JAGGED1/NOTCH3 activation promotes aortic hypermuscularization and stenosis in elastin deficiency.

Obstructive arterial diseases, including supravalvular aortic stenosis (SVAS), atherosclerosis, and restenosis, share 2 important features: an abnormal or disrupted elastic lamellae structure and excessive smooth muscle cells (SMCs). However, the relationship between these pathological features is poorly delineated. SVAS is caused by heterozygous loss-of-function, hypomorphic, or deletion mutations in the elastin gene (ELN), and SVAS patients and elastin-mutant mice display increased arterial wall cellularity and luminal obstructions. Pharmacological treatments for SVAS are lacking, as the underlying pathobiology is inadequately defined. Herein, using human aortic vascular cells, mouse models, and aortic samples and SMCs derived from induced pluripotent stem cells of ELN-deficient patients, we demonstrated that elastin insufficiency induced epigenetic changes, upregulating the NOTCH pathway in SMCs. Specifically, reduced elastin increased levels of γ-secretase, activated NOTCH3 intracellular domain, and downstream genes. Notch3 deletion or pharmacological inhibition of γ-secretase attenuated aortic hypermuscularization and stenosis in Eln-/- mutants. Eln-/- mice expressed higher levels of NOTCH ligand JAGGED1 (JAG1) in aortic SMCs and endothelial cells (ECs). Finally, Jag1 deletion in SMCs, but not ECs, mitigated the hypermuscular and stenotic phenotype in the aorta of Eln-/- mice. Our findings reveal that NOTCH3 pathway upregulation induced pathological aortic SMC accumulation during elastin insufficiency and provide potential therapeutic targets for SVAS.

LTBP4 affects renal fibrosis by influencing angiogenesis and altering mitochondrial structure.

Transforming growth factor beta (TGFß) signalling regulates extracellular matrix accumulation known to be essential for the pathogenesis of renal fibrosis; latent transforming growth factor beta binding protein 4 (LTBP4) is an important regulator of TGFß activity. To date, the regulation of LTBP4 in renal fibrosis remains unknown. Herein, we report that LTBP4 is upregulated in patients with chronic kidney disease and fibrotic mice kidneys created by unilateral ureteral obstruction (UUO). Mice lacking the short LTBP4 isoform (Ltbp4S-/-) exhibited aggravated tubular interstitial fibrosis (TIF) after UUO, indicating that LTBP4 potentially protects against TIF. Transcriptomic analysis of human proximal tubule cells overexpressing LTBP4 revealed that LTBP4 influences angiogenic pathways; moreover, these cells preserved better mitochondrial respiratory functions and expressed higher vascular endothelial growth factor A (VEGFA) compared to wild-type cells under hypoxia. Results of the tube formation assay revealed that additional LTBP4 in human umbilical vein endothelial cell supernatant stimulates angiogenesis with upregulated vascular endothelial growth factor receptors (VEGFRs). In vivo, aberrant angiogenesis, abnormal mitochondrial morphology and enhanced oxidative stress were observed in Ltbp4S-/- mice after UUO. These results reveal novel molecular functions of LTBP4 stimulating angiogenesis and potentially impacting mitochondrial structure and function. Collectively, our findings indicate that LTBP4 protects against disease progression and may be of therapeutic use in renal fibrosis.

LTBP4 in Health and Disease.

Latent transforming growth factor ß (TGFß)-binding protein (LTBP) 4, a member of the LTBP family, shows structural homology with fibrillins. Both these protein types are characterized by calcium-binding epidermal growth factor-like repeats interspersed with 8-cysteine domains. Based on its domain composition and distribution, LTBP4 is thought to adopt an extended structure, facilitating the linear deposition of tropoelastin onto microfibrils. In humans, mutations in LTBP4 result in autosomal recessive cutis laxa type 1C, characterized by redundant skin, pulmonary emphysema, and valvular heart disease. LTBP4 is an essential regulator of TGFß signaling and is related to development, immunity, injury repair, and diseases, playing a central role in regulating inflammation, fibrosis, and cancer progression. In this review, we focus on medical disorders or diseases that may be manipulated by LTBP4 in order to enhance the understanding of this protein.

