency, as seen in MGP-deficient mice, leads to mineral deposition in elastic fibers and a phenotypic shift in VSMCs due to increased bone-related protein expression and decreased smooth muscle markers [82,83]. This deficiency disrupts the balance between calcification-promoting and inhibiting factors, driving pathological remodeling of the vascular wall.

Tanimura et al. [84] first linked small, membrane-bound particles known as Matrix Vesicles (MVs) to vascular calcification. These vesicles, originating from vascular smooth muscle cells (VSMCs), are present in both the intimal and medial layers of the vascular wall [85,86,87]. Initially, the release of MVs by VSMCs was seen as a protective response to calcium overload and apoptosis [88]. However, further studies revealed their significant role in vascular conditions like atherosclerosis and hypertension [85,89]. In vitro research showed that MVs serve as nucleation sites for vascular calcification, aiding mineral deposition [90]. Elastin is crucial for arterial wall stability and VSMC function, with conditions like Marfan’s disease highlighting its importance. It is also a key substrate for vascular calcification, as seen in PXE, which features extensive calcification along elastic fibers, leading to vascular morbidity and premature mortality despite primarily cosmetic skin symptoms [91]. Elastin degradation is a hallmark of aging vasculature and aortic stiffening. While the exact mechanisms behind age-related elastin degradation are not fully understood, it was initially suggested that mechanical fatigue from repetitive cyclic stretching during heartbeats was a primary factor. This degradation may be worsened by conditions like systolic hypertension, where increased pulse pressure adds stress to the vascular wall, accelerating elastin deterioration. In vitro studies support this, showing a correlation between elastin structural deterioration and the cumulative number of heartbeats over time [92].

Vascular calcification is now viewed as a regulated process rather than a passive mineral deposition, influenced by VSMC phenotype switching and ECM degradation. This regulated calcification arises from an imbalance between promoting and inhibiting factors and is characteristic of various genetic disorders, including Keutel’s syndrome, PXE, PXE-like syndrome, and beta-thalassemia. Keutel’s syndrome, caused by a mutation in the matrix Gla-protein (MGP) gene, exemplifies an inherited disorder linked to abnormal vascular calcification. MGP, a vitamin K-dependent protein, is essential for inhibiting vascular calcification, and insights from this syndrome have illuminated mechanisms behind vitamin K antagonist-induced calcification [93,94,95]. In PXE, vascular calcification is associated with a loss-of-function mutation in the ABCC6 gene, which encodes the Multi Drug Resistant Protein 6 (MDRP-6). Although the specific substrate of MDRP-6 is unknown, research indicates that factors like VSMC phenotype switching, oxidative stress, and interference with MGP carboxylation may trigger elastin calcification in PXE [96,97,98,99,100]. PXE-like syndrome arises from mutations in the GGCX gene, responsible for γ-glutamylcarboxylase, leading to elastin fiber calcification and disrupting vitamin K-dependent coagulation factors, which increases bleeding risk [43,99]. This mutation reduces MGP activity, similarly impairing calcification inhibition as seen in Keutel’s syndrome [43,94].

Vitamin K-dependent proteins, particularly MGP, are crucial in regulating vascular calcification. GGCX mutations or vitamin K antagonists (e.g., warfarin) decrease MGP carboxylation, raising calcification risk [93,94]. Such antagonists inhibit the vitamin K cycle, further diminishing MGP activity and worsening calcification. Increasing vitamin K intake may help counteract this process and potentially reverse vascular calcification in certain cases. Overall, MGP plays a significant regulatory role in vascular calcification, particularly in genetic conditions like PXE, PXE-like syndrome, and Keutel’s syndrome. The complex interplay between VSMC phenotype switching, oxidative stress, and ECM degradation underscores the need for therapeutic strategies targeting MGP activity, such as vitamin K supplementation, to mitigate arterial calcification in both rare genetic disorders and common vascular diseases.

