Toward the quantification of α-synuclein aggregates with digital seed amplification assays.

The quantification and characterization of aggregated α-synuclein in clinical samples offer immense potential toward diagnosing, treating, and better understanding neurodegenerative synucleinopathies. Here, we developed digital seed amplification assays to detect single α-synuclein aggregates by partitioning the reaction into microcompartments. Using pre-formed α-synuclein fibrils as reaction seeds, we measured aggregate concentrations as low as 4 pg/mL. To improve our sensitivity, we captured aggregates on antibody-coated magnetic beads before running the amplification reaction. By first characterizing the pre-formed fibrils with transmission electron microscopy and size exclusion chromatography, we determined the specific aggregates targeted by each assay platform. Using brain tissue and cerebrospinal fluid samples collected from patients with Parkinson's Disease and multiple system atrophy, we demonstrated that the assay can detect endogenous pathological α-synuclein aggregates. Furthermore, as another application for these assays, we studied the inhibition of α-synuclein aggregation in the presence of small-molecule inhibitors and used a custom image analysis pipeline to quantify changes in aggregate growth and filament morphology.

Skin surface biomarkers are associated with future development of atopic dermatitis in children with family history of allergic disease.

BACKGROUND: Atopic dermatitis (AD) is a common childhood chronic inflammatory skin disorder that can significantly impact quality of life and has been linked to the subsequent development of food allergy, asthma, and allergic rhinitis, an association known as the "atopic march." OBJECTIVE: The aim of this study was to identify biomarkers collected non-invasively from the skin surface in order to predict AD before diagnosis across a broad age range of children. METHODS: Non-invasive skin surface measures and biomarkers were collected from 160 children (3-48 months of age) of three groups: (A) healthy with no family history of allergic disease, (B) healthy with family history of allergic disease, and (C) diagnosed AD. RESULTS: Eleven of 101 children in group B reported AD diagnosis in the subsequent 12 months following the measurements. The children who developed AD had increased skin immune markers before disease onset, compared to those who did not develop AD in the same group and to the control group. In those enrolled with AD, lesional skin was characterized by increased concentrations of certain immune markers and transepidermal water loss, and decreased skin surface hydration. CONCLUSIONS: Defining risk susceptibility before onset of AD through non-invasive methods may help identify children who may benefit from early preventative interventions.

Population Heterogeneity in the Epithelial to Mesenchymal Transition Is Controlled by NFAT and Phosphorylated Sp1.

Epithelial to mesenchymal transition (EMT) is an essential differentiation program during tissue morphogenesis and remodeling. EMT is induced by soluble transforming growth factor ß (TGF-ß) family members, and restricted by vascular endothelial growth factor family members. While many downstream molecular regulators of EMT have been identified, these have been largely evaluated individually without considering potential crosstalk. In this study, we created an ensemble of dynamic mathematical models describing TGF-ß induced EMT to better understand the operational hierarchy of this complex molecular program. We used ordinary differential equations (ODEs) to describe the transcriptional and post-translational regulatory events driving EMT. Model parameters were estimated from multiple data sets using multiobjective optimization, in combination with cross-validation. TGF-ß exposure drove the model population toward a mesenchymal phenotype, while an epithelial phenotype was enhanced following vascular endothelial growth factor A (VEGF-A) exposure. Simulations predicted that the transcription factors phosphorylated SP1 and NFAT were master regulators promoting or inhibiting EMT, respectively. Surprisingly, simulations also predicted that a cellular population could exhibit phenotypic heterogeneity (characterized by a significant fraction of the population with both high epithelial and mesenchymal marker expression) if treated simultaneously with TGF-ß and VEGF-A. We tested this prediction experimentally in both MCF10A and DLD1 cells and found that upwards of 45% of the cellular population acquired this hybrid state in the presence of both TGF-ß and VEGF-A. We experimentally validated the predicted NFAT/Sp1 signaling axis for each phenotype response. Lastly, we found that cells in the hybrid state had significantly different functional behavior when compared to VEGF-A or TGF-ß treatment alone. Together, these results establish a predictive mechanistic model of EMT susceptibility, and potentially reveal a novel signaling axis which regulates carcinoma progression through an EMT versus tubulogenesis response.

Organ chips with integrated multifunctional sensors enable continuous metabolic monitoring at controlled oxygen levels.

