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.

Polarized localization of phosphatidylserine in the endothelium regulates Kir2.1.

Lipid regulation of ion channels is largely explored using in silico modeling with minimal experimentation in intact tissue; thus, the functional consequences of these predicted lipid-channel interactions within native cellular environments remain elusive. The goal of this study is to investigate how lipid regulation of endothelial Kir2.1 - an inwardly rectifying potassium channel that regulates membrane hyperpolarization - contributes to vasodilation in resistance arteries. First, we show that phosphatidylserine (PS) localizes to a specific subpopulation of myoendothelial junctions (MEJs), crucial signaling microdomains that regulate vasodilation in resistance arteries, and in silico data have implied that PS may compete with phosphatidylinositol 4,5-bisphosphate (PIP2) binding on Kir2.1. We found that Kir2.1-MEJs also contained PS, possibly indicating an interaction where PS regulates Kir2.1. Electrophysiology experiments on HEK cells demonstrate that PS blocks PIP2 activation of Kir2.1 and that addition of exogenous PS blocks PIP2-mediated Kir2.1 vasodilation in resistance arteries. Using a mouse model lacking canonical MEJs in resistance arteries (Elnfl/fl/Cdh5-Cre), PS localization in endothelium was disrupted and PIP2 activation of Kir2.1 was significantly increased. Taken together, our data suggest that PS enrichment to MEJs inhibits PIP2-mediated activation of Kir2.1 to tightly regulate changes in arterial diameter, and they demonstrate that the intracellular lipid localization within the endothelium is an important determinant of vascular function.

A new mouse model of elastin haploinsufficiency highlights the importance of elastin to vascular development and blood pressure regulation.

Supravalvular aortic stenosis (SVAS) is an autosomal dominant disease resulting from elastin (ELN) haploinsufficiency. Individuals with SVAS typically develop a thickened arterial media with an increased number of elastic lamellae and smooth muscle cell (SMC) layers and stenosis superior to the aortic valve. A mouse model of SVAS (Eln+/-) was generated that recapitulates many aspects of the human disease, including increased medial SMC layers and elastic lamellae, large artery stiffness, and hypertension. The vascular changes in these mice were thought to be responsible for the hypertension phenotype. However, a renin gene (Ren) duplication in the original 129/Sv genetic background and carried through numerous strain backcrosses raised the possibility of renin-mediated effects on blood pressure. To exclude excess renin activity as a disease modifier, we utilized the Cre-LoxP system to rederive Eln hemizygous mice on a pure C57BL/6 background (Sox2-Cre;Elnf/f). Here we show that Sox2-Cre;Eln+/f mice, with a single Ren1 gene and normal renin levels, phenocopy the original global knockout line. Characteristic traits include an increased number of elastic lamellae and SMC layers, stiff elastic arteries, and systolic hypertension with widened pulse pressure. Importantly, small resistance arteries of Sox2-Cre;Eln+/f mice exhibit a significant change in endothelial cell function and hypercontractility to angiotensin II, findings that point to pathway-specific alterations in resistance arteries that contribute to the hypertensive phenotype. These data confirm that the cardiovascular changes, particularly systolic hypertension, seen in Eln+/- mice are due to Eln hemizygosity rather than Ren duplication.

SVEP1 is an endogenous ligand for the orphan receptor PEAR1.

Sushi, von Willebrand factor type A, EGF and pentraxin domain containing 1 (SVEP1) is an extracellular matrix protein that causally promotes vascular disease and associates with platelet reactivity in humans. Here, using a human genomic and proteomic approach, we identify a high affinity, disease-relevant, and potentially targetable interaction between SVEP1 and the orphan receptor Platelet and Endothelial Aggregation Receptor 1 (PEAR1). This interaction promotes PEAR1 phosphorylation and disease associated AKT/mTOR signaling in vascular cells and platelets. Mice lacking SVEP1 have reduced platelet activation, and exogenous SVEP1 induces PEAR1-dependent activation of platelets. SVEP1 and PEAR1 causally and concordantly relate to platelet phenotypes and cardiovascular disease in humans, as determined by Mendelian Randomization. Targeting this receptor-ligand interaction may be a viable therapeutic strategy to treat or prevent cardiovascular and thrombotic disease.

