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.

Constitutive Modeling of Mouse Arteries Suggests Changes in Directional Coupling and Extracellular Matrix Remodeling That Depend on Artery Type, Age, Sex, and Elastin Amounts.

Arterial stiffening occurs during natural aging, is associated with an increased risk of adverse cardiovascular events, and can follow different timelines in males and females. One mechanism of arterial stiffening includes remodeling of the extracellular matrix (ECM), which alters the wall material properties. We used elastin haploinsufficient (Eln+/-) and wildtype (Eln+/+) mice to investigate how material properties of two different arteries (ascending aorta and carotid artery) change with age, sex, and ECM composition. We used a constitutive model by Dong and Sun that is based on the Holzapfel-Gasser-Ogden (HGO) type, but does not require a discrete number of fibrous ECM families and allows varied deformation coupling. We find that the amount of deformation coupling for the best fit model depends on the artery type. We also find that remodeling to maintain homeostatic (i.e., young, wildtype) values of biomechanical parameters with age, sex, and ECM composition depends on the artery type, with ascending aorta being more adaptable than carotid artery. Fitted material constants indicate sex-dependent remodeling that may be important for determining the time course of arterial stiffening in males and females. We correlated fitted material constants with ECM composition measured by biochemical (ascending aorta) or histological (carotid artery) methods. We show significant correlations between ECM composition and material parameters for the mean values for each group, with biochemical measurements correlating more strongly than histological measurements. Understanding how arterial stiffening depends on age, sex, ECM composition, and artery type may help design effective, personalized clinical treatment strategies.

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.

Vascular Smooth Muscle Cell Subpopulations and Neointimal Formation in Mouse Models of Elastin Insufficiency.

OBJECTIVE: Using a mouse model of Eln (elastin) insufficiency that spontaneously develops neointima in the ascending aorta, we sought to understand the origin and phenotypic heterogeneity of smooth muscle cells (SMCs) contributing to intimal hyperplasia. We were also interested in exploring how vascular cells adapt to the absence of Eln. Approach and Results: We used single-cell sequencing together with lineage-specific cell labeling to identify neointimal cell populations in a noninjury, genetic model of neointimal formation. Inactivating Eln production in vascular SMCs results in rapid intimal hyperplasia around breaks in the ascending aorta's internal elastic lamina. Using lineage-specific Cre drivers to both lineage mark and inactivate Eln expression in the secondary heart field and neural crest aortic SMCs, we found that cells with a secondary heart field lineage are significant contributors to neointima formation. We also identified a small population of secondary heart field-derived SMCs underneath and adjacent to the internal elastic lamina. Within the neointima of SMC-Eln knockout mice, 2 unique SMC populations were identified that are transcriptionally different from other SMCs. While these cells had a distinct gene signature, they expressed several genes identified in other studies of neointimal lesions, suggesting that some mechanisms underlying neointima formation in Eln insufficiency are shared with adult vessel injury models. CONCLUSIONS: These results highlight the unique developmental origin and transcriptional signature of cells contributing to neointima in the ascending aorta. Our findings also show that the absence of Eln, or changes in elastic fiber integrity, influences the SMC biological niche in ways that lead to altered cell phenotypes.

Identification of the growth factor-binding sequence in the extracellular matrix protein MAGP-1.

Microfibril-associated glycoprotein-1 (MAGP-1) is a component of vertebrate extracellular matrix (ECM) microfibrils that, together with the fibrillins, contributes to microfibril function. Many of the phenotypes associated with MAGP-1 gene inactivation are consistent with dysregulation of the transforming growth factor ß (TGFß)/bone morphogenetic protein (BMP) signaling system. We have previously shown that full-length MAGP-1 binds active TGFß-1 and some BMPs. The work presented here further defines the growth factor-binding domain of MAGP-1. Using recombinant domains and synthetic peptides, along with surface plasmon resonance analysis to measure the kinetics of the MAGP-1-TGFß-1 interaction, we localized the TGFß- and BMP-binding site in MAGP-1 to a 19-amino acid-long, highly acidic sequence near the N terminus. This domain was specific for binding active, but not latent, TGFß-1. Growth factor activity experiments revealed that TGFß-1 retains signaling activity when complexed with MAGP-1. Furthermore, when bound to fibrillin, MAGP-1 retained the ability to interact with TGFß-1, and active TGFß-1 did not bind fibrillin in the absence of MAGP-1. The absence of MAGP was sufficient to raise the amount of total TGFß stored in the ECM of cultured cells, suggesting that the MAGPs compete with the TGFß large latent complex for binding to microfibrils. Together, these results indicate that MAGP-1 plays an active role in TGFß signaling in the ECM.