Despite remarkable advances in Organ-on-a-chip (Organ Chip) microfluidic culture technology, recreating tissue-relevant physiological conditions, such as the region-specific oxygen concentrations, remains a formidable technical challenge, and analysis of tissue functions is commonly carried out using one analytical technique at a time. Here, we describe two-channel Organ Chip microfluidic devices fabricated from polydimethylsiloxane and gas impermeable polycarbonate materials that are integrated with multiple sensors, mounted on a printed circuit board and operated using a commercially available Organ Chip culture instrument. The novelty of this system is that it enables the recreation of physiologically relevant tissue-tissue interfaces and oxygen tension as well as non-invasive continuous measurement of transepithelial electrical resistance, oxygen concentration and pH, combined with simultaneous analysis of cellular metabolic activity (ATP/ADP ratio), cell morphology, and tissue phenotype. We demonstrate the reliable and reproducible functionality of this system in living human Gut and Liver Chip cultures. Changes in tissue barrier function and oxygen tension along with their functional and metabolic responses to chemical stimuli (e.g., calcium chelation, oligomycin) were continuously and noninvasively monitored on-chip for up to 23 days. A physiologically relevant microaerobic microenvironment that supports co-culture of human intestinal cells with living Lactococcus lactis bacteria also was demonstrated in the Gut Chip. The integration of multi-functional sensors into Organ Chips provides a robust and scalable platform for the simultaneous, continuous, and non-invasive monitoring of multiple physiological functions that can significantly enhance the comprehensive and reliable evaluation of engineered tissues in Organ Chip models in basic research, preclinical modeling, and drug development.

Correlates of a caregiver-reported child sleep problem and variation by community disadvantage.

BACKGROUND: Previous studies of sleep patterns and perceived problems in early childhood indicate variation by family socioeconomic status. The purpose of this study was to examine variation in correlates of a caregiver-perceived child sleep problem across and within levels of community disadvantage in a large US sample. METHODS: Caregivers of 14,980 young children (ages 0-35.9 months) in the US completed the Brief Infant Sleep Questionnaire-Revised (BISQ-R) on the freely and publicly available Johnson's® Bedtime® Baby Sleep App. Zip code was used to identify a Distressed Communities Index (DCI) score, which represents community disadvantage based on neighborhood indicators. RESULTS: Across all levels of community disadvantage, caregivers who reported greater impact of child sleep on their own sleep, bedtime difficulty, more frequent and longer night wakings, and increased total nighttime sleep were more likely to endorse a child sleep problem. These associations varied by level of community disadvantage. For caregivers living in more disadvantaged communities, impact of child sleep on their own sleep and night wakings were the strongest correlates of endorsing a child sleep problem, whereas for those in more advantaged communities the impact of child sleep on their own sleep and night wakings as well as additional aspects of sleep health, such as short sleep duration, were associated with endorsement of a child sleep problem. CONCLUSIONS: Findings suggest that families living in more distressed communities are most likely to identify the impact of child sleep on their own sleep and night wakings in reporting a child sleep problem, whereas those from more prosperous communities consider these factors as well as other sleep parameters, including sleep duration. Clinicians should consider expanding screening questions for child sleep problems to include the perceived impact on caregiver sleep.

Socioeconomic disadvantage and sleep in early childhood: Real-world data from a mobile health application.

OBJECTIVES: To examine whether increased socioeconomic disadvantage, indexed using a measure of community distress, was associated with variation in caregiver-reported early childhood sleep patterns and problems in a large US sample using a mobile health application (app). DESIGN: Cross-sectional. SETTING: Data were collected using the free, publicly available Johnson's Bedtime© baby sleep app. PARTICIPANTS: A total of 14,980 caregivers (85.1% mothers) of children ages 6-35.9 months (M = 13.88 months; 52.6% boys) participated in this study. MEASURES: Caregivers reported on child sleep using the Brief Infant Sleep Questionnaire-Revised. Socioeconomic disadvantage was indexed by zip code using the Distressed Communities Index (DCI), which combines seven US census indicators of socioeconomic disadvantage. DCI scores range from prosperous (lowest quintile) to distressed (highest quintile). RESULTS: Socioeconomic disadvantage was significantly associated with later bedtimes, longer sleep onset latency, and shorter nighttime and 24-hour (total) sleep duration, with children living in distressed communities showing the poorest sleep. However, caregivers living in distressed communities reported a significantly lower prevalence of overall child sleep problems (43% vs 58% in prosperous communities), and more confidence in managing child sleep (42% vs 34% in prosperous communities). CONCLUSIONS: Children living in the most distressed communities have the poorest reported sleep patterns and bedtime behaviors; however, their caregivers are less likely to report problematic child sleep. These findings highlight the need for community-level sleep health promotion interventions, as well as further investigation of caregiver perceptions about child sleep and sleep health promotion among families living in socioeconomically disadvantaged contexts.