Exploring the Paradoxical Relationship of a Creb 3 Regulatory Factor Missense Variant With Body Mass Index and Diabetes Among Samoans: Protocol for the Soifua Manuia (Good Health) Observational Cohort Study.

BACKGROUND: The prevalence of obesity and diabetes in Samoa, like many other Pacific Island nations, has reached epidemic proportions. Although the etiology of these conditions can be largely attributed to the rapidly changing economic and nutritional environment, a recently identified genetic variant, rs373863828 (CREB 3 regulatory factor, CREBRF: c.1370G>A p.[R457Q]) is associated with increased odds of obesity, but paradoxically, decreased odds of diabetes. OBJECTIVE: The overarching goal of the Soifua Manuia (Good Health) study was to precisely characterize the association of the CREBRF variant with metabolic (body composition and glucose homeostasis) and behavioral traits (dietary intake, physical activity, sleep, and weight control behaviors) that influence energy homeostasis in 500 adults. METHODS: A cohort of adult Samoans who participated in a genome-wide association study of adiposity in Samoa in 2010 was followed up, based on the presence or absence of the CREBRF variant, between August 2017 and March 2019. Over a period of 7-10 days, each participant completed the main study protocol, which consisted of anthropometric measurements (weight, height, circumferences, and skinfolds), body composition assessment (bioelectrical impedance and dual-energy x-ray absorptiometry), point-of-care glycated hemoglobin measurement, a fasting blood draw and oral glucose tolerance test, urine collection, blood pressure measurement, hand grip strength measurement, objective physical activity and sleep apnea monitoring, and questionnaire measures (eg, health interview, cigarette and alcohol use, food frequency questionnaire, socioeconomic position, stress, social support, food and water insecurity, sleep, body image, and dietary preferences). In January 2019, a subsample of the study participants (n=118) completed a buttock fat biopsy procedure to collect subcutaneous adipose tissue samples. RESULTS: Enrollment of 519 participants was completed in March 2019. Data analyses are ongoing, with results expected in 2020 and 2021. CONCLUSIONS: While the genetic variant rs373863828, in CREBRF, has the largest known effect size of any identified common obesity gene, very little is currently understood about the mechanisms by which it confers increased odds of obesity but paradoxically lowered odds of type 2 diabetes. The results of this study will provide insights into how the gene functions on a whole-body level, which could provide novel targets to prevent or treat obesity, diabetes, and associated metabolic disorders. This study represents the human arm of a comprehensive and integrated approach involving humans as well as preclinical models that will provide novel insights into metabolic disease. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): RR1-10.2196/17329.

Novel defect in phosphatidylinositol 4-kinase type 2-alpha (PI4K2A) at the membrane-enzyme interface is associated with metabolic cutis laxa.

Inherited cutis laxa, or inelastic, sagging skin is a genetic condition of premature and generalised connective tissue ageing, affecting various elastic components of the extracellular matrix. Several cutis laxa syndromes are inborn errors of metabolism and lead to severe neurological symptoms. In a patient with cutis laxa, a choreoathetoid movement disorder, dysmorphic features and intellectual disability we performed exome sequencing to elucidate the underlying genetic defect. We identified the amino acid substitution R275W in phosphatidylinositol 4-kinase type IIα, caused by a homozygous missense mutation in the PI4K2A gene. We used lipidomics, complexome profiling and functional studies to measure phosphatidylinositol 4-phosphate synthesis in the patient and evaluated PI4K2A deficient mice to define a novel metabolic disorder. The R275W residue, located on the surface of the protein, is involved in forming electrostatic interactions with the membrane. The catalytic activity of PI4K2A in patient fibroblasts was severely reduced and lipid mass spectrometry showed that particular acyl-chain pools of PI4P and PI(4,5)P2 were decreased. Phosphoinositide lipids play a major role in intracellular signalling and trafficking and regulate the balance between proliferation and apoptosis. Phosphatidylinositol 4-kinases such as PI4K2A mediate the first step in the main metabolic pathway that generates PI4P, PI(4,5)P2 and PI(3,4,5)P3 . Although neurologic involvement is common, cutis laxa has not been reported previously in metabolic defects affecting signalling. Here we describe a patient with a complex neurological phenotype, premature ageing and a mutation in PI4K2A, illustrating the importance of this enzyme in the generation of inositol lipids with particular acylation characteristics.