In PXE, PXE-like syndrome, and Keutel’s syndrome, arterial calcification is a hallmark feature. However, this process is also prevalent in conditions like diabetes, hyperparathyroidism, chronic kidney disease, and vascular aging. Certain medications, especially those that disrupt the balance between pro-calcification and anti-calcification factors, can also induce vascular calcification; for example, chronic use of vitamin K antagonists such as warfarin is linked to peripheral artery calcification [93]. In coronary arteries, calcification often reflects atherosclerosis, which can be quantified using computed tomography (CT) to assess calcium scores. The calcium score, measured in Agatston units, is crucial for risk stratification in coronary artery disease and decision-making regarding revascularization and diagnostic angiography. A low calcium score indicates a low likelihood of atherosclerotic plaque, while a high score correlates with increased cardiovascular risk [101,102]. These findings highlight the importance of targeting calcification therapeutically. The challenge is to develop interventions that address modifiable factors in the calcification process. As seen in PXE and related syndromes, MGP and the vitamin K cycle are vital regulators of calcification and VSMC phenotype switching. MGP requires vitamin K-dependent carboxylation for biological activity, suggesting that vitamin K treatment might inhibit or reverse arterial calcification and slow arterial stiffness progression. Research has shown that in rats with extensive calcification from warfarin treatment, subsequent vitamin K administration reversed the calcification [94]. In humans, a three-year trial of daily 500 mcg vitamin K supplementation, alongside a multivitamin, halted the progression of vascular calcification [103]. The Rotterdam study found that higher dietary vitamin K intake was linked to better cardiovascular outcomes and reduced coronary artery calcification [104,105]. Additionally, post-menopausal women receiving vitamin K treatment showed improvements in vascular stiffness markers [106]. Westenfeld et al. [107] also demonstrated that vitamin K2 supplementation decreased plasma levels of inactive, undercarboxylated MGP, further supporting the potential of vitamin K in regulating vascular calcification.

5. Arterial Calcifications in PXE and PXE-like Conditions

PXE is an inherited autosomal recessive multisystem disorder that affects connective tissues, characterized by the fragmentation and mineralization of elastic fibers. This condition primarily affects the skin, retina (specifically the Bruch’s membrane), and vasculature, leading to a phenomenon known as elastorrhexis [91,107]. As previously written, the disorder is caused by mutations in the ABCC6 gene, which encodes a transmembrane ATP-binding cassette transporter (ABCC6/MRP6). This gene is primarily expressed in the liver and kidneys, with lower expression levels in tissues such as the skin, eyes, and arteries [108]. Despite the identification of the genetic mutation, the biological function of the ABCC6 transporter and its substrates remains unknown. The pathogenesis of PXE is believed to stem from a defect originating in the liver and kidneys, which leads to extracellular calcium deposition, likely influenced by intracellular processes [52]. This supports the notion that PXE is a prototypical genetic metabolic calcification disorder [109]. PXE is marked by a delayed onset and considerable variability in its phenotypic expression, suggesting that various cofactors contribute to its presentation. Classical risk factors for arteriosclerosis, such as smoking, hypertension, and dyslipidemia, are suspected to exacerbate the vascular manifestations of the disease, although these vascular complications typically occur later in life (usually after age 40), while skin and eye lesions may manifest earlier. Interestingly, there is an unexplained female preponderance in PXE, with a 2:1 female-to-male ratio, leading to a higher prevalence of arterial disease in females compared to the general population [91].

A PXE-like phenotype has also been reported in several other genetic disorders, such as beta-thalassemia, sickle cell anemia [110], cutis laxa, generalized arterial calcification in infancy, defects in gamma-glutamyl carboxylase, familial idiopathic basal ganglia, and rarely in association with pharmacological substances such as D-penicillamine. The overlap of PXE’s phenotype with other genetic diseases suggests that these conditions may share a common pathophysiological mechanism [25]. The main phenotypic differences between PXE and other genetic calcifying diseases are summarized in

Table 4. Main phenotypic differences between PXE and PXE-like conditions.