Effect of left atrial ligation-driven altered inflow hemodynamics on embryonic heart development: clues for prenatal progression of hypoplastic left heart syndrome.

Congenital heart defects (CHDs) are abnormalities in the heart structure present at birth. One important condition is hypoplastic left heart syndrome (HLHS) where severely underdeveloped left ventricle (LV) cannot support systemic circulation. HLHS usually initiates as localized tissue malformations with no underlying genetic cause, suggesting that disturbed hemodynamics contribute to the embryonic development of these defects. Left atrial ligation (LAL) is a surgical procedure on embryonic chick resulting in a phenotype resembling clinical HLHS. In this study, we investigated disturbed hemodynamics and deteriorated cardiac growth following LAL to investigate possible mechanobiological mechanisms for the embryonic development of HLHS. We integrated techniques such as echocardiography, micro-CT and computational fluid dynamics (CFD) for these analyses. Specifically, LAL procedure causes an immediate flow disturbance over atrioventricular (AV) cushions. At later stages after the heart septation, it causes hemodynamic disturbances in LV. As a consequence of the LAL procedure, the left-AV canal and LV volume decrease in size, and in the opposite way, the right-AV canal and right ventricle volume increase. According to our CFD analysis, LAL results in an immediate decrease in the left AV canal WSS levels for 3.5-day (HH21) pre-septated hearts. For 7-day post-septated hearts (HH30), LAL leads to further reduction in WSS levels in the left AV canal, and relatively increased WSS levels in the right AV canal. This study demonstrates the critical importance of the disturbed hemodynamics during the heart valve and ventricle development.

Effectiveness of an mHealth Intervention for Infant Sleep Disturbances.

Bedtime problems and night wakings are highly prevalent in infants. This study assessed the real-world effectiveness of an mHealth behavioral sleep intervention (Customized Sleep Profile; CSP). Caregivers (83.9% mothers) of 404 infants (age 6 to 11.9 m, M = 8.32 m, 51.2% male) used the CSP (free and publicly available behavioral sleep intervention delivered via smartphone application, Johnson's® Bedtime® Baby Sleep App). Caregivers completed the Brief Infant Sleep Questionnaire-Revised (BISQ-R) at baseline and again 4 to 28 days later. Changes in sleep patterns were analyzed, based on sleep problem status (problem versus no problem sleepers; PS; NPS). Sleep onset latency improved in both groups. Earlier bedtimes, longer continuous stretches of sleep, as well as decreased number and duration of night wakings, were evident in the PS group only. The BISQ-R Total score, total nighttime sleep, and total 24-hour sleep time improved for both groups, with a greater change for the PS group. Further, caregivers of infants in the PS group decreased feeding (bedtime and overnight) and picking up overnight, and perceived better sleep. Bedtime routine regularity, bedtime difficulty, sleep onset difficulty, and caregiver confidence improved for both groups, with the PS group showing a greater magnitude of change. Thus, a real-world, publicly available, mHealth behavioral sleep intervention was associated with improved outcomes for older infants. Intervention recommendations resulted in changes in caregivers' behavior and improvements in caregiver-reported sleep outcomes in infants, in as few as 4 days.

Norm-referenced scoring system for the Brief Infant Sleep Questionnaire - Revised (BISQ-R).

OBJECTIVES: To develop an age-based norm-referenced scoring system for the Brief Infant Sleep Questionnaire - Revised (BISQ-R). METHODS: In sum, 33,835 submissions (data sample 1) of the expanded and revised BISQ-R by caregivers of infants and toddlers (1-36 months) were analyzed in the US via a publicly-available smartphone application, Johnson's® Bedtime®. Three subscales were created: Infant Sleep (IS; 5 items), Parent Perception (PP; 3 items), and Parent Behavior (PB; 11 items). The scoring algorithm was based on an age-based normative system, and each subscale was scored using a weighted average of items. Primary application of the scoring model was performed on a follow up set of 16,531 submissions (data sample 2). Secondary application was tested on an original web-based ecology study (data sample 3). Tertiary application was tested using previously published datasets consisting of a longitudinal study (data sample 4) and randomized control trial behavioral intervention study (data sample 5). RESULTS: Overall application of the scoring algorithm was confirmed across multiple samples. Each subscale (IS, PP, PB) and total score (T) is age referenced (scores range from 0 to 100). Cross-comparison between subscales across studies reveal consistent and convergent relationships. CONCLUSIONS: The BISQ-R provides a comprehensive assessment of infant and toddler sleep patterns, as well as parent perception and parent behaviors that may contribute to sleep outcomes. The age-based norm-referenced scoring system is publicly available to be used by researchers and clinicians.