Loss of skin elasticity is associated with pulmonary emphysema, biomarkers of inflammation, and matrix metalloproteinase activity in smokers.

BACKGROUND: Elastin breakdown and the resultant loss of lung elastic recoil is a hallmark of pulmonary emphysema in susceptible individuals as a consequence of tobacco smoke exposure. Systemic alterations to the synthesis and degradation of elastin may be important to our understanding of disease phenotypes in chronic obstructive pulmonary disease. We investigated the association of skin elasticity with pulmonary emphysema, obstructive lung disease, and blood biomarkers of inflammation and tissue protease activity in tobacco-exposed individuals. METHODS: Two hundred and thirty-six Caucasian individuals were recruited into a sub-study of the University of Pittsburgh Specialized Center for Clinically Orientated Research in chronic obstructive pulmonary disease, a prospective cohort study of current and former smokers. The skin viscoelastic modulus (VE), a determinant of skin elasticity, was recorded from the volar forearm and facial wrinkling severity was determined using the Daniell scoring system. RESULTS: In a multiple regression analysis, reduced VE was significantly associated with cross-sectional measurement of airflow obstruction (FEV1/FVC) and emphysema quantified from computed tomography (CT) images, ß = 0.26, p = 0.001 and ß = 0.24, p = 0.001 respectively. In emphysema-susceptible individuals, elasticity-determined skin age was increased (median 4.6 years) compared to the chronological age of subjects without emphysema. Plasma biomarkers of inflammation (TNFR1, TNFR2, CRP, PTX3, and SAA) and matrix metalloproteinase activity (MMP1, TIMP1, TIMP2, and TIMP4) were inversely associated with skin elasticity. CONCLUSIONS: We report that an objective non-invasive determinant of skin elasticity is independently associated with measures of lung function, pulmonary emphysema, and biomarkers of inflammation and tissue proteolysis in tobacco-exposed individuals. Loss of skin elasticity is a novel observation that may link the common pathological processes that drive tissue elastolysis in the extracellular matrix of the skin and lung in emphysema-susceptible individuals.

Bi-allelic Mutations in the Mitochondrial Ribosomal Protein MRPS2 Cause Sensorineural Hearing Loss, Hypoglycemia, and Multiple OXPHOS Complex Deficiencies.

Biogenesis of the mitochondrial oxidative phosphorylation system, which produces the bulk of ATP for almost all eukaryotic cells, depends on the translation of 13 mtDNA-encoded polypeptides by mitochondria-specific ribosomes in the mitochondrial matrix. These mitoribosomes are dual-origin ribonucleoprotein complexes, which contain mtDNA-encoded rRNAs and tRNAs and ∼80 nucleus-encoded proteins. An increasing number of gene mutations that impair mitoribosomal function and result in multiple OXPHOS deficiencies are being linked to human mitochondrial diseases. Using exome sequencing in two unrelated subjects presenting with sensorineural hearing impairment, mild developmental delay, hypoglycemia, and a combined OXPHOS deficiency, we identified mutations in the gene encoding the mitochondrial ribosomal protein S2, which has not previously been implicated in disease. Characterization of subjects' fibroblasts revealed a decrease in the steady-state amounts of mutant MRPS2, and this decrease was shown by complexome profiling to prevent the assembly of the small mitoribosomal subunit. In turn, mitochondrial translation was inhibited, resulting in a combined OXPHOS deficiency detectable in subjects' muscle and liver biopsies as well as in cultured skin fibroblasts. Reintroduction of wild-type MRPS2 restored mitochondrial translation and OXPHOS assembly. The combination of lactic acidemia, hypoglycemia, and sensorineural hearing loss, especially in the presence of a combined OXPHOS deficiency, should raise suspicion for a ribosomal-subunit-related mitochondrial defect, and clinical recognition could allow for a targeted diagnostic approach. The identification of MRPS2 as an additional gene related to mitochondrial disease further expands the genetic and phenotypic spectra of OXPHOS deficiencies caused by impaired mitochondrial translation.

