Matrisome and Immune Pathways Contribute to Extreme Vascular Outcomes in Williams-Beuren Syndrome.

BACKGROUND: Supravalvar aortic stenosis (SVAS) is a characteristic feature of Williams-Beuren syndrome (WBS). Its severity varies: ~20% of people with Williams-Beuren syndrome have SVAS requiring surgical intervention, whereas ~35% have no appreciable SVAS. The remaining individuals have SVAS of intermediate severity. Little is known about genetic modifiers that contribute to this variability. METHODS AND RESULTS: We performed genome sequencing on 473 individuals with Williams-Beuren syndrome and developed strategies for modifier discovery in this rare disease population. Approaches include extreme phenotyping and nonsynonymous variant prioritization, followed by gene set enrichment and pathway-level association tests. We next used GTEx v8 and proteomic data sets to verify expression of candidate modifiers in relevant tissues. Finally, we evaluated overlap between the genes/pathways identified here and those ascertained through larger aortic disease/trait genome-wide association studies. We show that SVAS severity in Williams-Beuren syndrome is associated with increased frequency of common and rarer variants in matrisome and immune pathways. Two implicated matrisome genes (ACAN and LTBP4) were uniquely expressed in the aorta. Many genes in the identified pathways were previously reported in genome-wide association studies for aneurysm, bicuspid aortic valve, or aortic size. CONCLUSIONS: Smaller sample sizes in rare disease studies necessitate new approaches to detect modifiers. Our strategies identified variation in matrisome and immune pathways that are associated with SVAS severity. These findings suggest that, like other aortopathies, SVAS may be influenced by the balance of synthesis and degradation of matrisome proteins. Leveraging multiomic data and results from larger aorta-focused genome-wide association studies may accelerate modifier discovery for rare aortopathies like SVAS.

Age-related loss of Notch3 underlies brain vascular contractility deficiencies, glymphatic dysfunction, and neurodegeneration in mice.

Vascular aging affects multiple organ systems, including the brain, where it can lead to vascular dementia. However, a concrete understanding of how aging specifically affects the brain vasculature, along with molecular readouts, remains vastly incomplete. Here, we demonstrate that aging is associated with a marked decline in Notch3 signaling in both murine and human brain vessels. To clarify the consequences of Notch3 loss in the brain vasculature, we used single-cell transcriptomics and found that Notch3 inactivation alters regulation of calcium and contractile function and promotes a notable increase in extracellular matrix. These alterations adversely impact vascular reactivity, manifesting as dilation, tortuosity, microaneurysms, and decreased cerebral blood flow, as observed by MRI. Combined, these vascular impairments hinder glymphatic flow and result in buildup of glycosaminoglycans within the brain parenchyma. Remarkably, this phenomenon mirrors a key pathological feature found in brains of patients with CADASIL, a hereditary vascular dementia associated with NOTCH3 missense mutations. Additionally, single-cell RNA sequencing of the neuronal compartment in aging Notch3-null mice unveiled patterns reminiscent of those observed in neurodegenerative diseases. These findings offer direct evidence that age-related NOTCH3 deficiencies trigger a progressive decline in vascular function, subsequently affecting glymphatic flow and culminating in neurodegeneration.

Multi-organ phenotypes in mice lacking latent TGFß binding protein 2 (LTBP2).

BACKGROUND: Latent TGFß binding protein-2 (LTBP2) is a fibrillin 1 binding component of the microfibril. LTBP2 is the only LTBP protein that does not bind any isoforms of TGFß, although it may interfere with the function of other LTBPs or interact with other signaling pathways. RESULTS: Here, we investigate mice lacking Ltbp2 (Ltbp2-/- ) and identify multiple phenotypes that impact bodyweight and fat mass, and affect bone and skin development. The alterations in skin and bone development are particularly noteworthy since the strength of these tissues is differentially affected by loss of Ltbp2. Interestingly, some tissues that express high levels of Ltbp2, such as the aorta and lung, do not have a developmental or homeostatic phenotype. CONCLUSIONS: Analysis of these mice show that LTBP2 has complex effects on development through direct effects on the extracellular matrix (ECM) or on signaling pathways that are known to regulate the ECM.

Loss of Baz1b in mice causes perinatal lethality, growth failure, and variable multi-system outcomes.