Heterogeneous Cellular Contributions to Elastic Laminae Formation in Arterial Wall Development.

RATIONALE: Elastin is an important ECM (extracellular matrix) protein in large and small arteries. Vascular smooth muscle cells (SMCs) produce the layered elastic laminae found in elastic arteries but synthesize little elastin in muscular arteries. However, muscular arteries have a well-defined internal elastic lamina (IEL) that separates endothelial cells (ECs) from SMCs. The extent to which ECs contribute elastin to the IEL is unknown. OBJECTIVE: To use targeted elastin (Eln) deletion in mice to explore the relative contributions of SMCs and ECs to elastic laminae formation in different arteries. METHODS AND RESULTS: We used SMC- and EC-specific Cre recombinase transgenes with a novel floxed Eln allele to focus gene inactivation in mice. Inactivation of Eln in SMCs using Sm22aCre resulted in depletion of elastic laminae in the arterial wall with the exception of the IEL and SMC clusters in the outer media near the adventitia. Inactivation of elastin in ECs using Tie2Cre or Cdh5Cre resulted in normal medial elastin and a typical IEL in elastic arteries. In contrast, the IEL was absent or severely disrupted in muscular arteries. Interruptions in the IEL resulted in neointimal formation in the ascending aorta but not in muscular arteries. CONCLUSIONS: Combined with lineage-specific fate mapping systems, our knockout results document an unexpected heterogeneity in vascular cells that produce the elastic laminae. SMCs and ECs can independently form an IEL in most elastic arteries, whereas ECs are the major source of elastin for the IEL in muscular and resistance arteries. Neointimal formation at IEL disruptions in the ascending aorta confirms that the IEL is a critical physical barrier between SMCs and ECs in the large elastic arteries. Our studies provide new information about how SMCs and ECs contribute elastin to the arterial wall and how local elastic laminae defects may contribute to cardiovascular disease.

Elastin haploinsufficiency in mice has divergent effects on arterial remodeling with aging depending on sex.

Elastin is a primary structural protein in the arterial wall that contributes to vascular mechanical properties and degrades with aging. Aging is associated with arterial stiffening and an increase in blood pressure. There is evidence that arterial aging follows different timelines with sex. Our objective was to investigate how elastin content affects arterial remodeling in male and female mice with aging. We used male and female wild-type (Eln+/+) and elastin heterozygous (Eln+/-) mice at 6, 12, and 24 mo of age and measured their blood pressure and arterial morphology, wall structure, protein content, circumferential stress, stretch ratio, and stiffness. Two arteries were used with varying contents of elastin: the left common carotid and ascending aorta. We show that Eln+/- arteries start at a different homeostatic set point for circumferential wall stress, stretch, and material stiffness but show similar increases with aging to Eln+/+ mice. With aging, structural stiffness is greatly increased, while material stiffness and circumferential stress are only slightly increased, highlighting the importance of maintaining these homeostatic values. Circumferential stretch shows the smallest change with age and may be important for controlling cellular phenotype. Independent sex differences are mostly associated with males being larger than females; however, many of the measured factors show age × sex and/or genotype × sex interactions, indicating that males and females follow different cardiovascular remodeling timelines with aging and are differentially affected by reduced elastin content.NEW & NOTEWORTHY A comprehensive study on arterial mechanical behavior as a function of elastin content, aging, and sex in mice. Elastin haploinsufficient arteries start at a different homeostatic set point for mechanical parameters such as circumferential stress, stretch, and material stiffness. Structural stiffness of the arterial wall greatly increases with aging, as expected, but there are interactions between sex and aging for most of the mechanical parameters that are important to consider in future work.

