Biomechanical Remodeling of Aortic Valve Interstitial Cells During Calcified Lesion Formation In Vitro.

Healthy aortic heart valves are essential to the regulation of unidirectional blood flow. Calcific aortic valve disease (CAVD) is an actively progressive disease that involves the disorganization of valve cells and accumulation of calcium deposits on the aortic valve leaflets. CAVD involves disruption of cell environment homeostasis that prior cell culture models have found difficult to portray and model. As it is still poorly understood how tissue stiffening associates with lesion formation, here, we implement a novel 3D culture platform to characterize the relationship between mechanical stress and tissue remodeling and analyze how the application of pro-osteogenic stimulation dysregulates the native ability of valve cells to organize its matrix. Through a temporal study of macroscopic remodeling, we determine that aortic valve interstitial neo-tissues undergo varying stiffness and mechanical stress, demonstrate greater myofibroblastic gene expression, and show greater remodeling activity in the outer surface of the neo-tissue in a banding pattern when cultured in osteogenic growth medium. In human aortic valve interstitial cells cultured in osteogenic growth medium, we observed an increase in stress but significant decreases in myofibroblastic gene expression with the addition of growth factors. In summary, we are able to see the interplay of biochemical and biomechanical stimuli in valvular remodeling by using our platform to model dynamic stiffening of valve interstitial neo-tissues under different biochemical conditions.

Quantitative 4D imaging of biomechanical regulation of ventricular growth and maturation.

Abnormal cardiac development is intimately associated with congenital heart disease. During development, a sponge-like network of muscle fibers in the endocardium, known as trabeculation, becomes compacted. Biomechanical forces regulate myocardial differentiation and proliferation to form trabeculation, while the molecular mechanism is still enigmatic. Biomechanical forces, including intracardiac hemodynamic flow and myocardial contractile force, activate a host of molecular signaling pathways to mediate cardiac morphogenesis. While mechanotransduction pathways to initiate ventricular trabeculation is well studied, deciphering the relative importance of hemodynamic shear vs. mechanical contractile forces to modulate the transition from trabeculation to compaction requires advanced imaging tools and genetically tractable animal models. For these reasons, the advent of 4-D multi-scale light-sheet imaging and complementary multiplex live imaging via micro-CT in the beating zebrafish heart and live chick embryos respectively. Thus, this review highlights the complementary animal models and advanced imaging needed to elucidate the mechanotransduction underlying cardiac ventricular development.

Future prospects in the tissue engineering of heart valves: a focus on the role of stem cells.

INTRODUCTION: Heart valve disease is a growing burden on the healthcare system. Current solutions are insufficient for young patients and do not offer relief from reintervention. Tissue engineered heart valves (TEHVs) offer a solution that grows and responds to the native environment in a similar way to a healthy valve. Stem cells hold potential to populate these valves as a malleable source that can adapt to environmental cues. AREAS COVERED: This review covers current methods of recapitulating features of native heart valves with tissue engineering through use of stem cell populations with in situ and in vitro methods. EXPERT OPINION: In the field of TEHVs, we see a variety of approaches in cell source, biomaterial, and maturation methods. Choosing appropriate cell populations may be very patient specific; consistency and predictability will be key to long-term success. In situ methods are closer to translation but struggle with consistent cellularization. In vitro culture requires specialized methods but may recapitulate native valve cell populations with higher fidelity. Understanding how cell populations react to valve conditions and immune response is vital for success. Detrimental valve pathologies have proven to be difficult to avoid in early translation attempts.

Shear and hydrostatic stress regulate fetal heart valve remodeling through YAP-mediated mechanotransduction.