Arterial Phenotype	Beta-Thalassemia/Sickle Cell Disease	PXE-like + Cutis Laxa	PXE	GACI/ACDC (or CALJA)
Defective gene	HBB/HBF	GGCX	ABCC6	ENPP1, NT5E
Intima-media thickness	Increased	Unknown	Unknown	Unknown
Endothelial dysfunction	Present	Unknown	Unknown	Unknown
Stenosis	Yes (26.7% mostly cerebral)	Yes (PAD 50%)	Yes (mostly coronary and cerebral)	Yes (mostly coronary, cerebral, and renal)
Aneurysm	Sporadic	Yes (mostly cerebral)	Infrequent	Popliteal
Arterial calcifications	Present	Present	Present	Present
Coagulation defects	Present (50%)	Present	Secondary?	Present

GACI: generalized arterial calcification of infancy; ACDC (or CALJA): arterial calcification or calcification of joints and arteries due to deficiency of CD73; HBB/HBF: hemoglobinopathies beta and S; GGCX: gamma-glutamylcarboxylase; ABCC6: adenosine triphosphate-binding cassette (ABC) gene subfamily C; ENPP1: ectonucleotide pyrophosphatase/phosphodiesterase 1; PAD: peripheral artery disease.

[111].

The arterial lesions observed in PXE primarily involve the mineralization of elastic fibers within the medial layer of medium- and small-sized musculo-elastic arteries. These abnormal elastic fibers are thought to be produced by PXE skin fibroblasts, though they can also form when normal fibroblasts are exposed to PXE serum or elastin degradation products. This suggests that PXE is a disorder affecting the mechanisms responsible for matrix constituent production, with elastic fiber mineralization resulting from factors abnormally produced and trapped within the fibers during elastin synthesis [112,113,114]. This led to the development of the “elastosis hypothesis”, which proposes that the elastosis (abnormal formation of elastin) is the primary mechanism in PXE, while calcification is secondary. Smooth muscle cells and fibroblasts are suspected to play a key role in this process, as they are sources of regulatory proteins involved in calcification, such as alkaline phosphatase and matrix Gla-protein (MGP) [90,114]. Additionally, an abnormal balance between elevated proteolytic activity and increased levels of P-selectin, matrix metalloproteinases (MMP) 2 and 9, indicates improper remodeling of the ECM in PXE [115,116]. Although the exact sequence of mechanisms leading to elastosis and calcification in PXE remains unknown, the nature and location of the calcification help explain its functional consequences. Calcification along the arterial tree can be mapped using X-ray imaging, with 3D reconstructions via helicoidal X-rays providing more precise quantification and site identification. In PXE, calcification is most commonly found in the distal superficial femoral and below-knee arteries (such as the tibial and pedal arteries), similar to the distribution seen in age-related arteriosclerosis. Histological findings of vascular lesions in PXE are limited, typically obtained from rare autopsy samples. Ultrastructural analyses of the ascending aorta, iliac arteries, and vena cava in two male PXE patients (ages 36 and 80) revealed vascular damage with spotty alterations of elastic fibers and aggregates of amorphous elastin. Von Kossa staining confirmed the presence of calcium deposits in the medial layers of the arteries, particularly in medium-sized arteries like the carotids and small-sized arteries such as the radials. In the carotid arteries, calcification was observed extracellularly around the elastin fibers, though a slight increase in intracellular calcium was also noted. Additionally, proteoglycans accumulated preferentially in the media rather than the intima, compared to control vessels [117]. Qualitative and quantitative alterations in proteoglycan metabolism have also been reported in PXE patients. Urine analysis showed increased levels of heparan sulfate and decreased levels of chondroitin sulfate, further suggesting ECM remodeling disruptions in this disease [118].