Targeting Epidermal Growth Factor Receptor to Stimulate Elastic Matrix Regenerative Repair.

Abdominal aortic aneurysms (AAAs) represent a multifactorial, proteolytic disorder involving disintegration of the matrix structure within the AAA wall. Intrinsic deficiency of adult vascular cells to regenerate and repair the wall elastic matrix, which contributes to vessel stretch and recoil, is a major clinical challenge to therapeutic reversal of AAA growth. In this study, we investigate the involvement of epidermal growth factor receptor-mitogen activated protein kinase (EGFR-MAPK) pathway in the activation of aneurysmal smooth muscle cells (SMCs) by neutrophil elastase, and how EGFR can be targeted for elastic matrix regeneration. We have demonstrated that neutrophil elastase activates EGFR and downregulates expression level of key elastin homeostasis genes (elastin, crosslinking enzyme-lysyl oxidase, and fibulin4) between a dose range of 1-10 µg/mL (p < 0.05). It also incites downstream proteolytic outcomes by upregulating p-extracellular signal-regulated kinase (ERK)1/2 (p < 0.0001) and matrix metalloprotease 2 (MMP2) at a protein level, which is significantly downregulated upon EGFR-specific inhibition by tyrosine kinase inhibitor AG1478 (p-ERK1/2 and MMP2 [p < 0.05]). Moreover, we have shown that EGFR inhibition suppresses collagen amounts in aneurysmal SMCs (p < 0.05) and promotes robust formation of elastic fibers by enhancing its deposition in the extracellular space. Hence, the EGFR-MAPK pathway in aneurysmal cells can be targeted to provide therapeutic effects toward stimulating vascular matrix regeneration. Impact statement Proteolytic disorders such as aortal expansions, called abdominal aortic aneurysms (AAAs), are characterized by naturally irreversible enzymatic breakdown and loss of elastic fibers, a problem that has not yet been surmounted by existing tissue engineering approaches. In this work, we show, for the first time, how epidermal growth factor receptor (EGFR) inhibition provides downstream benefits in elastic fiber assembly and deposition in aneurysmal smooth muscle cell cultures. This work can open future possibilities for development of EGFR-targeted drug-based therapies not only for vessel wall repair in AAAs but also other proteolytically compromised elastic tissues.

Progerin induces a phenotypic switch in vascular smooth muscle cells and triggers replication stress and an aging-associated secretory signature.

Hutchinson-Gilford progeria syndrome is a premature aging disease caused by LMNA gene mutation and the production of a truncated prelamin A protein "progerin" that elicits cellular and organismal toxicity. Progerin accumulates in the vasculature, being especially detrimental for vascular smooth muscle cells (VSMC). Vessel stiffening and aortic atherosclerosis in HGPS patients are accompanied by VSMC depletion in the medial layer, altered extracellular matrix (ECM), and thickening of the adventitial layer. Mechanisms whereby progerin causes massive VSMC loss and vessel alterations remain poorly understood. Mature VSMC retain phenotypic plasticity and can switch to a synthetic/proliferative phenotype. Here, we show that progerin expression in human and mouse VSMC causes a switch towards the synthetic phenotype. This switch elicits some level of replication stress in normal cells, which is exacerbated in the presence of progerin, leading to telomere fragility, genomic instability, and ultimately VSMC death. Calcitriol prevents replication stress, telomere fragility, and genomic instability, reducing VSMC death. In addition, RNA-seq analysis shows induction of a profibrotic and pro-inflammatory aging-associated secretory phenotype upon progerin expression in human primary VSMC. Our data suggest that phenotypic switch-induced replication stress might be an underlying cause of VSMC loss in progeria, which together with loss of contractile features and gain of profibrotic and pro-inflammatory signatures contribute to vascular stiffness in HGPS.

Increases in hydraulic conductance and solute permeability in a mouse model of ascending thoracic aortic aneurysm.

