Identification of Circulating Inflammatory Proteins Associated with Calcific Aortic Valve Stenosis by Multiplex Analysis.

Calcific aortic valve stenosis (CAVS) is characterized by increasing inflammation and progressive calcification in the aortic valve leaflets and is a major cause of death in the aging population. This study aimed to identify the inflammatory proteins involved in CAVS and provide potential therapeutic targets. We investigated the observational and causal associations of 92 inflammatory proteins, which were measured using affinity-based proteomic assays. Firstly, the case-control cohort identified differential proteins associated with the occurrence and progression of CAVS. Subsequently, we delved into exploring the causal impacts of these associated proteins through Mendelian randomization. This involved utilizing genetic instruments derived from cis-protein quantitative loci identified in genome-wide association studies, encompassing a cohort of over 400,000 individuals. Finally, we investigated the gene transcription and protein expression levels of inflammatory proteins by single-cell and immunohistochemistry analysis. Multivariate logistic regression and spearman's correlation analysis showed that five proteins showed a significant positive correlation with disease severity. Mendelian randomization showed that elevated levels of two proteins, namely, matrix metallopeptidase-1 (MMP1) and sirtuin 2 (SIRT2), were associated with an increased risk of CAVS. Immunohistochemistry and single-cell transcriptomes showed that expression levels of MMP1 and SIRT2 at the tissue and cell levels were significantly higher in calcified valves than in non-calcified control valves. These findings indicate that MMP1 and SIRT2 are causally related to CAVS and open up the possibility for identifying novel therapeutic targets.

Novel findings in a Swedish primary familial brain calcification cohort.

INTRODUCTION: Brain calcifications are frequent findings on imaging. In a small proportion of cases, these calcifications are associated with pathogenic gene variants, hence termed primary familial brain calcification (PFBC). The clinical penetrance is incomplete and phenotypic variability is substantial. This paper aims to characterize a Swedish PFBC cohort including 25 patients: 20 from seven families and five sporadic cases. METHODS: Longitudinal clinical assessment and CT imaging were conducted, abnormalities were assessed using the total calcification score (TCS). Genetic analyses, including a panel of six known PFBC genes, were performed in all index and sporadic cases. Additionally, three patients carrying a novel pathogenic copy number variant in SLC20A2 had their cerebrospinal fluid phosphate (CSF-Pi) levels measured. RESULTS: Among the 25 patients, the majority (76%) displayed varying symptoms during the initial assessment including motor (60%), psychiatric (40%), and/or cognitive abnormalities (24%). Clinical progression was observed in most patients (78.6%), but there was no significant difference in calcification between the first and second scans, with mean scores of 27.3 and 32.8, respectively. In three families and two sporadic cases, pathogenic genetic variants were identified, including a novel finding, in the SLC20A2 gene. In the three tested patients, the CSF-Pi levels were normal. CONCLUSIONS: This report demonstrates the variable expressivity seen in PFBC and includes a novel pathogenic variant in the SLC20A2 gene. In four families and three sporadic cases, no pathogenic variants were found, suggesting that new PFBC genes remain to be discovered.

Fusion of Platelet Derived Growth Factor Receptor Alpha (PDGFRA) With Ubiquitin Specific Peptidase 8 (USP8) in a Calcified Chondroid Mesenchymal Neoplasm Harboring t(4;15)(q12;q21) as a Sole Aberration.

BACKGROUND/AIM: The term "calcified chondroid mesenchymal neoplasm" was introduced in 2021 to describe a group of tumors characterized by various morphological features, including the formation of cartilage or chondroid matrix. These tumors frequently carry chimeric genes where the 5'-end partner gene is fibronectin 1 and the 3'-end partner gene codes for receptor tyrosine kinase. Our study explores fusion of the genes platelet-derived growth factor receptor alpha (PDGFRA) and ubiquitin-specific peptidase 8 (USP8) in calcified chondroid mesenchymal neoplasm. CASE REPORT: Genetic investigations were conducted on a tumor located in the leg of a 71-year-old woman. G-banding analysis of short-term cultured tumor cells revealed the karyotype 46,XX,t(4;15)(q12;q21)[6]/46,XX[4]. RNA sequencing detected in-frame PDGFRA::USP8 and USP8::PDGFRA chimeric transcripts, which were validated by RT-PCR/Sanger sequencing. The PDGFRA::USP8 chimeric protein is predicted to have cell membrane location and functions as a chimeric ubiquitinyl hydrolase. The USP8::PDGFRA protein was predicted to be nuclear and function as a positive regulator of cellular metabolic process. CONCLUSION: We report, for the first time, a calcified chondroid mesenchymal neoplasm carrying a balanced t(4;15)(q12;q21) chromosomal translocation, resulting in the generation of both PDGFRA::USP8 and USP8::PDGFRA chimeras. The PDGFRA::USP8 protein is located on the cell membrane and functions as a chimeric ubiquitinyl hydrolase, activated by PDGFs. Conversely, USP8::PDGFRA is a nuclear protein regulating metabolic processes.