The functional impacts of arterial lesions in PXE, including elastocalcinosis and proteoglycan accumulation, have been explored in a limited number of studies. Most of the available data come from non-invasive imaging techniques, particularly ultrasound, examining the carotid and radial arteries, documenting an increased intima-media thickness (IMT) with inward remodeling [119]. Although these findings remain to be confirmed in larger cohorts, they suggest that arterial remodeling in PXE differs from other diseases such as aging, hypertension, or atherosclerosis. PXE typically exhibits intima-media thickening in large and medium-sized musculo-elastic arteries, unlike aging, hypertension, or atherosclerosis, which are more often associated with inward remodeling [120]. Fragmentation of elastic fibers and calcification are expected to impact the elasticity and distensibility of PXE arteries. Some studies found no significant change in distensibility in large arteries (e.g., carotid) compared to age- and sex-matched controls [118,119], while others observed an increase in medium-sized arteries like the radial artery. This suggests that the altered extracellular matrix, resulting from elastin fragmentation, proteoglycan replacement, and calcification, may mask the expected increase in arterial stiffness due to calcification.

The main features of calcification in PXE are summarized in

Table 5. PXE-related calcifications and their features.

Issues	Features
Primary Lesion	Mineralization of elastic fibers in the medial layer of medium- and small-sized arteries.
Source of Abnormal Fibers	Produced by PXE skin fibroblasts or normal fibroblasts exposed to PXE serum/degradation products.
Elastosis Hypothesis	Proposes abnormal elastin formation (elastosis) as the primary mechanism, with calcification being secondary.
Key Cells Involved	Smooth muscle cells and fibroblasts, which produce regulatory proteins like alkaline phosphatase and MGP.
ECM Remodeling Indicators	Abnormal balance of proteolytic activity and increased levels of P-selectin and MMPs (2 and 9).
Calcification Mapping	Utilizes X-ray imaging and 3D reconstructions for precise quantification and site identification.
Common Calcification Sites	Distal superficial femoral and below-knee arteries (tibial and pedal arteries).
Histological Findings	Limited; includes vascular damage, alterations of elastic fibers, and calcium deposits confirmed by Von Kossa staining.
Proteoglycan Changes	Increased heparan sulfate and decreased chondroitin sulfate levels in urine analysis.
Functional Impacts	Explored through ultrasound; increased intima-media thickness (IMT) and inward remodeling observed.
Comparison with Other Diseases	PXE shows different arterial remodeling compared to aging, hypertension, or atherosclerosis; typically exhibits intima-media thickening.
Impact on Elasticity	Fragmentation and calcification impact elasticity; mixed findings on distensibility in large vs. medium-sized arteries. Altered ECM from fragmentation, proteoglycan replacement, and calcification may mask expected arterial stiffness increase.

.

In β-thalassemia patients, vascular complications and myocardial damage are significant concerns. Iron overload, hemolysis-induced reductions in nitric oxide (NO) bioavailability, and increased lipid peroxidation have all been identified as contributing factors to endothelial dysfunction and arterial stiffness [121,122,123]. Additionally, inflammatory factors—such as those altered by transfusions, infectious agents, and cytokine levels of stored allogeneic blood—further exacerbate vascular damage. These factors, alongside stromal cells of hyperplastic bone marrow, also play a role in promoting vascular injury [124]. Arterial stiffness has been observed in both peripheral and central elastic arteries in β-thalassemia patients, with postmortem examinations revealing increased fibrosis and glycosaminoglycan content in the aorta, iliac, and pulmonary arteries. Radiological studies have also reported calcifications in arteries, such as the posterior tibial artery [125]. A study by Nassef et al. [126] highlighted that central vascular ischemia is more prevalent than peripheral ischemia in patients with β-thalassemia intermedia (β-TI). Interestingly, no significant correlation was found between IMT, a marker for subclinical atherosclerosis, and ferritin or cholesterol levels in β-TI patients. Regarding iron chelation therapy, although it is used to manage iron overload, patients with transfusion-dependent thalassemia (TDT) still experience high iron levels, leading to arterial structural alterations, damage to elastic tissues, and calcification. Several studies have demonstrated early signs of atherosclerosis in both TDT and non-transfusion-dependent thalassemia (NTDT) patients. In NTDT, the mechanisms of atherosclerosis remain unclear, as no correlation between IMT and ferritin or cholesterol levels has been observed. Conversely, in TDT patients, a positive correlation has been reported between carotid atherosclerosis and serum ferritin levels, indicating that iron overload plays a significant role in the development of vascular complications [127].