BAZ1B is one of 25-27 coding genes deleted in canonical Williams syndrome, a multi-system disorder causing slow growth, vascular stenosis, and gastrointestinal complaints, including constipation. BAZ1B is involved in (among other processes) chromatin organization, DNA damage repair, and mitosis, suggesting reduced BAZ1B may contribute to Williams syndrome symptoms. In mice, loss of Baz1b causes early neonatal death. 89.6% of Baz1b-/- mice die within 24 h of birth without vascular anomalies or congenital heart disease (except for patent ductus arteriosus). Some (<50%) Baz1b-/- were noted to have prolonged neonatal cyanosis, patent ductus arteriosus, or reduced lung aeration, and none developed a milk spot. Meanwhile, 35.5% of Baz1b+/- mice die over the first three weeks after birth. Surviving Baz1b heterozygotes grow slowly (with variable severity). 66.7% of Baz1b+/- mice develop bowel dilation, compared to 37.8% of wild-type mice, but small bowel and colon transit studies were normal. Additionally, enteric neuron density appeared normal in Baz1b-/- mice except in distal colon myenteric plexus, where neuron density was modestly elevated. Combined with several rare phenotypes (agnathia, microphthalmia, bowel dilation) recovered, our work confirms the importance of BAZ1B in survival and growth and suggests that reduced copy number of BAZ1B may contribute to the variability in Williams syndrome phenotypes.

A protocol for visualization of murine in situ neurovascular interfaces.

Mapping cranial vasculature and adjacent neurovascular interfaces in their entirety will enhance our understanding of central nervous system function in any physiologic state. We present a workflow to visualize in situ murine vasculature and surrounding cranial structures using terminal polymer casting of vessels, iterative sample processing and image acquisition, and automated image registration and processing. While this method does not obtain dynamic imaging due to mouse sacrifice, these studies can be performed before sacrifice and processed with other acquired images. For complete details on the use and execution of this protocol, please refer to Rosenblum et al.1.

Copper-Binding Domain Variation in a Novel Murine Lysyl Oxidase Model Produces Structurally Inferior Aortic Elastic Fibers Whose Failure Is Modified by Age, Sex, and Blood Pressure.

Lysyl oxidase (LOX) is a copper-binding enzyme that cross-links elastin and collagen. The dominant LOX variation contributes to familial thoracic aortic aneurysm. Previously reported murine Lox mutants had a mild phenotype and did not dilate without drug-induced provocation. Here, we present a new, more severe mutant, Loxb2b370.2Clo (c.G854T; p.Cys285Phe), whose mutation falls just N-terminal to the copper-binding domain. Unlike the other mutants, the C285F Lox protein was stably produced/secreted, and male C57Bl/6J Lox+/C285F mice exhibit increased systolic blood pressure (BP; p < 0.05) and reduced caliber aortas (p < 0.01 at 100mmHg) at 3 months that independently dilate by 6 months (p < 0.0001). Multimodal imaging reveals markedly irregular elastic sheets in the mutant (p = 2.8 × 10−8 for breaks by histology) that become increasingly disrupted with age (p < 0.05) and breeding into a high BP background (p = 6.8 × 10−4). Aortic dilation was amplified in males vs. females (p < 0.0001 at 100mmHg) and ameliorated by castration. The transcriptome of young Lox mutants showed alteration in dexamethasone (p = 9.83 × 10−30) and TGFß-responsive genes (p = 7.42 × 10−29), and aortas from older C57Bl/6J Lox+/C285F mice showed both enhanced susceptibility to elastase (p < 0.01 by ANOVA) and increased deposition of aggrecan (p < 0.05). These findings suggest that the secreted Lox+/C285F mutants produce dysfunctional elastic fibers that show increased susceptibility to proteolytic damage. Over time, the progressive weakening of the connective tissue, modified by sex and blood pressure, leads to worsening aortic disease.

Elastin Insufficiency Confers Proximal and Distal Pulmonary Vasculopathy in Mice, Partially Remedied by the KATP Channel Opener Minoxidil: Considerations and Cautions for the Treatment of People With Williams-Beuren Syndrome.