Clinically serious congenital heart valve defects arise from improper growth and remodeling of endocardial cushions into leaflets. Genetic mutations have been extensively studied but explain less than 20% of cases. Mechanical forces generated by beating hearts drive valve development, but how these forces collectively determine valve growth and remodeling remains incompletely understood. Here, we decouple the influence of those forces on valve size and shape, and study the role of YAP pathway in determining the size and shape. The low oscillatory shear stress promotes YAP nuclear translocation in valvular endothelial cells (VEC), while the high unidirectional shear stress restricts YAP in cytoplasm. The hydrostatic compressive stress activated YAP in valvular interstitial cells (VIC), whereas the tensile stress deactivated YAP. YAP activation by small molecules promoted VIC proliferation and increased valve size. Whereas YAP inhibition enhanced the expression of cell-cell adhesions in VEC and affected valve shape. Finally, left atrial ligation was performed in chick embryonic hearts to manipulate the shear and hydrostatic stress in vivo. The restricted flow in the left ventricle induced a globular and hypoplastic left atrioventricular (AV) valves with an inhibited YAP expression. By contrast, the right AV valves with sustained YAP expression grew and elongated normally. This study establishes a simple yet elegant mechanobiological system by which transduction of local stresses regulates valve growth and remodeling. This system guides leaflets to grow into proper sizes and shapes with the ventricular development, without the need of a genetically prescribed timing mechanism.

Soft, strong, tough, and durable protein-based fiber hydrogels.

Load-bearing soft tissues normally show J-shaped stress-strain behaviors with high compliance at low strains yet high strength at high strains. They have high water content but are still tough and durable. By contrast, naturally derived hydrogels are weak and brittle. Although hydrogels prepared from synthetic polymers can be strong and tough, they do not have the desired bioactivity for emerging biomedical applications. Here, we present a thermomechanical approach to replicate the combinational properties of soft tissues in protein-based photocrosslinkable hydrogels. As a demonstration, we create a gelatin methacryloyl fiber hydrogel with soft tissue-like mechanical properties, such as low Young's modulus (0.1 to 0.3 MPa), high strength (1.1 ± 0.2 MPa), high toughness (9,100 ± 2,200 J/m3), and high fatigue resistance (2,300 ± 500 J/m2). This hydrogel also resembles the biochemical and architectural properties of native extracellular matrix, which enables a fast formation of 3D interconnected cell meshwork inside hydrogels. The fiber architecture also regulates cellular mechanoresponse and supports cell remodeling inside hydrogels. The integration of tissue-like mechanical properties and bioactivity is highly desirable for the next-generation biomaterials and could advance emerging fields such as tissue engineering and regenerative medicine.

Biological Response to Sintered Titanium in Left Ventricular Assist Devices: Pseudoneointima, Neointima, and Pannus.

Titanium alloys have traditionally been used in blood-contacting cardiovascular devices, including left ventricular assist devices (LVADs). However, titanium surfaces are susceptible to adverse coagulation, leading to thrombogenesis and stroke. To improve hemocompatibility, LVAD manufacturers introduced powder sintering on blood-wetted surfaces in the 1980s to induce endothelialization. This technique has been employed in multiple contemporary LVADs on the pump housing, as well as the interior and exterior of the inflow cannula. Despite the wide adoption of sintered titanium, reported biologic response over the past several decades has been highly variable and apparently unpredictable-including combinations of neointima, pseudoneoimtima, thrombus, and pannus. We present a history of sintered titanium used in LVAD, a review of accumulated clinical outcomes, and a synopsis of gross appearance and composition of various depositions found clinically and in animal studies, which is unfortunately confounded by the variability and inconsistency in terminology. Therefore, this review endeavors to introduce a unified taxonomy to harmonize published observations of biologic response to sintered titanium in LVADs. From these data, we are able to deduce the natural history of the biologic response to sintered titanium, toward development of a deterministic model of the genesis of a hemocompatible neointima.

Spatial analysis of future climate risk to stormwater infrastructure.

Climate change is expected to result in more intense precipitation events that will affect the performance and design requirements of stormwater infrastructure. Such changes will vary spatially, and climate models provide a range of estimates of the effects on events of different intensities and recurrence. Infrastructure performance should be evaluated against the expected range of events, not just rare extremes. We present a national-scale, spatially detailed screening assessment of the potential effects of climatic change on precipitation, stormwater runoff, and associated design requirements. This is accomplished through adjustment relative to multiple future climate scenarios of precipitation intensity-duration-frequency analyses presented in NOAA Atlas 14, which are commonly used in infrastructure design. Future precipitation results are estimated for each Atlas 14 station (these currently omit the Pacific Northwest). Results are interpolated using a geographically conditioned regression kriging approach to provide information about potential climate change impacts in a format more directly useful to local stormwater managers. The intensity of 24-h events with 2-year or greater recurrence is likely to increase in most areas of the United States leading to increased runoff and potential need for increased storage volumes. Changes in more frequent events (e.g., the 90th percentile event) commonly used in design of green infrastructure are relatively less.