Calpain 9 as a therapeutic target in TGFß-induced mesenchymal transition and fibrosis.

Fibrosis is a common pathologic outcome of chronic disease resulting in the replacement of normal tissue parenchyma with a collagen-rich extracellular matrix produced by myofibroblasts. Although the progenitor cell types and cellular programs giving rise to myofibroblasts through mesenchymal transition can vary between tissues and diseases, their contribution to fibrosis initiation, maintenance, and progression is thought to be pervasive. Here, we showed that the ability of transforming growth factor-ß (TGFß) to efficiently induce myofibroblast differentiation of cultured epithelial cells, endothelial cells, or quiescent fibroblasts is dependent on the induced expression and activity of dimeric calpains, a family of non-lysosomal cysteine proteases that regulate a variety of cellular events through posttranslational modification of diverse substrates. siRNA-based gene silencing demonstrated that TGFß-induced mesenchymal transition of a murine breast epithelial cell line was dependent on induction of expression of calpain 9 (CAPN9), an isoform previously thought to be restricted to the gastrointestinal tract. Mice lacking functional CAPN9 owing to biallelic targeting of Capn9 were viable and fertile but showed overt protection from bleomycin-induced lung fibrosis, carbon tetrachloride-induced liver fibrosis, and angiotensin II-induced cardiac fibrosis and dysfunction. A predicted loss-of-function allele of CAPN9 is common in Southeast Asia, with the frequency of homozygosity matching the prediction of Hardy-Weinberg equilibrium. Together with the highly spatially restricted pattern of CAPN9 expression under physiologic circumstances and the heartiness of the murine knockout, these data provide a strong signature for tolerance of therapeutic strategies for fibrosis aimed at CAPN9 antagonism.

ROBO4 variants predispose individuals to bicuspid aortic valve and thoracic aortic aneurysm.

Bicuspid aortic valve (BAV) is a common congenital heart defect (population incidence, 1-2%)1-3 that frequently presents with ascending aortic aneurysm (AscAA)4. BAV/AscAA shows autosomal dominant inheritance with incomplete penetrance and male predominance. Causative gene mutations (for example, NOTCH1, SMAD6) are known for ≤1% of nonsyndromic BAV cases with and without AscAA5-8, impeding mechanistic insight and development of therapeutic strategies. Here, we report the identification of variants in ROBO4 (which encodes a factor known to contribute to endothelial performance) that segregate with disease in two families. Targeted sequencing of ROBO4 showed enrichment for rare variants in BAV/AscAA probands compared with controls. Targeted silencing of ROBO4 or mutant ROBO4 expression in endothelial cell lines results in impaired barrier function and a synthetic repertoire suggestive of endothelial-to-mesenchymal transition. This is consistent with BAV/AscAA-associated findings in patients and in animal models deficient for ROBO4. These data identify a novel endothelial etiology for this common human disease phenotype.

Candidate Gene Resequencing in a Large Bicuspid Aortic Valve-Associated Thoracic Aortic Aneurysm Cohort: SMAD6 as an Important Contributor.

Bicuspid aortic valve (BAV) is the most common congenital heart defect. Although many BAV patients remain asymptomatic, at least 20% develop thoracic aortic aneurysm (TAA). Historically, BAV-related TAA was considered as a hemodynamic consequence of the valve defect. Multiple lines of evidence currently suggest that genetic determinants contribute to the pathogenesis of both BAV and TAA in affected individuals. Despite high heritability, only very few genes have been linked to BAV or BAV/TAA, such as NOTCH1, SMAD6, and MAT2A. Moreover, they only explain a minority of patients. Other candidate genes have been suggested based on the presence of BAV in knockout mouse models (e.g., GATA5, NOS3) or in syndromic (e.g., TGFBR1/2, TGFB2/3) or non-syndromic (e.g., ACTA2) TAA forms. We hypothesized that rare genetic variants in these genes may be enriched in patients presenting with both BAV and TAA. We performed targeted resequencing of 22 candidate genes using Haloplex target enrichment in a strictly defined BAV/TAA cohort (n = 441; BAV in addition to an aortic root or ascendens diameter ≥ 4.0 cm in adults, or a Z-score ≥ 3 in children) and in a collection of healthy controls with normal echocardiographic evaluation (n = 183). After additional burden analysis against the Exome Aggregation Consortium database, the strongest candidate susceptibility gene was SMAD6 (p = 0.002), with 2.5% (n = 11) of BAV/TAA patients harboring causal variants, including two nonsense, one in-frame deletion and two frameshift mutations. All six missense mutations were located in the functionally important MH1 and MH2 domains. In conclusion, we report a significant contribution of SMAD6 mutations to the etiology of the BAV/TAA phenotype.