Background: Williams Beuren syndrome (WBS) is a recurrent microdeletion disorder that removes one copy of elastin (ELN), resulting in large artery vasculopathy. Early stenosis of the pulmonary vascular tree is common, but few data are available on longer-term implications of the condition. Methods: Computed tomography (CT) angiogram (n = 11) and echocardiogram (n = 20) were performed in children with WBS aged 3.4-17.8 years. Controls (n = 11, aged 4.4-16.8 years) also underwent echocardiogram. Eln +/- mice were analyzed by invasive catheter, echocardiogram, micro-CT (µCT), histology, and pressure myography. We subsequently tested whether minoxidil resulted in improved pulmonary vascular endpoints. Results: WBS participants with a history of main or branch pulmonary artery (PA) stenosis requiring intervention continued to exhibit increased right ventricular systolic pressure (RVSP, echocardiogram) relative to their peers without intervention (p < 0.01), with no clear difference in PA size. Untreated Eln +/- mice also show elevated RVSP by invasive catheterization (p < 0.0001), increased normalized right heart mass (p < 0.01) and reduced caliber branch PAs by pressure myography (p < 0.0001). Eln +/- main PA medias are thickened histologically relative to Eln +/+ (p < 0.0001). Most Eln +/- phenotypes are shared by both sexes, but PA medial thickness is substantially greater in Eln +/- males (p < 0.001). Eln +/- mice showed more acute proximal branching angles (p < 0.0001) and longer vascular segment lengths (p < 0.0001) (µCT), with genotype differences emerging by P7. Diminished PA acceleration time (p < 0.001) and systolic notching (p < 0.0001) were also observed in Eln +/- echocardiography. Vascular casting plus µCT revealed longer generation-specific PA arcade length (p < 0.0001), with increased PA branching detectable by P90 (p < 0.0001). Post-weaning minoxidil decreased RVSP (p < 0.01) and normalized PA caliber (p < 0.0001) but not early-onset proximal branching angle or segment length, nor later-developing peripheral branch number. Conclusions: Vascular deficiencies beyond arterial caliber persist in individuals with WBS who have undergone PA stenosis intervention. Evaluation of Eln +/- mice reveals complex vascular changes that affect the proximal and distal vasculatures. Minoxidil, given post-weaning, decreases RVSP and improves lumen diameter, but does not alter other earlier-onset vascular patterns. Our data suggest additional therapies including minoxidil could be a useful adjunct to surgical therapy, and future trials should be considered.

Airflow Obstruction in Adults with Williams Syndrome and Mice with Elastin Insufficiency.

Williams−Beuren syndrome (WS) results from the deletion of 25−27 coding genes, including elastin (ELN), on human chromosome 7q11.23. Elastin provides recoil to tissues; emphysema and chronic obstructive pulmonary disease have been linked to its destruction. Consequently, we hypothesized that elastin insufficiency would predispose to obstructive features. Twenty-two adults with WS (aged 18−55) and controls underwent pulmonary function testing, 6 min walk, and chest computed tomography (CT). Lung and airspace dimensions were assessed in Eln+/− and control mice via microCT and histology. The forced expiratory volume in 1 s (FEV1) and the ratio of FEV1 to forced vital capacity (FVC) were lower in adults with WS (p < 0.0001 and p < 0.05, respectively). The FEV1/FVC ratio was more frequently below the lower limit of normal in cases (p < 0.01). The ratio of residual volume to total lung capacity (RV/TLC, percent predicted) was higher in cases (p < 0.01), suggesting air trapping. People with WS showed reduced exercise capacity (p < 0.0001). In Eln+/− mice, ex vivo lung volumes were increased (p < 0.0001), with larger airspaces (p < 0.001). Together these data show that elastin insufficiency impacts lung physiology in the form of increased air trapping and obstruction, suggesting a role for lung function monitoring in adults with WS.

Non-invasive in situ Visualization of the Murine Cranial Vasculature.

Understanding physiologic and pathologic central nervous system function depends on our ability to map the entire in situ cranial vasculature and neurovascular interfaces. To accomplish this, we developed a non-invasive workflow to visualize murine cranial vasculature via polymer casting of vessels, iterative sample processing and micro-computed tomography, and automatic deformable image registration, feature extraction, and visualization. This methodology is applicable to any tissue and allows rapid exploration of normal and altered pathologic states.

Cardiac pathologies in mouse loss of imprinting models are due to misexpression of H19 long noncoding RNA.