Aessopos et al. [8] investigated the prevalence of arterial calcifications in patients with beta thalassemia as PXE-like phenotype. They included 40 patients diagnosed as having the major or intermediate form of beta-thalassemia older than 30 years and a control group represented by 40 healthy age- and sex-matched subjects among the first-degree relatives of the patients. In beta-thalassemia, PXE findings are age-related [4]. Tibial artery calcifications were investigated using radiographs. Among the 40 patients who were radiographically examined, 22 (55%) had arterial calcifications at the posterior tibial artery region. They all had the intermediate form of the disease, and only four of them had a smoking history. The mean age of the patients with calcifications was 41.2 years. None of these patients had symptoms or signs of arterial disease. In the control group, calcifications were observed in six (15%) subjects. The difference in the prevalence of arterial calcifications between the two groups was statistically significant (Fischer’s Exact Test, p = 0.0003). Dystrophic calcification is commonly observed in various conditions such as Ehlers–Danlos syndrome, pseudoxanthoma elasticum, arteriosclerosis obliterans, and Monckeberg’s medial arteriosclerosis [128]. The most notable connection in the present case is with pseudoxanthoma elasticum, as a significant proportion (62.5%) of patients also exhibited one of the two major clinical features of this disorder: angioid streaks and/or skin lesions. The authors concluded that the finding of a high prevalence (55%) of arterial calcifications supports a correlation between beta-thalassemia patients and the entire spectrum of clinical manifestations of the hereditary PXE. A genetic linkage should have been extremely strong, but it is a remote possibility in view of the fact that the genes for collagen I and elastin are located on chromosomes 17 and 7, respectively, while those for beta-globin are on chromosome 11 [129,130]. However, several factors may affect this phenotype. In fact, regular transfusion therapy, while essential for the adequate management of severely affected patients, is associated with a variety of iron overload-related complications, including endocrine disorders, myocardiopathy, and liver fibrosis, all of which contribute to increased morbidity and mortality. Approximately 16% of patients with major or intermediate β-thalassemia display progressive skin, eye, and vascular pathologies similar to those observed in PXE, a genetic disease in which soft connective tissues are specifically affected [2,3,4,5,6,7,8]. Ultrastructural studies of dermal biopsies in these β-thalassemia patients have demonstrated calcification of elastic fibers, deposition of abnormal matrix constituents in the extracellular space, and abnormal collagen fibrillogenesis, all of which closely resemble the changes seen in PXE.

Table 6. Key points regarding vascular complications in β-thalassemia and their associations with PXE-like conditions.

Issues	Features
Vascular Complications	Significant concerns in β-thalassemia include iron overload, nitric oxide (NO) reduction, and increased lipid peroxidation.
Contributing Factors	Inflammatory factors from transfusions, infections, cytokines, and stromal cells of hyperplastic bone marrow promote vascular injury.
Arterial Stiffness	Observed in both peripheral and central elastic arteries; postmortem exams show increased fibrosis and glycosaminoglycan content in various arteries.
Calcifications	Radiological studies report calcifications, particularly in the posterior tibial artery.
Ischemia Prevalence	Central vascular ischemia is more common than peripheral ischemia in β-thalassemia intermedia (β-TI) patients.
IMT and Iron Levels	No significant correlation was found between intima-media thickness (IMT) and ferritin or cholesterol levels in β-TI patients.
Iron Chelation Therapy	Used to manage iron overload; however, patients with transfusion-dependent thalassemia (TDT) still experience high iron levels and structural arterial changes.
Atherosclerosis Signs	Early signs were observed in both TDT and non-transfusion-dependent thalassemia (NTDT) patients; unclear mechanisms in NTDT.
Correlation in TDT Patients	Positive correlation between carotid atherosclerosis and serum ferritin levels, indicating iron’s role in vascular complications.
Dystrophic Calcification	Commonly seen in conditions like Ehlers–Danlos syndrome and PXE; 62.5% of β-thalassemia patients exhibited features of PXE.
Genetic Linkage	Remote possibility due to different chromosomal locations for relevant genes; factors affecting the phenotype include regular transfusion therapy complications.
Progressive Pathologies	16% of patients with major or intermediate β-thalassemia show skin, eye, and vascular pathologies similar to PXE.
Ultrastructural Studies	Show calcification of elastic fibers and abnormal matrix constituents, resembling changes seen in PXE.

summarizes the main features of PXE-like manifestations in β-thalassemia.

