Mucopolysaccharidosis IVA (MPS IVA; Morquio A syndrome) is an inherited, autosomal recessive disease caused by a deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS), which results in excessive lysosomal storage of keratan sulfate (KS) and chondroitin 6-sulfate (C6S) in many tissues and organs. This accumulation leads to chronic and progressive clinical findings including but not limited to systemic skeletal dysplasia, joint abnormalities, short stature, hearing and vision loss, coarse facial features, valvular heart disease, and pulmonary compromise (1–3). The extent of GALNS deficiency is highly variable thus resulting in a heterogenous clinical presentation (2, 3). Early diagnosis of Morquio A is important to employ disease modifying treatments to slow progression as no cure exists (4).

Currently available therapies for Morquio A include enzyme replacement therapy (ERT) with recombinant human GALNS enzyme, hematopoietic stem cell therapy (HSCT), and orthopedic surgeries. ERT is the most widely used therapeutic option in many lysosomal storage disorders including Morquio A. The enzyme, Elosulfase alfa, works within lysosomes to catabolize the glycosaminoglycans (GAGs) that have accumulated within. ERT has shown clinical improvement in somatic manifestations, activities of daily living, and quality of life (5–9). It has a low mortality risk with no limitations on use, however, the enzymes have a short half-life with rapid clearance from circulation resulting in poor delivery to bone and avascular tissues such as cartilage and cornea even when started at a young age. Other limitations include the need for weekly treatments, hypersensitivity reactions, and high cost (1). Another treatment option is HSCT which is a one-time permanent treatment allowing continuous production of the enzyme. HSCT has shown improvement in pulmonary function, activities of daily living, bone mineral density, cardiovascular involvement, and reduction in number of surgical interventions (10–12). However, it has many health and age limitations, requires a matched donor, carries a high risk of mortality and complications, and has limited effect on bone and cartilage (10, 11, 13). Morquio A patients often develop severe skeletal dysplasia requiring multiple orthopedic surgical interventions in the upper cervical spine and lower extremities as well as tracheal obstruction surgery. ERT and HSCT have limited to no impact on skeletal dysplasia (1, 3, 14).

Although these therapeutic treatments have extended life expectancy for Morquio A patients by attenuating disease progression, they have shown only a limited effect on heart disease which is the second leading cause of death in this population (1, 3, 15, 16). Cardiovascular disease (CVD) in these patients emerges silently and contributes significantly to early mortality (17). Cardiovascular involvement has been reported in Morquio patients as early as 3.6 months of age with subsequent progression of the disease (18). A wide range of cardiac findings have been associated with Morquio A including mitral and/or aortic insufficiency, aortic stenosis, tricuspid regurgitation, thickened interventricular septum, hypertrophic cardiomyopathy, and aortic root dilatation (15, 17–20). Furthermore, post-mortem histopathology on a 20-year-old patient has shown valvular infiltration by foam cells and macrophages along with GAG deposition (particularly C6S), and fragmented, attenuated elastic fibers in the aortic intima and media. These findings imply aortic intima thickening, atherosclerotic plaques, and aortic valve hypertrophy (21). A recent study of 12 Morquio A patients has revealed increased carotid intima media thickness and carotid hyperelasticity. These findings may be explained by alteration of arterial elasticity by GAG bearing proteoglycans (16). Limited therapeutic treatments exist for Morquio A that can address the CVD. ERT has poor delivery of the enzyme to cardiac valves and the aorta because of its short half-life in circulation, effect on cartilaginous tissues, and low density of mannose 6 phosphate receptors (1, 22). Another explanation of the slight therapeutic efficacy of ERT in heart has been attributed historically to the “avascular” nature of heart valves, which remains to be a debatable topic that needs further investigation in Morquio A patients (23). ERT studies with short term echocardiographic follow up have shown only a negligible therapeutic effect on cardiovascular findings (1, 15, 16, 20, 22, 24). The incidence of cardiac surgery in Morquio A patients is low (2%) compared to other surgeries. However, the most common cardiac surgery in adult Morquio A patients is aortic valve replacement (3, 25–27). Thus, development of a therapy to ameliorate CVD could be lifesaving in Morquio A patients.

Attenuated and fragmented elastin fibrils have been found not only in the aortic intima of a Morquio A patient (21) but also in patients and animal models of other MPS types, such as MPS I, VI, VII (28–30), and IVA (unpublished). Elastin fragmentation is the result of proteolysis by cathepsins and the matrix metalloproteinases secreted by GAG-activated macrophages (28, 31). It results in acquired CVD including atherosclerosis, calcification, and aneurysms. Elastic fiber degradation allows infiltration of lipids and immune cells into the aortic wall for plaque formation and rupture. The products from degradation further enhance atherogenesis. Calcification in the arterial medial wall and aneurysmal formation have also been linked to elastic fiber degradation and fragmentation. Fragmented elastic fibers may be more susceptible to protease digestion leading to aneurysmal dilation and stimulation and signaling of cytokine and inflammatory cell infiltration (32).