Maternal loss of imprinting (LOI) at the H19/IGF2 locus results in biallelic IGF2 and reduced H19 expression and is associated with Beckwith--Wiedemann syndrome (BWS). We use mouse models for LOI to understand the relative importance of Igf2 and H19 mis-expression in BWS phenotypes. Here we focus on cardiovascular phenotypes and show that neonatal cardiomegaly is exclusively dependent on increased Igf2. Circulating IGF2 binds cardiomyocyte receptors to hyperactivate mTOR signaling, resulting in cellular hyperplasia and hypertrophy. These Igf2-dependent phenotypes are transient: cardiac size returns to normal once Igf2 expression is suppressed postnatally. However, reduced H19 expression is sufficient to cause progressive heart pathologies including fibrosis and reduced ventricular function. In the heart, H19 expression is primarily in endothelial cells (ECs) and regulates EC differentiation both in vivo and in vitro. Finally, we establish novel mouse models to show that cardiac phenotypes depend on H19 lncRNA interactions with Mirlet7 microRNAs.

Inhibition of NOX1 Mitigates Blood Pressure Increases in Elastin Insufficiency.

Elastin (ELN) insufficiency leads to the cardiovascular hallmarks of the contiguous gene deletion disorder, Williams-Beuren syndrome, including hypertension and vascular stiffness. Previous studies showed that Williams-Beuren syndrome deletions, which extended to include the NCF1 gene, were associated with lower blood pressure (BP) and reduced vascular stiffness. NCF1 encodes for p47phox, the regulatory component of the NOX1 NADPH oxidase complex that generates reactive oxygen species (ROS) in the vascular wall. Dihydroethidium and 8-hydroxyguanosine staining of mouse aortas confirmed that Eln heterozygotes (Eln+/- ) had greater ROS levels than the wild-types (Eln+/+ ), a finding that was negated in vessels cultured without hemodynamic stressors. To analyze the Nox effect on ELN insufficiency, we used both genetic and chemical manipulations. Both Ncf1 haploinsufficiency (Ncf1+/- ) and Nox1 insufficiency (Nox1-/y ) decreased oxidative stress and systolic BP in Eln+/- without modifying vascular structure. Chronic treatment with apocynin, a p47phox inhibitor, lowered systolic BP in Eln+/- , but had no impact on Eln+/+ controls. In vivo dosing with phenylephrine (PE) produced an augmented BP response in Eln+/- relative to Eln+/+ , and genetic modifications or drug-based interventions that lower Nox1 expression reduced the hypercontractile response to PE in Eln+/- mice to Eln+/+ levels. These results indicate that the mechanical and structural differences caused by ELN insufficiency leading to oscillatory flow can perpetuate oxidative stress conditions, which are linked to hypertension, and that by lowering the Nox1-mediated capacity for vascular ROS production, BP differences can be normalized.

Developmental vascular malformations in EPAS1 gain-of-function syndrome.

Mutations in EPAS1, encoding hypoxia-inducible factor-2α (HIF-2α), were previously identified in a syndrome of multiple paragangliomas, somatostatinoma, and polycythemia. HIF-2α, when dimerized with HIF-1ß, acts as an angiogenic transcription factor. Patients referred to the NIH for new, recurrent, and/or metastatic paraganglioma or pheochromocytoma were confirmed for EPAS1 gain-of-function mutation; imaging was evaluated for vascular malformations. We evaluated the Epas1A529V transgenic syndrome mouse model, corresponding to the mutation initially detected in the patients (EPAS1A530V), for vascular malformations via intravital 2-photon microscopy of meningeal vessels, terminal vascular perfusion with Microfil silicate polymer and subsequent intact ex vivo 14T MRI and micro-CT, and histologic sectioning and staining of the brain and identified pathologies. Further, we evaluated retinas from corresponding developmental time points (P7, P14, and P21) and the adult dura via immunofluorescent labeling of vessels and confocal imaging. We identified a spectrum of vascular malformations in all 9 syndromic patients and in all our tested mutant mice. Patient vessels had higher variant allele frequency than adjacent normal tissue. Veins of the murine retina and intracranial dura failed to regress normally at the expected developmental time points. These findings add vascular malformation as a new clinical feature of EPAS1 gain-of-function syndrome.