FGF receptors control alveolar elastogenesis.

Alveologenesis, the final step of lung development, is characterized by the formation of millions of alveolar septa that constitute the vast gas-exchange surface area. The genetic network driving alveologenesis is poorly understood compared with earlier steps in lung development. FGF signaling through receptors Fgfr3 and Fgfr4 is crucial for alveologenesis, but the mechanisms through which they mediate this process remain unclear. Here we show that in Fgfr3;Fgfr4 (Fgfr3;4) global mutant mice, alveolar simplification is first observed at the onset of alveologenesis at postnatal day 3. This is preceded by disorganization of elastin, indicating defects in the extracellular matrix (ECM). Although Fgfr3 and Fgfr4 are expressed in the mesenchyme and epithelium, inactivation in the mesenchyme, but not the epithelium, recapitulated the defects. Expression analysis of components of the elastogenesis machinery revealed that Mfap5 (also known as Magp2), which encodes an elastin-microfibril bridging factor, is upregulated in Fgfr3;4 mutants. Mfap5 mutation in the Fgfr3;4 mutant background partially attenuated the alveologenesis defects. These data demonstrate that, during normal lung maturation, FGF signaling restricts expression of the elastogenic machinery in the lung mesenchyme to control orderly formation of the elastin ECM, thereby driving alveolar septa formation to increase the gas-exchange surface.

Sodium-activated potassium channels moderate excitability in vascular smooth muscle.

KEY POINTS: We report that a sodium-activated potassium current, IKNa , has been inadvertently overlooked in both conduit and resistance arterial smooth muscle cells. IKNa is a major K+ resting conductance and is absent in cells of IKNa knockout (KO) mice. The phenotype of the IKNa KO is mild hypertension, although KO mice react more strongly than wild-type with raised blood pressure when challenged with vasoconstrictive agents. IKNa is negatively regulated by angiotensin II acting through Gαq protein-coupled receptors. In current clamp, KO arterial smooth muscle cells have easily evoked Ca2+ -dependent action potentials. ABSTRACT: Although several potassium currents have been reported to play a role in arterial smooth muscle (ASM), we find that one of the largest contributors to membrane conductance in both conduit and resistance ASMs has been inadvertently overlooked. In the present study, we show that IKNa , a sodium-activated potassium current, contributes a major portion of macroscopic outward current in a critical physiological voltage range that determines intrinsic cell excitability; IKNa is the largest contributor to ASM cell resting conductance. A genetic knockout (KO) mouse strain lacking KNa channels (KCNT1 and KCNT2) shows only a modest hypertensive phenotype. However, acute administration of vasoconstrictive agents such as angiotensin II (Ang II) and phenylephrine results in an abnormally large increase in blood pressure in the KO animals. In wild-type animals Ang II acting through Gαq protein-coupled receptors down-regulates IKNa , which increases the excitability of the ASMs. The complete genetic removal of IKNa in KO mice makes the mutant animal more vulnerable to vasoconstrictive agents, thus producing a paroxysmal-hypertensive phenotype. This may result from the lowering of cell resting K+ conductance allowing the cells to depolarize more readily to a variety of excitable stimuli. Thus, the sodium-activated potassium current may serve to moderate blood pressure in instances of heightened stress. IKNa may represent a new therapeutic target for hypertension and stroke.

Decreased lysyl oxidase level protects against development of retinal vascular lesions in diabetic retinopathy.