Elastin degradation leads to an irreversible and difficult to halt cascade of arterial remodeling, and cytokine and inflammatory signaling pathway activation. Damaged and degraded elastic fibers are generally not repaired due to the limited timeframe of elastin synthesis (32). Elastin is a highly abundant hydrophobic extracellular matrix (ECM) protein within the arterial media, and it is arranged in tightly coiled laminae. It is responsible for the resilience and elasticity of large arteries allowing them to withstand blood pressure oscillations throughout the cardiac cycle (32, 33). Elastic fibers can be easily degraded by specific proteases called elastases. One of these elastases, cathepsin S, is a lysosomal cysteine protease highly expressed in GAG-bearing macrophages (32, 34). Secreted cathepsin S at the basal membrane of blood vessels cleaves several ECM proteins including laminin, collagen, and preferentially elastin which generates bioactive elastin peptides (35). This potent elastolytic and collagenolytic activity has been linked to vascular inflammation and calcification resulting in the acquired CVD atherosclerosis and aneurysmal formation (32, 34, 36). Although protein levels and/or gene expression of elastin and cathepsin S have not been explored in Morquio A patients, we have observed dysregulation with age (unpublished) in Morquio A mice. Thus elastin and cathepsin S may be potential biomarkers to predict the severity of heart disease and phenotype of this patient population.

Cardiovascular disease in Morquio A remains the second leading cause of death. It has been documented as early as infancy and it progresses with age. Current therapeutic options have shown limited to no impact on CVD progression. This study aimed to discover novel lifesaving biomarkers for the diagnosis, prognosis, and treatment of CVD in this patient population.

This study is the first to investigate cathepsin S and elastin levels as biomarkers to assess the severity of CVD, genotype, and phenotype in Morquio A Syndrome. We found that cathepsin S levels in Morquio A patients were significantly lower when compared to normal controls independent of patient’s severity. This reflects that cathepsin S activity may be impeded in Morquio A. Cathepsin S must be activated and secreted from the lysosome into the ECM to cleave its substrates and its remodeling (50). It has been previously shown that large amounts of GAGs, in particular C4S and C6S, within the lysosome can alter and affect the maturation of cathepsin S (51). Stability and enhanced activation of cathepsin S into its functioning form occurs in the presence of physiologic levels of GAGs (50). This may explain our findings of increased cathepsin S levels in normal controls, especially in the young pediatric age range. GAG levels in pediatric controls are the highest in early childhood and decrease with cessation of growth into adulthood (45, 46, 52). Our experiments displayed similar findings in our pediatric normal control population. Cathepsin S activity may be enhanced by the increased presence of GAGs in early normal control childhood resulting in higher cathepsin S levels during this time frame. This contrasts to young Morquio A children who tend to display large, excessive amounts of GAGs during childhood which may be altering cathepsin S activity with subsequent reflection in decreased levels.

Furthermore, our findings suggest that cathepsin S levels can be used to discriminate Morquio A children from normal controls of all ages, especially early childhood. To date, there is no published reference range for circulating cathepsin S in human plasma. Previous studies looking at cathepsin S in various human disease processes report serum and/or plasma levels in age matched healthy controls >20 (53–58). Cathepsin S levels in the pediatric age range have yet to be reported. Our study is the first to investigate cathepsin S levels in the pediatric population and in Morquio A disease. To the best of our knowledge, there exists no study on cathepsin S in serum and plasma of MPS patients. We note the existence of cathepsin studies in animal models of MPS I (59), II (60), III (59), or VII (28, 31, 61).

Cathepsin S has been associated with the pathogenesis of many conditions including but not limited to lung diseases (62), autoimmune diseases (56, 57, 63, 64), CVD (36, 65), type 2 diabetes (66, 67), obesity (58, 68), metabolic syndrome (55), neurodegenerative disease (69), and cancer (70, 71). The majority of studies report elevated cathepsin S levels in association with the specific disease process, however, a recent study has reported decreased cathepsin S levels in patients with systemic sclerosis associated interstitial lung disease (72).

In normal controls, we discovered that elastin increased throughout childhood then decreased until middle adulthood before elevating again. These findings may be explained by normal elastin fiber formation. Elastogenesis is a complex hierarchal process compromised of many distinct phases that begins with tropoelasin synthesis. Tropoelastin synthesis mainly occurs perinatally with elastin fiber formation and deposition occurring prior to birth and throughout childhood (73). De novo elastin synthesis normally terminates leading up to and following adolescence (32). While the majority of tropoelastin synthesis occurs perinatally, synthesis has also been shown to occur in response to tissue damage (73). Tissue damage occurs with the normal aging process (74). Our findings of increased elastin levels after age 40 could be explained by increasing tropoelastin synthesis in response to the normal tissue damage that occurs with aging.