6. Focus on Intracranial Arterial Calcifications

In general, in beta-thalassemia, intracranial vasculature, including arteries and venous sinuses, does not appear to be significantly involved, as recent literature has not confirmed an increased rate or progression of intracranial stenosis, venous thrombosis, or aneurysms [131,132,133]. However, a regionally specific vulnerability of the white matter has been identified in a large sample of anemia patients, including those with β-thalassemia [134]. Specifically, decreased white matter volume and microstructural integrity were observed in the watershed areas, which are the regions of white matter situated between two major cerebral artery territories [134,135]. This finding suggests an increased vulnerability of brain perfusion in these regions. A recent perfusion MRI study using arterial spin labeling (ASL) found that relative hyperperfusion of watershed territories represents a hemodynamic hallmark of beta-thalassemia anemia, challenging previous hypotheses of brain injury in hereditary anemias [136]. Unfortunately, the presence of intracranial arterial calcifications was not examined in this study, so the hypothesis that the perfusion alterations found may depend on an altered autoregulation due to greater stiffness of the intracranial arteries, whether or not they have parietal calcifications, cannot be further verified.

However, in patients with β-thalassemia intermedia (β-TI), silent cerebral infarcts (SCI) have been widely reported in various regions of the brain, with the most frequent involvement seen in the parietal and frontal white matter. Subcortical white matter is commonly affected. Nemtsas et al. [137] observed that cerebrovascular involvement in β-TI patients, when compared to β-thalassemia major (β-TM) patients, tends to be asymptomatic. Lesions in β-TI patients are typically smaller, and involvement is predominantly seen in deeper brain structures rather than the cortex. Some β-TI patients have numerous and multiple silent infarct lesions visible on imaging, with counts reaching up to 73 lesions [138]. Taher et al. [139] found that brain MRI can easily detect white matter lesions or brain atrophy resulting from ischemic events, particularly in splenectomized adults with β-TI. The spectrum of white matter lesions in these patients can range from mild perivascular changes to larger areas with multiple small cavitations, fiber loss, and arteriosclerosis. Irregular high signal intensity lesions around the ventricles are often indicative of microcytic and patchy infarcts in individuals with β-TI.

Conversely, both intracranial arterial calcifications and small vessel disease (SVD) have been recognized in PXE [140]. In a recent systematic review [140], the most frequently reported cerebrovascular complications of PXE were ischemic stroke and white matter lesions, which may be caused by SVD [141,142]. This cerebral SVD likely plays an important role in the brain involvement seen in PXE. It is hypothesized that the progressive calcification of arterial walls in PXE patients—caused by reduced plasma levels of inorganic pyrophosphate—might also occur in the medium and small intracerebral vessels [143]. The pathophysiology of PXE involves reduced pyrophosphate levels, which disrupt phosphate and calcium homeostasis, and this mechanism is shared by other rare diseases, such as generalized arterial calcification of infancy (GACI, OMIM#208000) and idiopathic basal ganglia calcification (IBGC). These rare diseases share a similar pathophysiology and provide deeper insight into the effects of calcium and phosphate homeostasis deficits. The findings suggest that subsequent cerebral arterial calcification is strongly linked to cerebrovascular disease.