Large elastic arteries, such as the aorta, contain concentric layers of elastic laminae composed mainly of the extracellular matrix protein elastin. The structure of the elastic laminae could affect transmural mass transport and contribute to aortic disease progression. We studied the effects of a genetic mutation (LoxM292R/+, referred to as MU) in mice associated with ascending thoracic aortic aneurysm (TAA) on the mass transport and elastic laminae structure. Solute absent fluid flux and hydraulic conductance through the ascending aortic wall were not significantly different between groups, however solute present fluid flux, hydraulic conductance, solute flux, and solute permeability of 4 kDa FITC-dextran were significantly increased in the MU group, indicating that movement of small molecules into the aortic wall is facilitated in MU mice. Quantification from light microscopy images of the ascending aorta showed no significant differences in wall thickness, or inner elastic lamina fenestration size and density, but an increase in the number of elastic laminae breaks in the MU group. Ultrastructural comparisons from transmission electron micrographs suggest less dense and disorganized elastic laminae in MU aorta that may also contribute to the transport differences. Our results provide an initial investigation into the connections between mass transport and elastic laminae structure, specifically in a genetic mouse aneurysm model, which can be further used to understand TAA pathology and develop treatment strategies.

LTBP1 promotes fibrillin incorporation into the extracellular matrix.

LTBP1 is a large extracellular matrix protein and an associated ligand of fibrillin-microfibrils. Knowledge of LTBP1 functions is largely limited to its role in targeting and sequestering TGFß growth factors within the extracellular matrix, thereby regulating their bioavailability. However, the recent description of a wide spectrum of phenotypes in multiple tissues in patients harboring LTBP1 pathogenic variants suggests a multifaceted role of the protein in the homeostasis of connective tissues. To better understand the human pathology caused by LTBP1 deficiency it is important to investigate its functional role in extracellular matrix formation. In this study, we show that LTBP1 coordinates the incorporation of fibrillin-1 and -2 into the extracellular matrix in vitro. We also demonstrate that this function is differentially exerted by the two isoforms, the short and long forms of LTBP1. Thereby our findings uncover a novel TGFß-independent LTBP1 function potentially contributing to the development of connective tissue disorders.

Passive biaxial mechanical behavior of newborn mouse aorta with and without elastin.

Aortic wall material properties are needed for computational models and for comparisons across developmental and disease states. There has been abundant work in comparing aortic material properties across disease states, but limited work across developmental states. We performed passive biaxial mechanical testing on newborn mouse aorta with (Eln+/+) and without (Eln-/-) elastin. Elastin provides elasticity to the aortic wall and is necessary for survival beyond birth in the mouse. Mechanically functional elastin is challenging to create in vitro and so Eln-/- aorta can be a comparison for tissue engineered arteries with limited elastin amounts. We found that a traditional arterial strain energy function provided reasonable fits to newborn mouse aorta and generally predicted lower material constants in Eln-/- compared to Eln+/+ aorta. At physiologic pressures, the circumferential stresses and moduli trended lower in Eln-/- compared to Eln+/+ aorta. Increased blood pressure in Eln-/- mice helps to alleviate the differences in stresses and moduli. Increased blood pressure also serves to partially offload stresses in the isotropic compared to the anisotropic component of the wall. The baseline material parameters can be used in computational models of growth and remodeling to improve understanding of developmental mechanobiology and tissue engineering strategies.

Loss of Angiotensin II Type 2 Receptor Improves Blood Pressure in Elastin Insufficiency.

There is ample evidence supporting a role for angiotensin II type 2 receptor (AT2R) in counterbalancing the effects of angiotensin II (ang II) through the angiotensin II type 1 receptor by promoting vasodilation and having anti-inflammatory effects. Elastin insufficiency in both humans and mice results in large artery stiffness and systolic hypertension. Unexpectedly, mesenteric arteries from elastin insufficient (Eln +/-) mice were shown to have significant vasoconstriction to AT2R agonism in vitro suggesting that AT2R may have vasoconstrictor effects in elastin insufficiency. Given the potential promise for the use of AT2R agonists clinically, the goal of this study was to determine whether AT2R has vasoconstrictive effects in elastin insufficiency in vivo. To avoid off-target effects of agonists and antagonists, mice lacking AT2R (Agtr2 -/Y ) were bred to Eln +/- mice and cardiovascular parameters were assessed in wild-type (WT), Agtr2 -/Y , Eln +/-, and Agtr2 -/Y ;Eln +/- littermates. As previously published, Agtr2 -/Y mice were normotensive at baseline and had no large artery stiffness, while Eln +/- mice exhibited systolic hypertension and large artery stiffness. Loss of AT2R in Eln +/- mice did not affect large artery stiffness or arterial structure but resulted in significant reduction of both systolic and diastolic blood pressure. These data support a potential vasocontractile role for AT2R in elastin insufficiency. Careful consideration and investigation are necessary to determine the patient population that might benefit from the use of AT2R agonists.