Histological Findings in the Eyes of Abcc6 Knockout Rat Model of Pseudoxanthoma Elasticum.

Purpose: To describe the ocular findings of murine pseudoxanthoma elasticum (PXE) models with ATP-binding cassette subfamily C member 6 (Abcc6) gene knockout. Methods: This experiment was conducted in four Abcc6-/- rats and compared with six wild-type Abcc6+/+ control rats. The animals underwent necropsy at 6 months of age. Histological examination of the eyes was performed. Results: Histological examination of eight eyes from four Abcc6-/- rats revealed multiple nodular foci of calcification in the uvea, sclera, and conjunctiva, focally in perivascular distribution, as well as linear and nodular calcification of Bruch's membrane. Calcific foci were not associated with inflammation in the knockout rats. There was no evidence of calcification in control eyes. Discussion: The Abcc6-/- rat model shows that PXE can affect multiple ocular tissues beyond the calcification in Bruch's membrane noted in human eyes. Nodular calcific foci probably correspond to comet lesions seen in patients with PXE. The presence of ectopic calcium without inflammation distinguishes it from inflammatory calcium deposition in atherosclerosis. Further studies are needed to determine why PXE does not cause inflammatory infiltration. Translational Relevance: The Abcc6-/- murine model may be suitable for studying ocular PXE pathophysiology and ectopic calcification and developing effective therapies.

Inhibition of BMP signaling pathway induced senescence and calcification in anaplastic meningioma.

PURPOSE: Meningiomas are the most common type of brain tumors and are generally benign, but malignant atypical meningiomas and anaplastic meningiomas frequently recur with poor prognosis. The metabolism of meningiomas is little known, so few effective treatment options other than surgery and radiation are available, and the targets for treatment of recurrence are not well defined. The Aim of this paper is to find the therapeutic target. METHODS: The effects of bone morphogenetic protein (BMP) signal inhibitor (K02288) and upstream regulator Gremlin2 (GREM2) on meningioma's growth and senescence were examined. In brief, we examined as follows: 1) Proliferation assay by inhibiting BMP signaling. 2) Comprehensive analysis of forced expression GREM2.3) Correlation between GREM2 mRNA expression and proliferation marker in 87 of our clinical samples. 4) Enrichment analysis between GREM2 high/low expressed groups using RNA-seq data (42 cases) from the public database GREIN. 5) Changes in metabolites and senescence markers associated with BMP signal suppression. RESULTS: Inhibitors of BMP receptor (BMPR1A) and forced expression of GREM2 shifted tryptophan metabolism from kynurenine/quinolinic acid production to serotonin production in malignant meningiomas, reduced NAD + /NADH production, decreased gene cluster expression involved in oxidative phosphorylation, and caused decrease in ATP. Finally, malignant meningiomas underwent cellular senescence, decreased proliferation, and eventually formed psammoma bodies. Reanalyzed RNA-seq data of clinical samples obtained from GREIN showed that increased expression of GREM2 decreased the expression of genes involved in oxidative phosphorylation, similar to our experimental results. CONCLUSIONS: The GREM2-BMPR1A-tryptophan metabolic pathway in meningiomas is a potential new therapeutic target.

Identification of hub genes in calcific aortic valve disease.