Corrigendum: Candidate Gene Resequencing in a Large Bicuspid Aortic Valve-Associated Thoracic Aortic Aneurysm Cohort: SMAD6 as an Important Contributor.

[This corrects the article on p. 400 in vol. 8, PMID: 28659821.].

Pathophysiology of aortic aneurysm: insights from human genetics and mouse models.

Aneurysms are local dilations of an artery that predispose the vessel to sudden rupture. They are often asymptomatic and undiagnosed, resulting in a high mortality rate. The predisposition to develop thoracic aortic aneurysms is often genetically inherited and associated with syndromes affecting connective tissue homeostasis. This review discusses how elucidation of the genetic causes of syndromic forms of thoracic aortic aneurysm has helped identify pathways that contribute to disease progression, including those activated by TGF-ß, angiotensin II and Notch ligands. We also discuss how pharmacological manipulation of these signaling pathways has provided further insight into the mechanism of disease and identified compounds with therapeutic potential in these and related disorders.

Cyclic Mechanical Loading Is Essential for Rac1-Mediated Elongation and Remodeling of the Embryonic Mitral Valve.

During valvulogenesis, globular endocardial cushions elongate and remodel into highly organized thin fibrous leaflets. Proper regulation of this dynamic process is essential to maintain unidirectional blood flow as the embryonic heart matures. In this study, we tested how mechanosensitive small GTPases, RhoA and Rac1, coordinate atrioventricular valve (AV) differentiation and morphogenesis. RhoA activity and its regulated GTPase-activating protein FilGAP are elevated during early cushion formation but decreased considerably during valve remodeling. In contrast, Rac1 activity was nearly absent in the early cushions but increased substantially as the valve matured. Using gain- and loss-of-function assays, we determined that the RhoA pathway was essential for the contractile myofibroblastic phenotype present in early cushion formation but was surprisingly insufficient to drive matrix compaction during valve maturation. The Rac1 pathway was necessary to induce matrix compaction in vitro through increased cell adhesion, elongation, and stress fiber alignment. Facilitating this process, we found that acute cyclic stretch was a potent activator of RhoA and subsequently downregulated Rac1 activity via FilGAP. On the other hand, chronic cyclic stretch reduced active RhoA and downstream FilGAP, which enabled Rac1 activation. Finally, we used partial atrial ligation experiments to confirm in vivo that altered cyclic mechanical loading augmented or restricted cushion elongation and thinning, directly through potentiation of active Rac1 and active RhoA, respectively. Together, these results demonstrate that cyclic mechanical signaling coordinates the RhoA to Rac1 signaling transition essential for proper embryonic mitral valve remodeling.

Hierarchical approaches for systems modeling in cardiac development.

Ordered cardiac morphogenesis and function are essential for all vertebrate life. The heart begins as a simple contractile tube, but quickly grows and morphs into a multichambered pumping organ complete with valves, while maintaining regulation of blood flow and nutrient distribution. Though not identical, cardiac morphogenesis shares many molecular and morphological processes across vertebrate species. Quantitative data across multiple time and length scales have been gathered through decades of reductionist single variable analyses. These range from detailed molecular signaling pathways at the cellular levels to cardiac function at the tissue/organ levels. However, none of these components act in true isolation from others, and each, in turn, exhibits short- and long-range effects in both time and space. With the absence of a gene, entire signaling cascades and genetic profiles may be shifted, resulting in complex feedback mechanisms. Also taking into account local microenvironmental changes throughout development, it is apparent that a systems level approach is an essential resource to accelerate information generation concerning the functional relationships across multiple length scales (molecular data vs physiological function) and structural development. In this review, we discuss relevant in vivo and in vitro experimental approaches, compare different computational frameworks for systems modeling, and the latest information about systems modeling of cardiac development. Finally, we conclude with some important future directions for cardiac systems modeling.