Retinal capillary basement membrane (BM) thickening is closely associated with the development of vascular lesions in diabetic retinopathy. Thickened capillary BM can compromise blood-retinal-barrier characteristics and contribute to retinal vascular permeability, a significant clinical manifestation of diabetic retinopathy. We have previously shown that high glucose increases the expression and activity of lysyl oxidase (LOX), a crosslinking enzyme, in retinal endothelial cells. Additionally, concomitant with overexpression of LOX, increased vascular permeability was observed in diabetic rat retinas. However, it is unknown whether decreasing LOX overexpression may have protective effects against development of retinal vascular lesions in diabetes. To investigate whether reduced LOX level protects against diabetes-induced development of retinal vascular lesions characteristic of diabetic retinopathy, four groups of mice: wild type (WT) control mice, streptozotocin (STZ)-induced diabetic mice, LOX +/- mice, and STZ-induced diabetic LOX +/- mice were used for this study. Diabetes was maintained for 16 weeks; at the end of the study, retinas were assessed for LOX protein level by Western Blot (WB) analysis, and retinal capillary networks were isolated using retinal trypsin digestion and stained with hematoxylin and periodic acid Schiff to identify the number of acellular capillaries (AC) and pericyte loss (PL). In parallel, TUNEL assay was performed on retinal trypsin digests (RTDs) to detect cells undergoing apoptosis in the retinal capillary networks. Retinal vascular permeability was analyzed following FITC-dextran injection in retinal whole mounts. A significant increase in LOX expression was detected in the diabetic retinas compared to those of the WT control retinas, and as expected, a significant decrease in LOX expression in the diabetic LOX +/- retinas was observed compared to those of the diabetic retinas. RTD images showed significantly increased AC and PL counts in the retinas of diabetic mice compared to those of the WT control mice. Importantly, the number of AC and PL was significantly decreased, as was retinal vascular permeability in the retinas of the diabetic LOX +/- mice compared to those of the diabetic mice. Results suggest that decreasing diabetes-induced LOX overexpression may have protective effects against the development of vascular lesions characteristic of diabetic retinopathy. Therefore, LOX overexpression may be a potential target in preventing retinal vascular cell loss and excess permeability associated with diabetic retinopathy.

Loss of function mutation in LOX causes thoracic aortic aneurysm and dissection in humans.

Thoracic aortic aneurysms and dissections (TAAD) represent a substantial cause of morbidity and mortality worldwide. Many individuals presenting with an inherited form of TAAD do not have causal mutations in the set of genes known to underlie disease. Using whole-genome sequencing in two first cousins with TAAD, we identified a missense mutation in the lysyl oxidase (LOX) gene (c.893T > G encoding p.Met298Arg) that cosegregated with disease in the family. Using clustered regularly interspaced short palindromic repeats (CRISPR)/clustered regularly interspaced short palindromic repeats-associated protein-9 nuclease (Cas9) genome engineering tools, we introduced the human mutation into the homologous position in the mouse genome, creating mice that were heterozygous and homozygous for the human allele. Mutant mice that were heterozygous for the human allele displayed disorganized ultrastructural properties of the aortic wall characterized by fragmented elastic lamellae, whereas mice homozygous for the human allele died shortly after parturition from ascending aortic aneurysm and spontaneous hemorrhage. These data suggest that a missense mutation in LOX is associated with aortic disease in humans, likely through insufficient cross-linking of elastin and collagen in the aortic wall. Mutation carriers may be predisposed to vascular diseases because of weakened vessel walls under stress conditions. LOX sequencing for clinical TAAD may identify additional mutation carriers in the future. Additional studies using our mouse model of LOX-associated TAAD have the potential to clarify the mechanism of disease and identify novel therapeutics specific to this genetic cause.

Deficient Circumferential Growth Is the Primary Determinant of Aortic Obstruction Attributable to Partial Elastin Deficiency.

OBJECTIVE: Williams syndrome is characterized by obstructive aortopathy attributable to heterozygous loss of ELN, the gene encoding elastin. Lesions are thought to result primarily from excessive smooth muscle cell (SMC) proliferation and consequent medial expansion, although an initially smaller caliber and increased stiffness of the aorta may contribute to luminal narrowing. The relative contributions of such abnormalities to the obstructive phenotype had not been defined. APPROACH AND RESULTS: We quantified determinants of luminal stenosis in thoracic aortas of Eln-/- mice incompletely rescued by human ELN. Moderate obstruction was largely because of deficient circumferential growth, most prominently of ascending segments, despite increased axial growth. Medial thickening was evident in these smaller diameter elastin-deficient aortas, with medial area similar to that of larger diameter control aortas. There was no difference in cross-sectional SMC number between mutant and wild-type genotypes at multiple stages of postnatal development. Decreased elastin content was associated with medial fibrosis and reduced aortic distensibility because of increased structural stiffness but preserved material stiffness. Elastin-deficient SMCs exhibited greater contractile-to-proliferative phenotypic modulation in vitro than in vivo. We confirmed increased medial collagen without evidence of increased medial area or SMC number in a small ascending aorta with thickened media of a Williams syndrome subject. CONCLUSIONS: Deficient circumferential growth is the predominant mechanism for moderate obstructive aortic disease resulting from partial elastin deficiency. Our findings suggest that diverse aortic manifestations in Williams syndrome result from graded elastin content, and SMC hyperplasia causing medial expansion requires additional elastin loss superimposed on ELN haploinsufficiency.