Deletion of type VIII collagen reduces blood pressure, increases carotid artery functional distensibility and promotes elastin deposition.

Arterial stiffening is a significant predictor of cardiovascular disease development and mortality. In elastic arteries, stiffening refers to the loss and fragmentation of elastic fibers, with a progressive increase in collagen fibers. Type VIII collagen (Col-8) is highly expressed developmentally, and then once again dramatically upregulated in aged and diseased vessels characterized by arterial stiffening. Yet its biophysical impact on the vessel wall remains unknown. The purpose of this study was to test the hypothesis that Col-8 functions as a matrix scaffold to maintain vessel integrity during extracellular matrix (ECM) development. These changes are predicted to persist into the adult vasculature, and we have tested this in our investigation. Through our in vivo and in vitro studies, we have determined a novel interaction between Col-8 and elastin. Mice deficient in Col-8 (Col8-/-) had reduced baseline blood pressure and increased arterial compliance, indicating an enhanced Windkessel effect in conducting arteries. Differences in both the ECM composition and VSMC activity resulted in Col8-/- carotid arteries that displayed increased crosslinked elastin and functional distensibility, but enhanced catecholamine-induced VSMC contractility. In vitro studies revealed that the absence of Col-8 dramatically increased tropoelastin mRNA and elastic fiber deposition in the ECM, which was decreased with exogenous Col-8 treatment. These findings suggest a causative role for Col-8 in reducing mRNA levels of tropoelastin and the presence of elastic fibers in the matrix. Moreover, we also found that Col-8 and elastin have opposing effects on VSMC phenotype, the former promoting a synthetic phenotype, whereas the latter confers quiescence. These studies further our understanding of Col-8 function and open a promising new area of investigation related to elastin biology.

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.

Tissue-specific smooth muscle cell subtypes identified by transcriptional profiling.

Smooth muscle cells (SMCs) are specialized cells present in many organs where they serve diverse tissue-specific functions. Using the Tabula Muris compendium of single-cell RNA sequencing data, we extracted individual SMC transcriptomes from eight mouse tissues to investigate the transcriptomic landscape of tissue-specific SMCs. We identified marker genes, signaling pathways, and biological processes enriched in tissue-specific SMCs, and inferred potential ligand-receptor interaction between SMC and other cell types. Our analysis also identified sex differences in SMC gene expression in different tissues. Lastly, we used unsupervised clustering to identify novel SMC subtypes based on their downstream targets of transcription factors. Our results highlight the variable SMC phenotypes and underscore this cell's remarkable adaptability to contribute to diverse tissue function.

Early Aberrant Angiogenesis Due to Elastic Fiber Fragmentation in Aortic Valve Disease.

Elastic fiber fragmentation (EFF) is a hallmark of aortic valve disease (AVD), and neovascularization has been identified as a late finding related to inflammation. We sought to characterize the relationship between early EFF and aberrant angiogenesis. To examine disease progression, regional anatomy and pathology of aortic valve tissue were assessed using histochemistry, immunohistochemistry, and electron microscopy from early-onset (<40 yo) and late-onset (≥40 yo) non-syndromic AVD specimens. To assess the effects of EFF on early AVD processes, valve tissue from Williams and Marfan syndrome patients was also analyzed. Bicuspid aortic valve was more common in early-onset AVD, and cardiovascular comorbidities were more common in late-onset AVD. Early-onset AVD specimens demonstrated angiogenesis without inflammation or atherosclerosis. A distinct pattern of elastic fiber components surrounded early-onset AVD neovessels, including increased emilin-1 and decreased fibulin-5. Different types of EFF were present in Williams syndrome (WS) and Marfan syndrome (MFS) aortic valves; WS but not MFS aortic valves demonstrated angiogenesis. Aberrant angiogenesis occurs in early-onset AVD in the absence of inflammation, implicating EFF. Elucidation of underlying mechanisms may inform the development of new pharmacologic treatments.