Method for non-optical quantification of in situ local soft tissue biomechanics.

Soft tissues exhibit significant biomechanical changes as they grow, adapt, and remodel under a variety of normal and pathogenic stimuli. Biomechanical measurement of intact soft tissues is challenging because of its large strain and nonlinear behavior. Tissue distention through applied vacuum pressure is an attractive method for acquiring local biomechanical information minimally invasive and non-destructive, but the current requirement for optical strain measurement limits its use. In this study, we implemented a novel flexible micro-electrode array placed within a cylindrical probe tip. We hypothesized that upon tissue distention, contact with each electrode would result in a precipitous voltage drop (from the resistive connection formed between input and output electrodes) across the array. Hence, tissue distention (strain) can be derived directly from the electrode array geometry. In pilot studies, we compared the electrode array measurements directly against optical deformation measurements in-situ of agar tissue phantoms and freshly isolated porcine tissue. Our results demonstrate that the probe derived stress-strain profiles and modulus measurements were statistically indistinguishable from optical measurement. We further show that electrode geometry can be scaled down to 50µm in size (length and width) and spaced 50µm apart without impairing measurement accuracy. These results establish a promising new method for minimally invasive local soft tissue biomechanical measurement, which may be useful for applications such as disease diagnosis and health monitoring.

Quantification of embryonic atrioventricular valve biomechanics during morphogenesis.

Tissue assembly in the developing embryo is a rapid and complex process. While much research has focused on genetic regulatory machinery, understanding tissue level changes such as biomechanical remodeling remains a challenging experimental enigma. In the particular case of embryonic atrioventricular valves, micro-scale, amorphous cushions rapidly remodel into fibrous leaflets while simultaneously interacting with a demanding mechanical environment. In this study we employ two microscale mechanical measurement systems in conjunction with finite element analysis to quantify valve stiffening during valvulogenesis. The pipette aspiration technique is compared to a uniaxial load deformation, and the analytic expression for a uniaxially loaded bar is used to estimate the nonlinear material parameters of the experimental data. Effective modulus and strain energy density are analyzed as potential metrics for comparing mechanical stiffness. Avian atrioventricular valves from globular Hamburger-Hamilton stages HH25-HH34 were tested via the pipette method, while the planar HH36 leaflets were tested using the deformable post technique. Strain energy density between HH25 and HH34 septal leaflets increased 4.6±1.8 fold (±SD). The strain energy density of the HH36 septal leaflet was four orders of magnitude greater than the HH34 pipette result. Our results establish morphological thresholds for employing the micropipette aspiration and deformable post techniques for measuring uniaxial mechanical properties of embryonic tissues. Quantitative biomechanical analysis is an important and underserved complement to molecular and genetic experimentation of embryonic morphogenesis.

Cyclic strain anisotropy regulates valvular interstitial cell phenotype and tissue remodeling in three-dimensional culture.

Many planar connective tissues exhibit complex anisotropic matrix fiber arrangements that are critical to their biomechanical function. This organized structure is created and modified by resident fibroblasts in response to mechanical forces in their environment. The directionality of applied strain fields changes dramatically during development, aging, and disease, but the specific effect of strain direction on matrix remodeling is less clear. Current mechanobiological inquiry of planar tissues is limited to equibiaxial or uniaxial stretch, which inadequately simulates many in vivo environments. In this study, we implement a novel bioreactor system to demonstrate the unique effect of controlled anisotropic strain on fibroblast behavior in three-dimensional (3-D) engineered tissue environments, using aortic valve interstitial fibroblast cells as a model system. Cell seeded 3-D collagen hydrogels were subjected to cyclic anisotropic strain profiles maintained at constant areal strain magnitude for up to 96 h at 1 Hz. Increasing anisotropy of biaxial strain resulted in increased cellular orientation and collagen fiber alignment along the principal directions of strain and cell orientation was found to precede fiber reorganization. Cellular proliferation and apoptosis were both significantly enhanced under increasing biaxial strain anisotropy (P<0.05). While cyclic strain reduced both vimentin and alpha-smooth muscle actin compared to unstrained controls, vimentin and alpha-smooth muscle actin expression increased with strain anisotropy and correlated with direction (P<0.05). Collectively, these results suggest that strain field anisotropy is an independent regulator of fibroblast cell phenotype, turnover, and matrix reorganization, which may inform normal and pathological remodeling in soft tissues.