Vascular Casting of Adult and Early Postnatal Mouse Lungs for Micro-CT Imaging.

Blood vessels form intricate networks in 3-dimensional space. Consequently, it is difficult to visually appreciate how vascular networks interact and behave by observing the surface of a tissue. This method provides a means to visualize the complex 3-dimensional vascular architecture of the lung. To accomplish this, a catheter is inserted into the pulmonary artery and the vasculature is simultaneously flushed of blood and chemically dilated to limit resistance. Lungs are then inflated through the trachea at a standard pressure and the polymer compound is infused into the vascular bed at a standard flow rate. Once the entire arterial network is filled and allowed to cure, the lung vasculature may be visualized directly or imaged on a micro-CT (µCT) scanner. When performed successfully, one can appreciate the pulmonary arterial network in mice ranging from early postnatal ages to adults. Additionally, while demonstrated in the pulmonary arterial bed, this method can be applied to any vascular bed with optimized catheter placement and endpoints.

Whole exome sequencing in patients with Williams-Beuren syndrome followed by disease modeling in mice points to four novel pathways that may modify stenosis risk.

Supravalvular aortic stenosis (SVAS) is a narrowing of the aorta caused by elastin (ELN) haploinsufficiency. SVAS severity varies among patients with Williams-Beuren syndrome (WBS), a rare disorder that removes one copy of ELN and 25-27 other genes. Twenty percent of children with WBS require one or more invasive and often risky procedures to correct the defect while 30% have no appreciable stenosis, despite sharing the same basic genetic lesion. There is no known medical therapy. Consequently, identifying genes that modify SVAS offers the potential for novel modifier-based therapeutics. To improve statistical power in our rare-disease cohort (N = 104 exomes), we utilized extreme-phenotype cohorting, functional variant filtration and pathway-based analysis. Gene set enrichment analysis of exome-wide association data identified increased adaptive immune system variant burden among genes associated with SVAS severity. Additional enrichment, using only potentially pathogenic variants known to differ in frequency between the extreme phenotype subsets, identified significant association of SVAS severity with not only immune pathway genes, but also genes involved with the extracellular matrix, G protein-coupled receptor signaling and lipid metabolism using both SKAT-O and RQTest. Complementary studies in Eln+/-; Rag1-/- mice, which lack a functional adaptive immune system, showed improvement in cardiovascular features of ELN insufficiency. Similarly, studies in mixed background Eln+/- mice confirmed that variations in genes that increase elastic fiber deposition also had positive impact on aortic caliber. By using tools to improve statistical power in combination with orthogonal analyses in mice, we detected four main pathways that contribute to SVAS risk.

Impaired angiogenesis and extracellular matrix metabolism in autosomal-dominant hyper-IgE syndrome.

There are more than 7000 described rare diseases, most lacking specific treatment. Autosomal-dominant hyper-IgE syndrome (AD-HIES, also known as Job's syndrome) is caused by mutations in STAT3. These patients present with immunodeficiency accompanied by severe nonimmunological features, including skeletal, connective tissue, and vascular abnormalities, poor postinfection lung healing, and subsequent pulmonary failure. No specific therapies are available for these abnormalities. Here, we investigated underlying mechanisms in order to identify therapeutic targets. Histological analysis of skin wounds demonstrated delayed granulation tissue formation and vascularization during skin-wound healing in AD-HIES patients. Global gene expression analysis in AD-HIES patient skin fibroblasts identified deficiencies in a STAT3-controlled transcriptional network regulating extracellular matrix (ECM) remodeling and angiogenesis, with hypoxia-inducible factor 1α (HIF-1α) being a major contributor. Consistent with this, histological analysis of skin wounds and coronary arteries from AD-HIES patients showed decreased HIF-1α expression and revealed abnormal organization of the ECM and altered formation of the coronary vasa vasorum. Disease modeling using cell culture and mouse models of angiogenesis and wound healing confirmed these predicted deficiencies and demonstrated therapeutic benefit of HIF-1α-stabilizing drugs. The study provides mechanistic insights into AD-HIES pathophysiology and suggests potential treatment options for this rare disease.

Neuraxial dysraphism in EPAS1-associated syndrome due to improper mesenchymal transition.