A SOX17-PDGFB signaling axis regulates aortic root development.

Developmental etiologies causing complex congenital aortic root abnormalities are unknown. Here we show that deletion of Sox17 in aortic root endothelium in mice causes underdeveloped aortic root leading to a bicuspid aortic valve due to the absence of non-coronary leaflet and mispositioned left coronary ostium. The respective defects are associated with reduced proliferation of non-coronary leaflet mesenchyme and aortic root smooth muscle derived from the second heart field cardiomyocytes. Mechanistically, SOX17 occupies a Pdgfb transcriptional enhancer to promote its transcription and Sox17 deletion inhibits the endothelial Pdgfb transcription and PDGFB growth signaling to the non-coronary leaflet mesenchyme. Restoration of PDGFB in aortic root endothelium rescues the non-coronary leaflet and left coronary ostium defects in Sox17 nulls. These data support a SOX17-PDGFB axis underlying aortic root development that is critical for aortic valve and coronary ostium patterning, thereby informing a potential shared disease mechanism for concurrent anomalous aortic valve and coronary arteries.

Tri-Layered and Gel-Like Nanofibrous Scaffolds with Anisotropic Features for Engineering Heart Valve Leaflets.

3D heterogeneous and anisotropic scaffolds that approximate native heart valve tissues are indispensable for the successful construction of tissue engineered heart valves (TEHVs). In this study, novel tri-layered and gel-like nanofibrous scaffolds, consisting of poly(lactic-co-glycolic) acid (PLGA) and poly(aspartic acid) (PASP), are fabricated by a combination of positive/negative conjugate electrospinning and bioactive hydrogel post-processing. The nanofibrous PLGA-PASP scaffolds present tri-layered structures, resulting in anisotropic mechanical properties that are comparable with native heart valve leaflets. Biological tests show that nanofibrous PLGA-PASP scaffolds with high PASP ratios significantly promote the proliferation and collagen and glycosaminoglycans (GAGs) secretions of human aortic valvular interstitial cells (HAVICs), compared to PLGA scaffolds. Importantly, the nanofibrous PLGA-PASP scaffolds are found to effectively inhibit the osteogenic differentiation of HAVICs. Two types of porcine VICs, from young and adult age groups, are further seeded onto the PLGA-PASP scaffolds. The adult VICs secrete higher amounts of collagens and GAGs and undergo a significantly higher level of osteogenic differentiation than young VICs. RNA sequencing analysis indicates that age has a pivotal effect on the VIC behaviors. This study provides important guidance and a reference for the design and development of 3D tri-layered, gel-like nanofibrous PLGA-PASP scaffolds for TEHV applications.

Rac1 mediates cadherin-11 induced cellular pathogenic processes in aortic valve calcification.

BACKGROUND: Calcific aortic valve disease (CAVD), a major cause for surgical aortic valve replacement, currently lacks available pharmacological treatments. Cadherin-11 (Cad11), a promising therapeutic target, promotes aortic valve calcification in vivo, but direct Cad11 inhibition in clinical trials has been unsuccessful. Targeting of downstream Cad11 effectors instead may be clinically useful; however, the downstream effectors that mediate Cad11-induced aortic valve cellular pathogenesis have not been investigated. APPROACH AND RESULTS: Immunofluorescence of calcified human aortic valves revealed that GTP-Rac1 is highly upregulated in calcified leaflets and is 2.15 times more co-localized with Cad11 in calcified valves than GTP-RhoA. Using dominant negative mutants in porcine aortic valve interstitial cells (PAVICs), we show that Cad11 predominantly regulates Runx2 nuclear localization via Rac1. Rac1-GEF inhibition via NSC23766 effectively reduces calcification in ex vivo porcine aortic valve leaflets treated with osteogenic media by 2.8-fold and also prevents Cad11-induced cell migration, compaction, and calcification in PAVICs. GTP-Rac1 and Trio, a known Cad11 binding partner and Rac1-GEF, are significantly upregulated in Nfatc1Cre; R26-Cad11Tg/Tg (Cad11 OX) mice that conditionally overexpress Cad11 in the heart valves by 3.1-fold and 6.3-fold, respectively. Finally, we found that the Trio-specific Rac1-GEF inhibitor, ITX3, effectively prevents Cad11-induced calcification and Runx2 induction in osteogenic conditions. CONCLUSION: Here we show that Cad11 induces many cellular pathogenic processes via Rac1 and that Rac1 inhibition effectively prevents many Cad11-induced aortic disease phenotypes. These findings highlight the therapeutic potential of blocking Rac1-GEFs in CAVD.