Acromelia-oligodontia syndrome.

This case report describes a patient with ankyloglossia, oligodontia, unilateral hypoplasia of the zygoma and mandible, along with bilateral distal reduction anomalies of his limbs without long bone abnormalities. This may represent a mild variant of oromandibular limb hypogenesis syndrome, expanding the phenotypic spectrum, or a previously unrecognized malformation syndrome.

Novel ELN mutation in a family with supravalvular aortic stenosis and intracranial aneurysm.

Pathogenic germline mutations in ELN can be detected in patients with supravalvular aortic stenosis. The mutation might occur de novo or be inherited following an autosomal dominant pattern of inheritance. In this report we describe a three-generation family suffering from supravalvular aortic stenosis, various other arterial stenoses, sudden death, and intracranial aneurysms. A frameshift mutation in exon 12, not described before, was detected in the affected family members. This report emphasises the importance of family history, genetic counselling, and demonstrates the great variability in the phenotype within a single SVAS family.

A thrifty variant in CREBRF strongly influences body mass index in Samoans.

Samoans are a unique founder population with a high prevalence of obesity, making them well suited for identifying new genetic contributors to obesity. We conducted a genome-wide association study (GWAS) in 3,072 Samoans, discovered a variant, rs12513649, strongly associated with body mass index (BMI) (P = 5.3 × 10(-14)), and replicated the association in 2,102 additional Samoans (P = 1.2 × 10(-9)). Targeted sequencing identified a strongly associated missense variant, rs373863828 (p.Arg457Gln), in CREBRF (meta P = 1.4 × 10(-20)). Although this variant is extremely rare in other populations, it is common in Samoans (frequency of 0.259), with an effect size much larger than that of any other known common BMI risk variant (1.36-1.45 kg/m(2) per copy of the risk-associated allele). In comparison to wild-type CREBRF, the Arg457Gln variant when overexpressed selectively decreased energy use and increased fat storage in an adipocyte cell model. These data, in combination with evidence of positive selection of the allele encoding p.Arg457Gln, support a 'thrifty' variant hypothesis as a factor in human obesity.

Integrin ß3 inhibition is a therapeutic strategy for supravalvular aortic stenosis.

The aorta is the largest artery in the body, yet processes underlying aortic pathology are poorly understood. The arterial media consists of circumferential layers of elastic lamellae and smooth muscle cells (SMCs), and many arterial diseases are characterized by defective lamellae and excess SMCs; however, a mechanism linking these pathological features is lacking. In this study, we use lineage and genetic analysis, pharmacological inhibition, explant cultures, and induced pluripotent stem cells (iPSCs) to investigate supravalvular aortic stenosis (SVAS) patients and/or elastin mutant mice that model SVAS. These experiments demonstrate that multiple preexisting SMCs give rise to excess aortic SMCs in elastin mutants, and these SMCs are hyperproliferative and dedifferentiated. In addition, SVAS iPSC-derived SMCs and the aortic media of elastin mutant mice and SVAS patients have enhanced integrin ß3 levels, activation, and downstream signaling, resulting in SMC misalignment and hyperproliferation. Reduced ß3 gene dosage in elastin-null mice mitigates pathological aortic muscularization, SMC misorientation, and lumen loss and extends survival, which is unprecedented. Finally, pharmacological ß3 inhibition in elastin mutant mice and explants attenuates aortic hypermuscularization and stenosis. Thus, integrin ß3-mediated signaling in SMCs links elastin deficiency and pathological stenosis, and inhibiting this pathway is an attractive therapeutic strategy for SVAS.