Mechanical behavior and matrisome gene expression in the aneurysm-prone thoracic aorta of newborn lysyl oxidase knockout mice.

Mutations in lysyl oxidase (LOX) are associated with thoracic aortic aneurysm and dissection (TAAD). Mice that do not express Lox (Lox-/- ) die soon after birth and have 60% and 40% reductions in elastin- and collagen-specific cross-links, respectively. LOX inactivation could also change the expression of secreted factors, the structural matrix, and matrix-associated proteins that constitute the aortic matrisome. We hypothesized that absence of Lox will change the mechanical behavior of the aortic wall because of reduced elastin and collagen cross-linking and alter the expression levels of matrisome and smooth muscle cell (SMC) genes in a vascular location-specific manner. Using fluorescence microscopy, pressure myography, and gene set enrichment analysis, we visualized the microarchitecture, quantified the mechanical behavior, and examined matrisome and SMC gene expression from ascending aortas (AAs) and descending aortas (DAs) from newborn Lox+/+ and Lox-/- mice. Even though Lox-/- AAs and DAs have fragmented elastic laminae and disorganized SMCs, the unloaded outer diameter and wall thickness were similar to Lox+/+ AAs and DAs. Lox-/- AAs and DAs have altered opening angles, circumferential stresses, and circumferential stretch ratios; however, only Lox-/- AAs have increased pressurized diameters and tangent moduli. Gene set enrichment analysis showed upregulation of the extracellular matrix (ECM) regulator gene set in Lox-/- AAs and DAs as well as differential expression of secreted factors, collagens, ECM-affiliated proteins, ECM glycoproteins, and SMC cell cycle gene sets that depend on the Lox genotype and vascular location. These results provide insights into the local chemomechanical changes induced by Lox inactivation that may be important for TAAD pathogenesis.NEW & NOTEWORTHY Absence of lysyl oxidase (Lox) causes thoracic aortic aneurysms. The aortic mechanical behavior of Lox-/- mice is consistent with reduced elastin and collagen cross-linking but demonstrates vascular location-specific differences. Lox-/- aortas show upregulation of matrix remodeling genes and location-specific differential expression of other matrix and smooth muscle cell gene sets.

Functionally Distinct Tendons From Elastin Haploinsufficient Mice Exhibit Mild Stiffening and Tendon-Specific Structural Alteration.

Elastic fibers are present in low quantities in tendon, where they are located both within fascicles near tenocytes and more broadly in the interfascicular matrix (IFM). While elastic fibers have long been known to be significant in the mechanics of elastin-rich tissue (i.e., vasculature, skin, lungs), recent studies have suggested a mechanical role for elastic fibers in tendons that is dependent on specific tendon function. However, the exact contribution of elastin to properties of different types of tendons (e.g., positional, energy-storing) remains unknown. Therefore, this study purposed to evaluate the role of elastin in the mechanical properties and collagen alignment of functionally distinct supraspinatus tendons (SSTs) and Achilles tendons (ATs) from elastin haploinsufficient (HET) and wild type (WT) mice. Despite the significant decrease in elastin in HET tendons, a slight increase in linear stiffness of both tendons was the only significant mechanical effect of elastin haploinsufficiency. Additionally, there were significant changes in collagen nanostructure and subtle alteration to collagen alignment in the AT but not the SST. Hence, elastin may play only a minor role in tendon mechanical properties. Alternatively, larger changes to tendon mechanics may have been mitigated by developmental compensation of HET tendons and/or the role of elastic fibers may be less prominent in smaller mouse tendons compared to the larger bovine and human tendons evaluated in previous studies. Further research will be necessary to fully elucidate the influence of various elastic fiber components on structure-function relationships in functionally distinct tendons.