Local fluid shear stress operates a molecular switch to drive fetal semilunar valve extension.

BACKGROUND: While much is known about the genetic regulation of early valvular morphogenesis, mechanisms governing fetal valvular growth and remodeling remain unclear. Hemodynamic forces strongly influence morphogenesis, but it is unknown whether or how they interact with valvulogenic signaling programs. Side-specific activity of valvulogenic programs motivates the hypothesis that shear stress pattern-specific endocardial signaling controls the elongation of leaflets. RESULTS: We determined that extension of the semilunar valve occurs via fibrosa sided endocardial proliferation. Low OSS was necessary and sufficient to induce canonical Wnt/ß-catenin activation in fetal valve endothelium, which in turn drives BMP receptor/ligand expression, and pSmad1/5 activity essential for endocardial proliferation. In contrast, ventricularis endocardial cells expressed active Notch1 but minimal pSmad1/5. Endocardial monolayers exposed to LSS attenuate Wnt signaling in a Notch1 dependent manner. CONCLUSIONS: Low OSS is transduced by endocardial cells into canonical Wnt signaling programs that regulate BMP signaling and endocardial proliferation. In contrast, high LSS induces Notch signaling in endocardial cells, inhibiting Wnt signaling and thereby restricting growth on the ventricular surface. Our results identify a novel mechanically regulated molecular switch, whereby fluid shear stress drives the growth of valve endothelium, orchestrating the extension of the valve in the direction of blood flow.

Incorporating nanocrystalline cellulose into a multifunctional hydrogel for heart valve tissue engineering applications.

Functional tissue engineered heart valves (TEHV) have been an elusive goal for nearly 30 years. Among the persistent challenges are the requirements for engineered valve leaflets that possess nonlinear elastic tissue biomechanical properties, support quiescent fibroblast phenotype, and resist osteogenic differentiation. Nanocellulose is an attractive tunable biological material that has not been employed to this application. In this study, we fabricated a series of photocrosslinkable composite hydrogels mNCC-MeGel (mNG) by conjugating TEMPO-modified nanocrystalline cellulose (mNCC) onto the backbone of methacrylated gelatin (MeGel). Their structures were characterized by FTIR, 1 HNMR and uniaxial compression testing. Human adipose-derived mesenchymal stem cells (HADMSC) were encapsulated within the material and evaluated for valve interstitial cell phenotypes over 14 days culture in both normal and osteogenic media. Compared to the MeGel control group, the HADMSC encapsulated within mNG showed decreased alpha smooth muscle actin (αSMA) expression and increased vimentin and aggrecan expression, suggesting the material supports a quiescent fibroblastic phenotype. Under osteogenic media conditions, HADMSC within mNG hydrogels showed lower expression of osteogenic genes, including Runx2 and osteocalcin, indicating resistance toward calcification. As a proof of principle, the mNG hydrogel, combined with a viscosity enhancing agent, was used to 3D bioprint a tall, self-standing tubular structure that sustained cell viability. Together, these results identify mNG as an attractive biomaterial for TEHV applications.

A review of climate change effects on practices for mitigating water quality impacts.