Calcific aortic valve disease (CAVD) is a heart valve disorder characterized primarily by calcification of the aortic valve, resulting in stiffness and dysfunction of the valve. CAVD is prevalent among aging populations and is linked to factors such as hypertension, dyslipidemia, tobacco use, and genetic predisposition, and can result in becoming a growing economic and health burden. Once aortic valve calcification occurs, it will inevitably progress to aortic stenosis. At present, there are no medications available that have demonstrated effectiveness in managing or delaying the progression of the disease. In this study, we mined four publicly available microarray datasets (GSE12644 GSE51472, GSE77287, GSE233819) associated with CAVD from the GEO database with the aim of identifying hub genes associated with the occurrence of CAVD and searching for possible biological targets for the early prevention and diagnosis of CAVD. This study provides preliminary evidence for therapeutic and preventive targets for CAVD and may provide a solid foundation for subsequent biological studies.

Familial primary calcific band-shaped keratopathy with late onset systemic disease: a case series and review of the literature.

BACKGROUND: Familial calcific band-shaped keratopathy (BSK) is a very rare disease, with no underlying cause. There is no underlying disease in this form of the disease. This article introduces a family with seven children, three of whom were diagnosed with familial primary calcific BSK. One of them developed a systemic disease 38 years after ocular manifestation. CASE PRESENTATION: In this case report, three Iranian siblings from a family with familial calcific band-shaped keratopathy (BSK) are introduced. Systemic and ocular examinations performed on these patients indicated the occurrence of chronic kidney disease in the older child, a 41-year-old woman, 38 years after ocular manifestation. The examinations conducted on the other two siblings revealed no pathological findings. The 41-year-old sister and 37-year-old brother underwent unilateral deep anterior lamellar keratoplasty (DALK), while the 33-year-old sister underwent bilateral superficial keratectomy (SK). CONCLUSION: Considering the late onset of systemic disease in one of the siblings diagnosed with familial calcific band-shaped keratopathy (BSK), it is crucial to emphasize the necessity of long-term follow-up for these patients and their families.

Successful treatment approaches for tumoral calcinosis in children and young people: A condition of diverse pathogenesis.

BACKGROUND: Ectopic calcification is inappropriate biomineralization of soft tissues occurring due to genetic or acquired causes of hyperphosphataemia and rarely in normophosphataemic individuals. Tumoral Calcinosis (TC) is a rare metabolic bone disorder commonly presenting in childhood and adolescence with periarticular extra-capsular calcinosis. Three subtypes of TC have been recognised: primary hyperphosphataemic familial TC (HFTC), primary normophosphataemic familial TC and secondary TC most commonly seen in chronic renal failure. In the absence of established treatment, management is challenging due to variable success rates with medical therapies and recurrence following surgery. AIM: We outline the successful treatment approaches in four children with TC (2 normophosphatemic TC, 2 HFTC) aged 2.5-10 years at initial presentation. CASES: Patient 1 (P1) presented at 10 years with a painless lump behind the right knee, P2 with swelling of the right knee anteriorly at 9 years, P3 and P4 with pain and swelling over the right elbow at 5 and 2.5 years respectively. All patients were of Black African-Caribbean origin and were previously reported to be fit and well with no family history of TC. RESULTS: P1, P2 had normophosphataemic TC and P3, P4 had HFTC with genetically confirmed GALNT3 mutation. All four patients had initial surgical resection with TC confirmed on histology. P1 had complete surgical resection with no recurrence at 27 months post-operatively. P2 had significant overgrowth of the tumour following surgery and was subsequently successfully managed with 25 % topical sodium metabisulphite (total duration of 8 months with a 4 month gap during which there was recurrence). P3 had post-surgical recurrence of TC on the right elbow and a new lesion on left elbow which resolved with oral acetazolamide monotherapy (15-20 mg/kg/day). P4 had recurrence of right elbow lesion following surgery and developed an extensive new hip lesion on sevelamer therapy which resolved completely with additional acetazolamide therapy (18-33 mg/kg/day). Acetazolamide was well tolerated with normal growth for 5 years in P3 and 6.5 years in P4 and no recurrence of lesions. CONCLUSION: The frequent post-surgical recurrence in TC and successful medical therapy on the other hand indicates that medical management as first line therapy should be adopted. Monotherapies with topical 25 % sodium metabisulphite in normophosphataemic and oral acetazolamide in HFTC are effective treatment strategies which are well tolerated.

High glucose promotes osteogenic differentiation of human lens epithelial cells through hypoxia-inducible factor (HIF) activation.