Latent transforming growth factor binding protein 4 regulates transforming growth factor beta receptor stability.

Mutations in the gene for the latent transforming growth factor beta binding protein 4 (LTBP4) cause autosomal recessive cutis laxa type 1C. To understand the molecular disease mechanisms of this disease, we investigated the impact of LTBP4 loss on transforming growth factor beta (TGFß) signaling. Despite elevated extracellular TGFß activity, downstream signaling molecules of the TGFß pathway, including pSMAD2 and pERK, were down-regulated in LTBP4 mutant human dermal fibroblasts. In addition, TGFß receptors 1 and 2 (TGFBR1 and TGFBR2) were reduced at the protein but not at the ribonucleic acid level. Treatment with exogenous TGFß1 led to an initially rapid increase in SMAD2 phosphorylation followed by a sustained depression of phosphorylation and receptor abundance. In mutant cells TGFBR1 was co-localized with lysosomes. Treatment with a TGFBR1 kinase inhibitor, endocytosis inhibitors or a lysosome inhibitor, normalized the levels of TGFBR1 and TGFBR2. Co-immunoprecipitation demonstrated a molecular interaction between LTBP4 and TGFBR2. Knockdown of LTBP4 reduced TGFß receptor abundance and signaling in normal cells and supplementation of recombinant LTBP4 enhanced these measures in mutant cells. In a mouse model of Ltbp4 deficiency, reduced TGFß signaling and receptor levels were normalized upon TGFBR1 kinase inhibitor treatment. Our results show that LTBP4 interacts with TGFBR2 and stabilizes TGFß receptors by preventing their endocytosis and lysosomal degradation in a ligand-dependent and receptor kinase activity-dependent manner. These findings identify LTBP4 as a key molecule required for the stability of the TGFß receptor complex, and a new mechanism by which the extracellular matrix regulates cytokine receptor signaling.

A novel elastin gene mutation in a Vietnamese patient with cutis laxa.

We report a 3-year-old girl from Vietnam with severe congenital cutis laxa; no cardiovascular, pulmonary, neurologic, or visceral involvement; and no family history of cutis laxa. Mutational analysis of the elastin gene identified heterozygosity for a previously unreported de novo c.2184delT mutation in exon 30 not present in either parent.

Cutis laxa: intersection of elastic fiber biogenesis, TGFß signaling, the secretory pathway and metabolism.

Cutis laxa (CL), a disease characterized by redundant and inelastic skin, displays extensive locus heterogeneity. Together with geroderma osteodysplasticum and arterial tortuosity syndrome, which show phenotypic overlap with CL, eleven CL-related genes have been identified to date, which encode proteins within 3 groups. Elastin, fibulin-4, fibulin-5 and latent transforming growth factor-ß-binding protein 4 are secreted proteins which form elastic fibers and are involved in the sequestration and subsequent activation of transforming growth factor-ß (TGFß). Proteins within the second group, localized to the secretory pathway, perform transport and membrane trafficking functions necessary for the modification and secretion of elastic fiber components. Key proteins include a subunit of the vacuolar-type proton pump, which ensures the efficient secretion of tropoelastin, the precursor or elastin. A copper transporter is required for the activity of lysyl oxidases, which crosslink collagen and elastin. A Rab6-interacting goglin recruits kinesin motors to Golgi-vesicles facilitating the transport from the Golgi to the plasma membrane. The Rab and Ras interactor 2 regulates the activity of Rab5, a small guanosine triphosphatase essential for the endocytosis of various cell surface receptors, including integrins. Proteins of the third group related to CL perform metabolic functions within the mitochondria, inhibiting the accumulation of reactive oxygen species. Two of these proteins catalyze subsequent steps in the conversion of glutamate to proline. The third transports dehydroascorbate into mitochondria. Recent studies on CL-related proteins highlight the intricate connections among membrane trafficking, metabolism, extracellular matrix assembly, and TGFß signaling.

Comprehensive clinical and molecular analysis of 12 families with type 1 recessive cutis laxa.