Water quality practices are commonly implemented to reduce human impacts on land and water resources. In series or parallel in a landscape, systems of practices can reduce local and downstream pollution delivery. Many practices function via physical, chemical, and biological processes that are dependent on weather and climate. Climate change will alter the function of many such systems, though effects will vary in different hydroclimatic and watershed settings. Reducing the risk of impacts will require risk-based, adaptive planning. Here, we review the literature addressing climate change effects on practices commonly used to mitigate the water quality impacts of urban stormwater, agriculture, and forestry. Information from the general literature review is used to make qualitative inferences about the resilience of different types of practices. We discuss resilience in the context of two factors: the sensitivity of practice function to changes in climatic drivers, and the adaptability, or relative ease with which a practice can be modified as change occurs. While only a first step in addressing a complex topic, our aim is to help communities incorporate consideration of resilience to climate change as an additional factor in decisions about water quality practices to meet long-term goals.

OCT4-mediated inflammation induces cell reprogramming at the origin of cardiac valve development and calcification.

Cell plasticity plays a key role in embryos by maintaining the differentiation potential of progenitors. Whether postnatal somatic cells revert to an embryonic-like naïve state regaining plasticity and redifferentiate into a cell type leading to a disease remains intriguing. Using genetic lineage tracing and single-cell RNA sequencing, we reveal that Oct4 is induced by nuclear factor κB (NFκB) at embyronic day 9.5 in a subset of mouse endocardial cells originating from the anterior heart forming field at the onset of endocardial-to-mesenchymal transition. These cells acquired a chondro-osteogenic fate. OCT4 in adult valvular aortic cells leads to calcification of mouse and human valves. These calcifying cells originate from the Oct4 embryonic lineage. Genetic deletion of Pou5f1 (Pit-Oct-Unc, OCT4) in the endocardial cell lineage prevents aortic stenosis and calcification of ApoE−/− mouse valve. We established previously unidentified self-cell reprogramming NFκB- and OCT4-mediated inflammatory pathway triggering a dose-dependent mechanism of valve calcification.

An Efficient Statistical Approach to Develop Intensity-Duration-Frequency Curves for Precipitation and Runoff under Future Climate.

Ongoing and potential future changes in precipitation will affect water management infrastructure. Urban drainage systems are particularly vulnerable. Design standards for many stormwater practices rely on precipitation intensity-duration-frequency (IDF) curves based on extreme value analysis. General Circulation Models (GCMs) project increases in future average temperature but are less clear on changes in precipitation. In many areas, climate projections suggest relatively small changes in total precipitation volume, but also suggest increased magnitude of extreme events. Model skill in predicting extreme precipitation events, however, is limited. We develop an approach for estimating future IDF curves that is efficient, uses widely available statistically downscaled GCM output, and is consistent with published IDF curves for the United States that are often incorporated into local stormwater regulations and design guides (and are GCM model agnostic). The method provides a relatively simple way to develop scenarios in a format directly useful to assessing risk to stormwater management infrastructure. Model biases are addressed through equidistant quantile mapping, in which the modeled change in the cumulative distribution of storm events from historical to future conditions is used to adjust the extreme value fit used for IDF curve development. The approach is efficient because it requires only annual maxima and is readily automated, allowing rapid examination of results across projections. We estimate future IDF curves at locations throughout the United States and link IDF-derived design storms to a rainfall-runoff model to evaluate the potential change in storage volume requirements for capture-based stormwater management practices by 2065.

Hydrostatic mechanical stress regulates growth and maturation of the atrioventricular valve.

During valvulogenesis, cytoskeletal, secretory and transcriptional events drive endocardial cushion growth and remodeling into thin fibrous leaflets. Genetic disorders play an important role in understanding valve malformations but only account for a minority of clinical cases. Mechanical forces are ever present, but how they coordinate molecular and cellular decisions remains unclear. In this study, we used osmotic pressure to interrogate how compressive and tensile stresses influence valve growth and shape maturation. We found that compressive stress drives a growth phenotype, whereas tensile stress increases compaction. We identified a mechanically activated switch between valve growth and maturation, by which compression induces cushion growth via BMP-pSMAD1/5, while tension induces maturation via pSer-19-mediated MLC2 contractility. The compressive stress acts through BMP signaling to increase cell proliferation and decrease cell contractility, and MEK-ERK is essential for both compressive stress and BMP mediation of compaction. We further showed that the effects of osmotic stress are conserved through the condensation and elongation stages of development. Together, our results demonstrate that compressive/tensile stress regulation of BMP-pSMAD1/5 and MLC2 contractility orchestrates valve growth and remodeling.