OBJECTIVE: To investigate the effect of somatic, postzygotic, gain-of-function mutation of Endothelial Per-Arnt-Sim (PAS) domain protein 1 (EPAS1) encoding hypoxia-inducible factor-2α (HIF-2α) on posterior fossa development and spinal dysraphism in EPAS1 gain-of-function syndrome, which consists of multiple paragangliomas, somatostatinoma, and polycythemia. METHODS: Patients referred to our institution for evaluation of new, recurrent, and/or metastatic paragangliomas/pheochromocytoma were confirmed for EPAS1 gain-of-function syndrome by identification of the EPAS1 gain-of-function mutation in resected tumors and/or circulating leukocytes. The posterior fossa, its contents, and the spine were evaluated retrospectively on available MRI and CT images of the head and neck performed for tumor staging and restaging. The transgenic mouse model underwent Microfil vascular perfusion and subsequent intact ex vivo 14T MRI and micro-CT as well as gross dissection, histology, and immunohistochemistry to assess the role of EPAS1 in identified malformations. RESULTS: All 8 patients with EPAS1 gain-of-function syndrome demonstrated incidental posterior fossa malformations-one Dandy-Walker variant and 7 Chiari malformations without syringomyelia. These findings were not associated with a small posterior fossa; rather, the posterior fossa volume exceeded that of its neural contents. Seven of 8 patients demonstrated spinal dysraphism; 4 of 8 demonstrated abnormal vertebral segmentation. The mouse model similarly demonstrated features of neuraxial dysraphism, including cervical myelomeningocele and spinal dysraphism, and cerebellar tonsil displacement through the foramen magnum. Histology and immunohistochemistry demonstrated incomplete mesenchymal transition in the mutant but not the control mouse. CONCLUSIONS: This study characterized posterior fossa and spinal malformations seen in EPAS1 gain-of-function syndrome and suggests that gain-of-function mutation in HIF-2α results in improper mesenchymal transition.

Minoxidil improves vascular compliance, restores cerebral blood flow, and alters extracellular matrix gene expression in a model of chronic vascular stiffness.

Increased vascular stiffness correlates with a higher risk of cardiovascular complications in aging adults. Elastin (ELN) insufficiency, as observed in patients with Williams-Beuren syndrome or with familial supravalvular aortic stenosis, also increases vascular stiffness and leads to arterial narrowing. We used Eln+/- mice to test the hypothesis that pathologically increased vascular stiffness with concomitant arterial narrowing leads to decreased blood flow to end organs such as the brain. We also hypothesized that drugs that remodel arteries and increase lumen diameter would improve flow. To test these hypotheses, we compared carotid blood flow using ultrasound and cerebral blood flow using MRI-based arterial spin labeling in wild-type (WT) and Eln+/- mice. We then studied how minoxidil, an ATP-sensitive K+ channel opener and vasodilator, affects vessel mechanics, blood flow, and gene expression. Both carotid and cerebral blood flows were lower in Eln+/- mice than in WT mice. Treatment of Eln+/- mice with minoxidil lowered blood pressure and reduced functional arterial stiffness to WT levels. Minoxidil also improved arterial diameter and restored carotid and cerebral blood flows in Eln+/- mice. The beneficial effects persisted for weeks after drug removal. RNA-Seq analysis revealed differential expression of 127 extracellular matrix-related genes among the treatment groups. These results indicate that ELN insufficiency impairs end-organ perfusion, which may contribute to the increased cardiovascular risk. Minoxidil, despite lowering blood pressure, improves end-organ perfusion. Changes in matrix gene expression and persistence of treatment effects after drug withdrawal suggest arterial remodeling. Such remodeling may benefit patients with genetic or age-dependent ELN insufficiency. NEW & NOTEWORTHY Our work with a model of chronic vascular stiffness, the elastin ( Eln)+/- mouse, shows reduced brain perfusion as measured by carotid ultrasound and MRI arterial spin labeling. Vessel caliber, functional stiffness, and blood flow improved with minoxidil. The ATP-sensitive K+ channel opener increased Eln gene expression and altered 126 other matrix-associated genes.

Albumin contributes to kidney disease progression in Alport syndrome.