However, intracranial arterial calcifications are a common incidental finding on computed tomography (CT) imaging in the general population. While some calcifications are considered benign, others are significant indicators of adverse clinical outcomes, including stroke, transient ischemic attacks, epileptic seizures, and cognitive decline [143,144,145,146]. The distribution of calcifications within different large and small arterial beds, as well as their location in either the intimal or medial layers of the arterial wall, can influence their associated risk factors and clinical outcomes. Eccentric calcifications, often found in atherosclerotic plaques, act as markers of plaque activity, with microcalcifications indicating a high risk of plaque rupture. In contrast, larger calcifications are typically seen in more stable plaques, contributing to plaque stability [147]. Concentric calcifications, which occur in the tunica media of the arteries, are associated with arterial stiffening, a process often observed with aging [148]. An example is illustrated in Figure 1.

Conditions such as diabetes mellitus and chronic kidney disease are known to accelerate the process of arterial calcification. Additionally, several rare monogenetic disorders that affect calcification homeostasis lead to extensive intracranial arterial calcification [149]. While the generalizability of findings from these model diseases must be interpreted with caution, they provide valuable insights into the etiology of arterial calcification at the extreme end of the calcification spectrum. This review aims to improve understanding of the significance of intracranial calcifications in both health and disease, offering a comprehensive overview of their prevalence on CT and histopathology in the general population, as well as their relationship to risk factors and clinical outcomes in both large and small intracranial arteries. The presence of both intimal and medial calcification in the carotid siphon has been linked to age, pulse pressure, and a family history of vascular diseases. Furthermore, intimal calcification is connected to smoking and hypertension, while medial calcification is associated with diabetes mellitus and a history of vascular conditions [150,151]. PXE serves as a model for medial calcification in large and medium-sized arteries, particularly in the carotid siphon and legs [150]. Recent studies have revealed that in PXE, carotid siphon calcification correlates with increased arterial pulsatility, which in turn is linked to lacunar infarctions, white matter lesions, reduced gray matter volumes, and lower processing speed scores [151]. The cerebrovascular phenotype of PXE includes SVD in different populations [152]. In addition, PXE has more structural brain disease and increased intracranial arterial flow pulsatility compared with healthy controls. Arterial calcification of the carotid siphon is associated with increased flow pulsatility and microvascular brain damage, including infarctions and WML [153,154].

7. Treatment Strategies

Currently, there are no curative therapies available for ectopic calcification disorders. Existing treatments primarily focus on reducing extracellular calcification to mitigate the effects of arteriopathy and its associated consequences. Most therapeutic strategies target the low levels of inorganic pyrophosphate, which are characteristic of these disorders. However, the rarity of conditions like GACI and PXE has resulted in limited clinical trials, often relying on retrospective data without standardized protocols, complicating the establishment of optimal treatment guidelines [155]. Dietary management and supplementation have seen limited exploration in GACI. While increased dietary magnesium during pregnancy has been shown to prevent aortic mineralization in offspring of Enpp1asj mice [156], no clinical trials for dietary manipulation in GACI are currently registered. In contrast, studies in Abcc6^−/− knockout mice indicated that dietary magnesium supplementation improved carotid artery wall thickness and connective tissue mineralization [157,158]. A randomized controlled trial (RCT) in humans with PXE showed promising results, where magnesium supplementation led to a decrease in skin elastic fiber calcification, although the difference was not statistically significant [159]. Phosphate binders have also been investigated, as increased dietary phosphate levels may accelerate mineralization [158]. A small case series suggested that aluminum hydroxide could improve histological appearances of skin lesions in PXE [160], but a larger RCT found no significant effects with sevelamer hydrochloride [161]. Bisphosphonates, traditionally used to prevent bone demineralization, have been the first-line treatment for GACI for over 50 years [162]. Recent studies indicate their effectiveness in reducing calcification in PXE as well [163]. Although many studies show benefits from bisphosphonate treatment, there are concerns regarding selection bias in studies correlating treatment with improved survival rates [164]. Some evidence suggests that dietary magnesium may enhance the effects of bisphosphonates in GACI patients who do not respond adequately [165]. TNAP inhibitors have emerged as a potential approach since TNAP facilitates the degradation of inorganic pyrophosphate, a key inhibitor of calcification [166]. Initial studies in mice indicated that TNAP inhibition could reduce abnormal mineralization [167,168]. A small RCT found that lansoprazole increased pyrophosphate levels, while another trial of the TNAP inhibitor DS-1211b is currently underway [169]. Furthermore, a trial about oral supplementation of PPi in PXE is ongoing [170]. ENPP1 enzyme replacement therapy aims to normalize extracellular inorganic pyrophosphate levels. In studies of Enpp1asj mice, administering recombinant ENPP1 protein reduced arterial calcification and improved cardiovascular function [162,171,172]. This therapy may also address myointimal proliferation associated with GACI, potentially improving outcomes [166]. Investigations into allele-specific therapies are ongoing, targeting specific mutations such as premature termination codon suppressors and chemical chaperones, although clinical trials have yet to commence [173]. Gene therapies aimed at ABCC6 mutations have shown effectiveness in animal models, significantly reducing ectopic mineralization [174]. Additionally, upregulating fetuin-A expression has been explored for its anti-mineralization effects [175]. However, challenges such as vector immunogenicity hinder clinical translation. Calcium crystallization inhibitors like SNF472, which bind to hydroxyapatite crystals, are currently undergoing trials to assess their efficacy in reducing calcification progression in conditions like calciphylaxis [176]. Antibody-targeted treatments are also being investigated, utilizing nanoparticles to deliver therapeutic agents directly to areas of elastin damage. This approach aims to enhance localized drug efficacy while minimizing off-target effects [177,178,179,180]. Preclinical studies have shown that such targeted therapies can effectively reduce arterial calcification and improve vascular function. Lastly, cell engineering techniques have demonstrated potential in reducing heterotopic ossification through modified osteoclasts, although further validation in appropriate animal models is required.