Assessing Early Cardiac Outflow Tract Adaptive Responses Through Combined Experimental-Computational Manipulations.

Mechanical forces are essential for proper growth and remodeling of the primitive pharyngeal arch arteries (PAAs) into the great vessels of the heart. Despite general acknowledgement of a hemodynamic-malformation link, the direct correlation between hemodynamics and PAA morphogenesis remains poorly understood. The elusiveness is largely due to difficulty in performing isolated hemodynamic perturbations and quantifying changes in-vivo. Previous in-vivo arch artery occlusion/ablation experiments either did not isolate the effects of hemodynamics, did not analyze the results in a 3D context or did not consider the effects of varying degrees of occlusion. Here, we overcome these limitations by combining minimally invasive occlusion experiments in the avian embryo with 3D anatomical models of development and in-silico testing of experimental phenomenon. We detail morphological and hemodynamic changes 24 hours post vessel occlusion. 3D anatomical models showed that occlusion geometries had more circular cross-sectional areas and more elongated arches than their control counterparts. Computational fluid dynamics revealed a marked change in wall shear stress-morphology trends. Instantaneous (in-silico) occlusion models provided mechanistic insights into the dynamic vessel adaptation process, predicting pressure-area trends for a number of experimental occlusion arches. We follow the propagation of small defects in a single embryo Hamburger Hamilton (HH) Stage 18 embryo to a more serious defect in an HH29 embryo. Results demonstrate that hemodynamic perturbation of the presumptive aortic arch, through varying degrees of vessel occlusion, overrides natural growth mechanisms and prevents it from becoming the dominant arch of the aorta.

Uncovering transcriptional dark matter via gene annotation independent single-cell RNA sequencing analysis.

Conventional scRNA-seq expression analyses rely on the availability of a high quality genome annotation. Yet, as we show here with scRNA-seq experiments and analyses spanning human, mouse, chicken, mole rat, lemur and sea urchin, genome annotations are often incomplete, in particular for organisms that are not routinely studied. To overcome this hurdle, we created a scRNA-seq analysis routine that recovers biologically relevant transcriptional activity beyond the scope of the best available genome annotation by performing scRNA-seq analysis on any region in the genome for which transcriptional products are detected. Our tool generates a single-cell expression matrix for all transcriptionally active regions (TARs), performs single-cell TAR expression analysis to identify biologically significant TARs, and then annotates TARs using gene homology analysis. This procedure uses single-cell expression analyses as a filter to direct annotation efforts to biologically significant transcripts and thereby uncovers biology to which scRNA-seq would otherwise be in the dark.

Inflammatory and Biomechanical Drivers of Endothelial-Interstitial Interactions in Calcific Aortic Valve Disease.

Calcific aortic valve disease is dramatically increasing in global burden, yet no therapy exists outside of prosthetic replacement. The increasing proportion of younger and more active patients mandates alternative therapies. Studies suggest a window of opportunity for biologically based diagnostics and therapeutics to alleviate or delay calcific aortic valve disease progression. Advancement, however, has been hampered by limited understanding of the complex mechanisms driving calcific aortic valve disease initiation and progression towards clinically relevant interventions.

Spatiotemporal single-cell RNA sequencing of developing chicken hearts identifies interplay between cellular differentiation and morphogenesis.

Single-cell RNA sequencing is a powerful tool to study developmental biology but does not preserve spatial information about tissue morphology and cellular interactions. Here, we combine single-cell and spatial transcriptomics with algorithms for data integration to study the development of the chicken heart from the early to late four-chambered heart stage. We create a census of the diverse cellular lineages in developing hearts, their spatial organization, and their interactions during development. Spatial mapping of differentiation transitions in cardiac lineages defines transcriptional differences between epithelial and mesenchymal cells within the epicardial lineage. Using spatially resolved expression analysis, we identify anatomically restricted expression programs, including expression of genes implicated in congenital heart disease. Last, we discover a persistent enrichment of the small, secreted peptide, thymosin beta-4, throughout coronary vascular development. Overall, our study identifies an intricate interplay between cellular differentiation and morphogenesis.