Alport syndrome is a familial kidney disease caused by defects in the collagen type IV network of the glomerular basement membrane. Lack of collagen-α3α4α5(IV) changes the glomerular basement membrane morphologically and functionally, rendering it leaky to albumin and other plasma proteins. Filtered albumin has been suggested to be a cause of the glomerular and tubular injuries observed at advanced stages of Alport syndrome. To directly investigate the role that albumin plays in the progression of disease in Alport syndrome, we generated albumin knockout (Alb(-/-)) mice to use as a tool for removing albuminuria as a component of kidney disease. Mice lacking albumin were healthy and indistinguishable from control littermates, although they developed hypertriglyceridemia. Dyslipidemia was observed in Alb(+/-) mice, which displayed half the normal plasma albumin concentration. Alb mutant mice were bred to collagen-α3(IV) knockout (Col4a3(-/-)) mice, which are a model for human Alport syndrome. Lack of circulating and filtered albumin in Col4a3(-/-);Alb(-/-) mice resulted in dramatically improved kidney disease outcomes, as these mice lived 64% longer than did Col4a3(-/-);Alb(+/+) and Col4a3(-/-);Alb(+/-) mice, despite similar blood pressures and serum triglyceride levels. Further investigations showed that the absence of albumin correlated with reduced transforming growth factor-ß1 signaling as well as reduced tubulointerstitial, glomerular, and podocyte pathology. We conclude that filtered albumin is injurious to kidney cells in Alport syndrome and perhaps in other proteinuric kidney diseases, including diabetic nephropathy.

Chronic antihypertensive treatment improves pulse pressure but not large artery mechanics in a mouse model of congenital vascular stiffness.

Increased arterial stiffness is a common characteristic of humans with Williams-Beuren syndrome and mouse models of elastin insufficiency. Arterial stiffness is associated with multiple negative cardiovascular outcomes, including myocardial infarction, stroke, and sudden death. Therefore, identifying therapeutic interventions that improve arterial stiffness in response to changes in elastin levels is of vital importance. The goal of this study was to determine the effect of chronic pharmacologic therapy with different classes of antihypertensive medications on arterial stiffness in elastin insufficiency. Elastin-insufficient mice 4-6 wk of age and wild-type littermates were subcutaneously implanted with osmotic micropumps delivering a continuous dose of one of the following: vehicle, losartan, nicardipine, or propranolol for 8 wk. At the end of treatment period, arterial blood pressure and large artery compliance and remodeling were assessed. Our results show that losartan and nicardipine treatment lowered blood pressure and pulse pressure in elastin-insufficient mice. Elastin and collagen content of abdominal aortas as well as ascending aorta and carotid artery biomechanics were not affected by any of the drug treatments in either genotype. By reducing pulse pressure and shifting the working pressure range of an artery to a more compliant region of the pressure-diameter curve, antihypertensive medications may mitigate the consequences of arterial stiffness, an effect that is drug class independent. These data emphasize the importance of early recognition and long-term management of hypertension in Williams-Beuren syndrome and elastin insufficiency.

Skin findings in Williams syndrome.

Previous examination in a small number of individuals with Williams syndrome (also referred to as Williams-Beuren syndrome) has shown subtly softer skin and reduced deposition of elastin, an elastic matrix protein important in tissue recoil. No quantitative information about skin elasticity in individuals with Williams syndrome is available; nor has there been a complete report of dermatologic findings in this population. To fill this knowledge gap, 94 patients with Williams syndrome aged 7-50 years were recruited as part of the skin and vascular elasticity (WS-SAVE) study. They underwent either a clinical dermatologic assessment by trained dermatologists (2010 WSA family meeting) or measurement of biomechanical properties of the skin with the DermaLab™ suction cup (2012 WSA family meeting). Clinical assessment confirmed that soft skin is common in this population (83%), as is premature graying of the hair (80% of those 20 years or older), while wrinkles (92%), and abnormal scarring (33%) were detected in larger than expected proportions. Biomechanical studies detected statistically significant differences in dP (the pressure required to lift the skin), dT (the time required to raise the skin through a prescribed gradient), VE (viscoelasticity), and E (Young's modulus) relative to matched controls. The RT (retraction time) also trended longer but was not significant. The biomechanical differences noted in these patients did not correlate with the presence of vascular defects also attributable to elastin insufficiency (vascular stiffness, hypertension, and arterial stenosis) suggesting the presence of tissue specific modifiers that modulate the impact of elastin insufficiency in each tissue.