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.

SVEP1 is a human coronary artery disease locus that promotes atherosclerosis.

A low-frequency variant of sushi, von Willebrand factor type A, EGF, and pentraxin domain-containing protein 1 (SVEP1), an extracellular matrix protein, is associated with risk of coronary disease in humans independent of plasma lipids. Despite a robust statistical association, if and how SVEP1 might contribute to atherosclerosis remained unclear. Here, using Mendelian randomization and complementary mouse models, we provide evidence that SVEP1 promotes atherosclerosis in humans and mice and is expressed by vascular smooth muscle cells (VSMCs) within the atherosclerotic plaque. VSMCs also interact with SVEP1, causing proliferation and dysregulation of key differentiation pathways, including integrin and Notch signaling. Fibroblast growth factor receptor transcription increases in VSMCs interacting with SVEP1 and is further increased by the coronary disease-associated SVEP1 variant p.D2702G. These effects ultimately drive inflammation and promote atherosclerosis. Together, our results suggest that VSMC-derived SVEP1 is a proatherogenic factor and support the concept that pharmacological inhibition of SVEP1 should protect against atherosclerosis in humans.

Complex consequences of Cantu syndrome SUR2 variant R1154Q in genetically modified mice.

Cantu syndrome (CS) is caused by gain-of-function (GOF) mutations in pore-forming (Kir6.1, KCNJ8) and accessory (SUR2, ABCC9) ATP-sensitive potassium (KATP) channel subunits, the most common mutations being SUR2[R1154Q] and SUR2[R1154W], carried by approximately 30% of patients. We used CRISPR/Cas9 genome engineering to introduce the equivalent of the human SUR2[R1154Q] mutation into the mouse ABCC9 gene. Along with minimal CS disease features, R1154Q cardiomyocytes and vascular smooth muscle showed much lower KATP current density and pinacidil activation than WT cells. Almost complete loss of SUR2-dependent protein and KATP in homozygous R1154Q ventricles revealed underlying diazoxide-sensitive SUR1-dependent KATP channel activity. Surprisingly, sequencing of SUR2 cDNA revealed 2 distinct transcripts, one encoding full-length SUR2 protein; and the other with an in-frame deletion of 93 bases (corresponding to 31 amino acids encoded by exon 28) that was present in approximately 40% and approximately 90% of transcripts from hetero- and homozygous R1154Q tissues, respectively. Recombinant expression of SUR2A protein lacking exon 28 resulted in nonfunctional channels. CS tissue from SUR2[R1154Q] mice and human induced pluripotent stem cell-derived (hiPSC-derived) cardiomyocytes showed only full-length SUR2 transcripts, although further studies will be required in order to fully test whether SUR2[R1154Q] or other CS mutations might result in aberrant splicing and variable expressivity of disease features in human CS.

Biological Preparation and Mechanical Technique for Determining Viscoelastic Properties of Zonular Fibers.

Elasticity is essential to the function of tissues such as blood vessels, muscles, and lungs. This property is derived mostly from the extracellular matrix (ECM), the protein meshwork that binds cells and tissues together. How the elastic properties of an ECM network relate to its composition, and whether the relaxation properties of the ECM play a physiological role, are questions that have yet to be fully addressed. Part of the challenge lies in the complex architecture of most ECM systems and the difficulty in isolating ECM components without compromising their structure. One exception is the zonule, an ECM system found in the eye of vertebrates. The zonule comprises fibers hundreds to thousands of micrometers in length that span the cell-free space between the lens and the eyewall. In this report, we describe a mechanical technique that takes advantage of the highly organized structure of the zonule to quantify its viscoelastic properties and to determine the contribution of individual protein components. The method involves dissection of a fixed eye to expose the lens and the zonule and employs a pull-up technique that stretches the zonular fibers equally while their tension is monitored. The technique is relatively inexpensive yet sensitive enough to detect alterations in viscoelastic properties of zonular fibers in mice lacking specific zonular proteins or with aging. Although the method presented here is designed primarily for studying ocular development and disease, it could also serve as an experimental model for exploring broader questions regarding the viscoelastic properties of elastic ECM's and the role of external factors such as ionic concentration, temperature, and interactions with signaling molecules.