In contrast to the normal control findings, elastin levels in Morquio A patients tended to increase with age with highest levels after 40 years of age. This too may be explained by the presence of large amounts of GAGs in Morquio A childhood. Elastogenesis or the formation of elastic fibers occurs very early in life and disappears by the onset of puberty (75). The presence of high amounts of GAGs in the ECM in early Morquio A childhood could prevent the proper assembly of elastic fibers or cause irreversible damage. For example, it has been speculated that marked accumulated GAGs in arteries cause swelling and separation of elastic lamella (76). As GAG levels decrease into adolescence and adulthood, the remaining elastic fibers may assemble properly reflecting the increase in elastin levels. Cell culture studies have shown that GAGs induce production of tropoelastin in cells and upregulate microfibrillar associated genes leading to the formation of elastic fibers (77). Thus, the presence of accumulated GAGs in early Morquio childhood could be leading to induction of tropoelastin, a soluble precursor of elastin essential to elastin fiber assembly. Tropoelastin then must be chaperoned outside of the cell to form elastin in the ECM (73). These same intracellular GAGs that induce tropoleastin production may affect the actual elastin fiber synthesis pathway. This has been shown in vitro in MPS I in which increased dermatan sulfate leads to a deficiency of elastic binding protein which is a key chaperone for elastin binding synthesis (78). As GAGs decrease throughout childhood into adulthood, proper elastin fiber assembly can be achieved from the remaining intracellular tropoelastin which would explain gradual increase in elastin levels observed in our study.

Our study suggests that elastin levels can be used to discriminate adolescent and adult Morquio patients from normal controls. Like cathepsin S, there is no published reference ranges for circulating plasma and/or serum elastin. Previous studies have mainly explored levels of elastin derived peptides or anti-elastin antibodies in pathological disease processes (79–83). The relationship of elastin protein levels to elastin derived peptides and anti-elastin antibodies has yet to be explored. Our study is the first to explore plasma elastin protein levels in the normal population and in a pathological disease. In addition, our findings confirm that urinary KS is associated with clinical severity of Morquio A (52, 84).

Our experiments show that sVCAM-1 levels in Morquio A patients are lower than in normal controls, which can be explained by the dysregulation of the vascular endothelial glycocalyx in MPS patients. In CVD, inflammatory activation of endothelial cells triggers the expression of some leukocyte adhesion molecules including sVCAM-1 followed by the migration of monocytes in the tunica intima of the arterial wall thus amplifying the inflammatory response (85, 86). Chondroitin sulfate (CS) represents the largest population of arterial GAGs, therefore it actively participates in the development of arterial disease (87). Patients with Morquio A disease showed increased cIMT (16) and their endothelial dysfunction/dysregulation may be primed by the accumulation of C6S developing atherosclerotic lesions. Since CS proteoglycans play a key role in the organization and assembly of ECM, the continuous accumulation of CS and KS substrate cause widespread dysregulation including inhibition of elastic fiber assembly and cell adhesion molecules (88).

C-reactive protein (CRP) and α2M are biomarkers widely used to predict severity of CVD. CRP is known to modulate innate immune response, promote platelet activation, vascular remodeling, and angiogenesis (89). Recent studies showed that CS intake is associated with a reduction in CRP concentration in blood, indicating decrease in inflammation (90–92). These findings, along with the significantly lower CRP levels in Morquio A patients found in this study, open a new direction in this field to gain insights in the understanding of vascular endothelial glycocalyx dysregulation in this patient population.

Identification of life-saving novel biomarkers remains the main therapeutic target in Morquio A CVD. This novel study is the first to explore the relationship of cathepsin S and elastin in Morquio A and the normal population. We discovered that cathepsin S has promising attributes as a biomarker in Morquio A children. Elastin, on the other hand, has promising attributes as a biomarker in Morquio A adolescents and adults. Further studies are needed to understand how cathepsin S and elastin correlate with Morquio A severity and treatment outcomes.

Our study also had several limitations. First, our study was underpowered in several age groups which may have impacted results of age group dependent analyses. Second, urine was not collected from the normal pediatric patients limiting comparisons of urinary GAGs and urinary KS in age dependent analyses. Similarly, plasma GAGs were not analyzed on Morquio A patients or normal adults further limiting comparisons. Importantly, patients’ database was de-identified so we could not reach out to patients to obtain echocardiograms or ultrasounds to have additional clinical information on the CVD status in patients without reported known cardiovascular symptoms. Lastly, we were unable to compare cathepsin S and elastin levels found in this study to previous studies as this is the first study to explore pediatric and adult aged Morquio A patients and healthy normal controls.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

The studies involving human participants were reviewed and approved by the Institutional Review Board (IRB) at Saint Louis University. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.

AM conceived and supervised the study. BM and AM designed the study and wrote the manuscript. BM, LW, and QG performed laboratory analysis. AA contributed to the analysis of CVD biomarkers. BM, AA, and AM performed statistical analysis. BM, LW, QG, AA, and AM critically reviewed the manuscript. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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