Autosomal recessive cutis laxa type I (ARCL type I) is characterized by generalized cutis laxa with pulmonary emphysema and/or vascular complications. Rarely, mutations can be identified in FBLN4 or FBLN5. Recently, LTBP4 mutations have been implicated in a similar phenotype. Studying FBLN4, FBLN5, and LTBP4 in 12 families with ARCL type I, we found bi-allelic FBLN5 mutations in two probands, whereas nine probands harbored biallelic mutations in LTBP4. FBLN5 and LTBP4 mutations cause a very similar phenotype associated with severe pulmonary emphysema, in the absence of vascular tortuosity or aneurysms. Gastrointestinal and genitourinary tract involvement seems to be more severe in patients with LTBP4 mutations. Functional studies showed that most premature termination mutations in LTBP4 result in severely reduced mRNA and protein levels. This correlated with increased transforming growth factor-beta (TGFß) activity. However, one mutation, c.4127dupC, escaped nonsense-mediated decay. The corresponding mutant protein (p.Arg1377Alafs(*) 27) showed reduced colocalization with fibronectin, leading to an abnormal morphology of microfibrils in fibroblast cultures, while retaining normal TGFß activity. We conclude that LTBP4 mutations cause disease through both loss of function and gain of function mechanisms.

The complexity of elastic fibre biogenesis in the skin--a perspective to the clinical heterogeneity of cutis laxa.

Elastic fibres are critical connective tissue components providing elasticity and resilience to skin and other tissues. These fibres are composed of elastin and a number of elastin-associated microfibrillar proteins that assemble in a complex fibre network in a multi-step process. Multiple cellular processes, including mitochondrial function, specific molecules in the secretory pathways and temporally and spatially ordered production of elastic fibre components, are required for the biogenesis of functional elastic fibres. Abnormalities in these processes can lead to loss of functional elastic fibres manifesting phenotypically as a skin disease. The paradigm of elastic fibre diseases affecting the skin is cutis laxa, a clinically and genetically heterogeneous group of disorders characterized by loose and sagging skin, frequently associated with extracutaneous manifestations in the lungs and the arterial blood vessels. The complexity of cutis laxa is emphasized by the fact that as many as 10 distinct genes can harbour mutations in this and related disorders. Understanding of the pathomechanistic pathways involved in perturbed elastic fibre assembly in cutis laxa provides information potentially helpful for the development of molecular strategies towards treatment of these, currently intractable, diseases.

Alternative splicing and tissue-specific elastin misassembly act as biological modifiers of human elastin gene frameshift mutations associated with dominant cutis laxa.

Elastin is the extracellular matrix protein in vertebrates that provides elastic recoil to blood vessels, the lung, and skin. Because the elastin gene has undergone significant changes in the primate lineage, modeling elastin diseases in non-human animals can be problematic. To investigate the pathophysiology underlying a class of elastin gene mutations leading to autosomal dominant cutis laxa, we engineered a cutis laxa mutation (single base deletion) into the human elastin gene contained in a bacterial artificial chromosome. When expressed as a transgene in mice, mutant elastin was incorporated into elastic fibers in the skin and lung with adverse effects on tissue function. In contrast, only low levels of mutant protein incorporated into aortic elastin, which explains why the vasculature is relatively unaffected in this disease. RNA stability studies found that alternative exon splicing acts as a modifier of disease severity by influencing the spectrum of mutant transcripts that survive nonsense-mediated decay. Our results confirm the critical role of the C-terminal region of tropoelastin in elastic fiber assembly and suggest tissue-specific differences in the elastin assembly pathway.

Cutis laxa: a review.

Cutis laxa is a rare disorder of elastic tissue resulting in loose, redundant, hypoelastic skin. Both acquired and inherited forms exist, some of which have significant systemic manifestations. Here, we review the various forms of cutis laxa, with focus on the inherited forms. Recent molecular studies have provided many new insights into the causes of cutis laxa and revealed greater genetic heterogeneity than previously appreciated.