Table 7. Main treatment perspectives for ectopic calcification disorders.

Therapeutic Approach	Main Features
Current Status	No curative therapies; focus on reducing extracellular calcification and addressing low inorganic pyrophosphate levels.
Dietary Management and Supplementation	Limited studies; magnesium supplementation shows promise in PXE. A trial indicated decreased skin elastic fiber calcification.
Phosphate Binders	May slow progression in PXE; a small case series showed improvement in skin lesions, but larger RCTs yielded no significant results.
Bisphosphonates	First-line treatment for GACI; effective in reducing calcification in PXE. Evidence is largely from case reports; etidronate shows efficacy in PXE.
TNAP Inhibitors	TNAP inhibition increases inorganic pyrophosphate levels; initial studies showed promise in mice. Human trials are ongoing.
ENPP1 Enzyme Replacement Therapy	Aims to normalize inorganic pyrophosphate levels; studies in mice show reduced calcification and improved cardiovascular function.
Allele-Specific Therapies	Targeting of specific mutations; premature termination codon suppressors and chemical chaperones show variable effects in animal models.
Gene Therapies	Aimed at ABCC6 mutations; effective in animal models. Challenges include vector immunogenicity.
Calcium Crystallization Inhibitors	SNF472 is being tested to reduce calcification in conditions like calciphylaxis; potential for broader application in GACI and PXE.
Antibody-Targeted Treatment	Uses nanoparticles to deliver drugs to areas of elastin damage; shows promise in reducing arterial calcification and improving vascular function.
Cell Engineering	Modification of osteoclasts shows potential in reducing heterotopic ossification; applicability in PXE and GACI needs further investigation.

summarizes the therapeutic approaches for ectopic calcification disorders.

However, while significant progress has been made in understanding and developing therapies for ectopic calcification disorders, many therapeutic approaches remain in the experimental stages, and comprehensive clinical trials are essential to establish their efficacy and safety.

8. Conclusions

PXE-like syndrome in thalassemia highlights the evolving landscape of complications as patients live longer. Arterial calcifications are a relevant component of the PXE-like spectrum of manifestations, but data on intracranial arterial calcifications are lacking in beta-thalassemia patients. Early recognition and management of these manifestations are crucial to improving outcomes in this population.