Cataract, a leading cause of blindness, is characterised by lens opacification. Type 2 diabetes is associated with a two- to fivefold higher prevalence of cataracts. The risk of cataract formation increases with the duration of diabetes and the severity of hyperglycaemia. Hydroxyapatite deposition is present in cataractous lenses that could be the consequence of osteogenic differentiation and calcification of lens epithelial cells (LECs). We hypothesised that hyperglycaemia might promote the osteogenic differentiation of human LECs (HuLECs). Osteogenic medium (OM) containing excess phosphate and calcium with normal (1 g/L) or high (4.5 g/L) glucose was used to induce HuLEC calcification. High glucose accelerated and intensified OM-induced calcification of HuLECs, which was accompanied by hyperglycaemia-induced upregulation of the osteogenic markers Runx2, Sox9, alkaline phosphatase and osteocalcin, as well as nuclear translocation of Runx2. High glucose-induced calcification was abolished in Runx2-deficient HuLECs. Additionally, high glucose stabilised the regulatory alpha subunits of hypoxia-inducible factor 1 (HIF-1), triggered nuclear translocation of HIF-1α and increased the expression of HIF-1 target genes. Gene silencing of HIF-1α or HIF-2α attenuated hyperglycaemia-induced calcification of HuLECs, while hypoxia mimetics (desferrioxamine, CoCl2) enhanced calcification of HuLECs under normal glucose conditions. Overall, this study suggests that high glucose promotes HuLEC calcification via Runx2 and the activation of the HIF-1 signalling pathway. These findings may provide new insights into the pathogenesis of diabetic cataracts, shedding light on potential factors for intervention to treat this sight-threatening condition.

A new perspective on intervertebral disc calcification-from bench to bedside.

Disc degeneration primarily contributes to chronic low back and neck pain. Consequently, there is an urgent need to understand the spectrum of disc degeneration phenotypes such as fibrosis, ectopic calcification, herniation, or mixed phenotypes. Amongst these phenotypes, disc calcification is the least studied. Ectopic calcification, by definition, is the pathological mineralization of soft tissues, widely studied in the context of conditions that afflict vasculature, skin, and cartilage. Clinically, disc calcification is associated with poor surgical outcomes and back pain refractory to conservative treatment. It is frequently seen as a consequence of disc aging and progressive degeneration but exhibits unique molecular and morphological characteristics: hypertrophic chondrocyte-like cell differentiation; TNAP, ENPP1, and ANK upregulation; cell death; altered Pi and PPi homeostasis; and local inflammation. Recent studies in mouse models have provided a better understanding of the mechanisms underlying this phenotype. It is essential to recognize that the presentation and nature of mineralization differ between AF, NP, and EP compartments. Moreover, the combination of anatomic location, genetics, and environmental stressors, such as aging or trauma, govern the predisposition to calcification. Lastly, the systemic regulation of calcium and Pi metabolism is less important than the local activity of PPi modulated by the ANK-ENPP1 axis, along with disc cell death and differentiation status. While there is limited understanding of this phenotype, understanding the molecular pathways governing local intervertebral disc calcification may lead to developing disease-modifying drugs and better clinical management of degeneration-related pathologies.

Generation of induced pluripotent stem cell line from a patient suffering from arterial calcification due to deficiency of CD73 (ACDC).

Arterial calcification due to deficiency of CD73 (ACDC) is an adult onset, rare genetic vascular disorder signified by calcium deposition in lower extremity arteries and joints of hands and feet. Mutations in NT5E gene has been shown to be responsible for the inactivation of enzyme CD73 causing calcium buildup. Here, we report a iPSC line generated from a patient showing signs of ACDC and carrying a missense mutation in NT5E (c.1126A→G,p.T376A) gene. This iPSC line shows normal morphology, pluripotency, karyotype, and capability to differentiate into three germ layers, making it useful for disease modeling and investigating pathological mechanisms of ACDC.

AVCAPIR: A Novel Procalcific PIWI-Interacting RNA in Calcific Aortic Valve Disease.