Fibulin-4 E57K Knock-in Mice Recapitulate Cutaneous, Vascular and Skeletal Defects of Recessive Cutis Laxa 1B with both Elastic Fiber and Collagen Fibril Abnormalities.

Fibulin-4 is an extracellular matrix protein essential for elastic fiber formation. Frameshift and missense mutations in the fibulin-4 gene (EFEMP2/FBLN4) cause autosomal recessive cutis laxa (ARCL) 1B, characterized by loose skin, aortic aneurysm, arterial tortuosity, lung emphysema, and skeletal abnormalities. Homozygous missense mutations in FBLN4 are a prevalent cause of ARCL 1B. Here we generated a knock-in mouse strain bearing a recurrent fibulin-4 E57K homozygous missense mutation. The mutant mice survived into adulthood and displayed abnormalities in multiple organ systems, including loose skin, bent forelimb, aortic aneurysm, tortuous artery, and pulmonary emphysema. Biochemical studies of dermal fibroblasts showed that fibulin-4 E57K mutant protein was produced but was prone to dimer formation and inefficiently secreted, thereby triggering an endoplasmic reticulum stress response. Immunohistochemistry detected a low level of fibulin-4 E57K protein in the knock-in skin along with altered expression of selected elastic fiber components. Processing of a precursor to mature lysyl oxidase, an enzyme involved in cross-linking of elastin and collagen, was compromised. The knock-in skin had a reduced level of desmosine, an elastin-specific cross-link compound, and ultrastructurally abnormal elastic fibers. Surprisingly, structurally aberrant collagen fibrils and altered organization into fibers were characteristics of the knock-in dermis and forelimb tendons. Type I collagen extracted from the knock-in skin had decreased amounts of covalent intermolecular cross-links, which could contribute to the collagen fibril abnormalities. Our studies provide the first evidence that fibulin-4 plays a role in regulating collagen fibril assembly and offer a preclinical platform for developing treatments for ARCL 1B.

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.

Fibroblast growth factor 2 is required for epithelial recovery, but not for pulmonary fibrosis, in response to bleomycin.

The pathogenesis of pulmonary fibrosis involves lung epithelial injury and aberrant proliferation of fibroblasts, and results in progressive pulmonary scarring and declining lung function. In vitro, fibroblast growth factor (FGF) 2 promotes myofibroblast differentiation and proliferation in cooperation with the profibrotic growth factor, transforming growth factor-ß1, but the in vivo requirement for FGF2 in the development of pulmonary fibrosis is not known. The bleomycin model of lung injury and pulmonary fibrosis was applied to Fgf2 knockout (Fgf2(-/-)) and littermate control mice. Weight loss, mortality, pulmonary fibrosis, and histology were analyzed after a single intranasal dose of bleomycin. Inflammation was evaluated in bronchoalveolar lavage (BAL) fluid, and epithelial barrier integrity was assessed by measuring BAL protein and Evans Blue dye permeability. Fgf2 is expressed in mouse and human lung epithelial and inflammatory cells, and, in response to bleomycin, Fgf2(-/-) mice have significantly increased mortality and weight loss. Analysis of BAL fluid and histology show that pulmonary fibrosis is unaltered, but Fgf2(-/-) mice fail to efficiently resolve inflammation, have increased BAL cellularity, and, importantly, deficient recovery of epithelial integrity. Fgf2(-/-) mice similarly have deficient recovery of club cell secretory protein(+) bronchial epithelium in response to naphthalene. We conclude that FGF2 is not required for bleomycin-induced pulmonary fibrosis, but rather is essential for epithelial repair and maintaining epithelial integrity after bleomycin-induced lung injury in mice. These data identify that FGF2 acts as a protective growth factor after lung epithelial injury, and call into question the role of FGF2 as a profibrotic growth factor in vivo.

Greater impairments in cerebral artery compared with skeletal muscle feed artery endothelial function in a mouse model of increased large artery stiffness.