BACKGROUND: Calcification of the aortic valve leads to increased leaflet stiffness and consequently results in the development of calcific aortic valve disease (CAVD). However, the underlying molecular and cellular mechanisms of calcification remain unclear. Here, we identified a novel aortic valve calcification-associated PIWI-interacting RNA (piRNA; AVCAPIR) that increases valvular calcification and promotes CAVD progression. METHODS: Using piRNA sequencing, we identified piRNAs contributing to the pathogenesis of CAVD that we termed AVCAPIRs. High-cholesterol diet-fed ApoE-/- mice with AVCAPIR knockout were used to examine the role of AVCAPIR in aortic valve calcification (AVC). Gain- and loss-of-function assays were conducted to determine the role of AVCAPIR in the induced osteogenic differentiation of human valvular interstitial cells. To dissect the mechanisms underlying AVCAPIR-elicited procalcific effects, we performed various analyses, including an RNA pulldown assay followed by liquid chromatography-tandem mass spectrometry, methylated RNA immunoprecipitation sequencing, and RNA sequencing. RNA pulldown and RNA immunoprecipitation assays were used to study piRNA interactions with proteins. RESULTS: We found that AVCAPIR was significantly upregulated during AVC and exhibited potential diagnostic value for CAVD. AVCAPIR deletion markedly ameliorated AVC in high-cholesterol diet-fed ApoE-/- mice, as shown by reduced thickness and calcium deposition in the aortic valve leaflets, improved echocardiographic parameters (decreased peak transvalvular jet velocity and mean transvalvular pressure gradient, as well as increased aortic valve area), and diminished levels of osteogenic markers (Runx2 and Osterix) in aortic valves. These results were confirmed in osteogenic medium-induced human valvular interstitial cells. Using unbiased protein-RNA screening and molecular validation, we found that AVCAPIR directly interacts with FTO (fat mass and obesity-associated protein), subsequently blocking its N6-methyladenosine demethylase activity. Further transcriptomic and N6-methyladenosine modification epitranscriptomic screening followed by molecular validation confirmed that AVCAPIR hindered FTO-mediated demethylation of CD36 mRNA transcripts, thus enhancing CD36 mRNA stability through the N6-methyladenosine reader IGF2BP1 (insulin-like growth factor 2 mRNA binding protein 1). In turn, the AVCAPIR-dependent increase in CD36 stabilizes its binding partner PCSK9 (proprotein convertase subtilisin/kexin type 9), a procalcific gene, at the protein level, which accelerates the progression of AVC. CONCLUSIONS: We identified a novel piRNA that induced AVC through an RNA epigenetic mechanism and provide novel insights into piRNA-directed theranostics in CAVD.

Klf10 is involved in extracellular matrix calcification of chondrocytes alleviating chondrocyte senescence.

Osteoarthritis (OA) is a chronic degenerative disease resulting joint disability and pain. Accumulating evidences suggest that chondrocyte extracellular matrix calcification plays an important role in the development of OA. Here, we showed that Krüppel-like factor 10 (Klf10) was involved in the regulation of chondrocyte extracellular matrix calcification by regulating the expression of Frizzled9. Knockdown of Klf10 attenuated TBHP induced calcification and reduced calcium content in chondrocytes. Restoring extracellular matrix calcification of chondrocytes could aggravate chondrocyte senescence. Destabilization of a medial meniscus (DMM) mouse model of OA, in vivo experiments revealed that knockdown Klf10 improved the calcification of articular cartilage and ameliorated articular cartilage degeneration. These findings suggested that knockdown Klf10 inhibited extracellular matrix calcification-related changes in chondrocytes and alleviated chondrocyte senescence.

Novel de novo mutation in ZBTB20 in a Chinese Primrose syndrome family and a review of the literature.

BACKGROUND: Primrose syndrome is an autosomal dominant disorder characterized by craniofacial dysmorphism, mental retardation, developmental delay, progressive muscle atrophy and calcification of the earlobe due to a mutation in the ZBTB20. METHOD: We reported a case of a Chinese boy with clinical symptoms resembling Primrose Syndrome, and performed genetic etiology analysis of the proband's family through Trio whole exome sequencing. RESULT: A novel missense variant c.1927T>A(p.F643I) in exon 14 of the ZBTB20 (NM_001348803) was identified in the proband. This is the first report case of primrose syndrome in China, and our case extends the variant spectrum of ZBTB20 and further strengthens the understanding of primrose syndrome. CONCLUSION: However, there are no formal clinical guidelines for the management of this disease, and research on treatment and prognosis remains a challenge and focus in future.

Multimodal Analytical Tools to Enhance Mechanistic Understanding of Aortic Valve Calcification.

This review focuses on technologies at the core of calcific aortic valve disease (CAVD) and drug target research advancement, including transcriptomics, proteomics, and molecular imaging. We examine how bulk RNA sequencing and single-cell RNA sequencing have engendered organismal genomes and transcriptomes, promoting the analysis of tissue gene expression profiles and cell subpopulations, respectively. We bring into focus how the field is also largely influenced by increasingly accessible proteome profiling techniques. In unison, global transcriptional and protein expression analyses allow for increased understanding of cellular behavior and pathogenic pathways under pathologic stimuli including stress, inflammation, low-density lipoprotein accumulation, increased calcium and phosphate levels, and vascular injury. We also look at how direct investigation of protein signatures paves the way for identification of targetable pathways for pharmacologic intervention. Here, we note that imaging techniques, once a clinical diagnostic tool for late-stage CAVD, have since been refined to address a clinical need to identify microcalcifications using positron emission tomography/computed tomography and even detect in vivo cellular events indicative of early stage CAVD and map the expression of identified proteins in animal models. Together, these techniques generate a holistic approach to CAVD investigation, with the potential to identify additional novel regulatory pathways.

GNAQ/GNA11 Mosaicism Is Associated with Abnormal Serum Calcium Indices and Microvascular Neurocalcification.

Mosaic mutations in genes GNAQ or GNA11 lead to a spectrum of diseases including Sturge-Weber syndrome and phakomatosis pigmentovascularis with dermal melanocytosis. The pathognomonic finding of localized "tramlining" on plain skull radiography, representing medium-sized neurovascular calcification and associated with postnatal neurological deterioration, led us to study calcium metabolism in a cohort of 42 children. In this study, we find that 74% of patients had at least one abnormal measurement of calcium metabolism, the commonest being moderately low serum ionized calcium (41%) or high parathyroid hormone (17%). Lower levels of ionized calcium even within the normal range were significantly associated with seizures, and with specific antiepileptics despite normal vitamin D levels. Successive measurements documented substantial intrapersonal fluctuation in indices over time, and DEXA scans were normal in patients with hypocalcemia. Neurohistology from epilepsy surgery in five patients revealed not only intravascular, but perivascular and intraparenchymal mineral deposition and intraparenchymal microvascular disease in addition to previously reported findings. Neuroradiology review clearly demonstrated progressive calcium deposition in individuals over time. These findings and those of the adjoining paper suggest that calcium deposition in the brain of patients with GNAQ/GNA11 mosaicism may not be a nonspecific sign of damage as was previously thought, but may instead reflect the central postnatal pathological process in this disease spectrum.

TREX-1 related Aicardi-Goutières syndrome improved by Janus kinase inhibitor.

Aicardi-Goutières syndrome (AGS) is a genetic interferonopathy classically characterized by early onset of severe neurologic injury with basal ganglia calcifications, white matter abnormalities, and progressive cerebral atrophy, along with lymphocytosis and raised interferon alpha (INFα) in the cerebrospinal fluid (CSF). Here, we report a 31/2 year-old patient born with prenatal onset AGS, first manifesting as intra-uterine growth retardation. Cranial ultrasonography and cerebral MRI revealed ventriculomegaly and periventricular and basal ganglia calcifications, along with cerebral atrophy. Perinatal infections and known metabolic disorders were excluded. Both CSF lymphocytosis and raised INFα were present. Molecular analysis disclosed two already described compound heterozygous pathogenic variants in TREX1 (c. 309dup, p.(Thr104Hisfs*53) and c. 506G > A, p.(Arg169His)). The evolution was marked by severe global developmental delay with progressive microcephaly. Promptly, the patient developed irritability, quadri-paretic dyskinetic movements, and subsequently tonic seizures. Sensorineural hearing loss was detected as well as glaucoma. Initially, he was symptomatically treated with trihexyphenidyl followed by levetiracetam and topiramate. At age 22 months, baricitinib (0.4 mg/kg/day) was introduced, leading to normal serum INFα levels. Clinically, dyskinetic movements significantly decreased as well as irritability and sleep disturbance. We confirmed that baricitinib was a useful treatment with no major side effect.