KEY POINTS: Increased large artery stiffness is a hallmark of arterial dysfunction with advancing age and is also present in other disease conditions such as diabetes. Increased large artery stiffness is correlated with resistance artery dysfunction in humans. Using a mouse model of altered arterial elastin content, this is the first study to examine the cause-and-effect relationship between large artery stiffness and peripheral resistance artery function. Our results indicate that mice with genetically greater large artery stiffness have impaired cerebral artery endothelial function, but generally preserved skeletal muscle feed artery endothelial function. The mechanisms for impaired cerebral artery endothelial function are reduced nitric oxide bioavailability and increased oxidative stress. These findings suggest that interventions that target large artery stiffness may be important to reduce disease risk associated with cerebral artery dysfunction in conditions such as advancing age. ABSTRACT: Advancing age as well as diseases such as diabetes are characterized by both increased large artery stiffness and impaired peripheral artery function. It has been hypothesized that greater large artery stiffness causes peripheral artery dysfunction; however, a cause-and-effect relationship has not previously been established. We used elastin heterozygote mice (Eln(+/-) ) as a model of increased large artery stiffness without co-morbidities unrelated to the large artery properties. Aortic stiffness, measured by pulse wave velocity, was ∼35% greater in Eln(+/-) mice than in wild-type (Eln(+/+) ) mice (P = 0.04). Endothelium-dependent dilatation (EDD), assessed by the maximal dilatation to acetylcholine, was ∼40% lower in Eln(+/-) than Eln(+/+) mice in the middle cerebral artery (MCA, P < 0.001), but was similar between groups in the gastrocnemius feed arteries (GFA, P = 0.79). In the MCA, EDD did not differ between groups after incubation with the nitric oxide (NO) synthase inhibitor N(ω) -nitro-l-arginine methyl ester (P > 0.05), indicating that lower NO bioavailability contributed to the impaired EDD in Eln(+/-) mice. Superoxide production and content of the oxidative stress marker nitrotyrosine was higher in MCAs from Eln(+/-) compared with Eln(+/+) mice (P < 0.05). In the MCA, after incubation with the superoxide scavenger TEMPOL, maximal EDD improved by ∼65% in Eln(+/-) (P = 0.002), but was unchanged in Eln(+/+) mice (P = 0.17). These results indicate that greater large artery stiffness has a more profound effect on endothelial function in cerebral arteries compared with skeletal muscle feed arteries. Greater large artery stiffness can cause cerebral artery endothelial dysfunction by reducing NO bioavailability and increasing oxidative stress.

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.

Microfibril-associated glycoprotein 2 (MAGP2) loss of function has pleiotropic effects in vivo.

Microfibril-associated glycoprotein (MAGP) 1 and 2 are evolutionarily related but structurally divergent proteins that are components of microfibrils of the extracellular matrix. Using mice with a targeted inactivation of Mfap5, the gene for MAGP2 protein, we demonstrate that MAGPs have shared as well as unique functions in vivo. Mfap5(-/-) mice appear grossly normal, are fertile, and have no reduction in life span. Cardiopulmonary development is typical. The animals are normotensive and have vascular compliance comparable with age-matched wild-type mice, which is indicative of normal, functional elastic fibers. Loss of MAGP2 alone does not significantly alter bone mass or architecture, and loss of MAGP2 in tandem with loss of MAGP1 does not exacerbate MAGP1-dependent osteopenia. MAGP2-deficient mice are neutropenic, which contrasts with monocytopenia described in MAGP1-deficient animals. This suggests that MAGP1 and MAGP2 have discrete functions in hematopoiesis. In the cardiovascular system, MAGP1;MAGP2 double knockout mice (Mfap2(-/-);Mfap5(-/-)) show age-dependent aortic dilation. These findings indicate that MAGPs have shared primary functions in maintaining large vessel integrity. In solid phase binding assays, MAGP2 binds active TGFß1, TGFß2, and BMP2. Together, these data demonstrate that loss of MAGP2 expression in vivo has pleiotropic effects potentially related to the ability of MAGP2 to regulate growth factors or participate in cell signaling.