Hippo Signaling Mediates TGFß-Dependent Transcriptional Inputs in Cardiac Cushion Mesenchymal Cells to Regulate Extracellular Matrix Remodeling.

The transforming growth factor beta (TGFß) and Hippo signaling pathways are evolutionarily conserved pathways that play a critical role in cardiac fibroblasts during embryonic development, tissue repair, and fibrosis. TGFß signaling and Hippo signaling are also important for cardiac cushion remodeling and septation during embryonic development. Loss of TGFß2 in mice causes cardiac cushion remodeling defects resulting in congenital heart disease. In this study, we used in vitro molecular and pharmacologic approaches in the cushion mesenchymal cell line (tsA58-AVM) and investigated if the Hippo pathway acts as a mediator of TGFß2 signaling. Immunofluorescence staining showed that TGFß2 induced nuclear translocation of activated SMAD3 in the cushion mesenchymal cells. In addition, the results indicate increased nuclear localization of Yes-associated protein 1 (YAP1) following a similar treatment of TGFß2. In collagen lattice formation assays, the TGFß2 treatment of cushion cells resulted in an enhanced collagen contraction compared to the untreated cushion cells. Interestingly, verteporfin, a YAP1 inhibitor, significantly blocked the ability of cushion cells to contract collagen gel in the absence or presence of exogenously added TGFß2. To confirm the molecular mechanisms of the verteporfin-induced inhibition of TGFß2-dependent extracellular matrix (ECM) reorganization, we performed a gene expression analysis of key mesenchymal genes involved in ECM remodeling in heart development and disease. Our results confirm that verteporfin significantly decreased the expression of α-smooth muscle actin (Acta2), collagen 1a1 (Col1a1), Ccn1 (i.e., Cyr61), and Ccn2 (i.e., Ctgf). Western blot analysis indicated that verteporfin treatment significantly blocked the TGFß2-induced activation of SMAD2/3 in cushion mesenchymal cells. Collectively, these results indicate that TGFß2 regulation of cushion mesenchymal cell behavior and ECM remodeling is mediated by YAP1. Thus, the TGFß2 and Hippo pathway integration represents an important step in understanding the etiology of congenital heart disease.

Self-Assembling Toroidal Cell Constructs for Tissue Engineering Applications.

Developing tissues have intricate, three-dimensional (3D) organizations of cells and extracellular matrix (ECM) that provide the framework necessary to meet morphogenic and necessary demands. Migrating cells, in vivo, are exposed to numerous conflicting signals: chemokines, ECM, growth factors, and physical forces. While most of these have been studied individually in vivo or in vitro, our understanding of how cells integrate these various signals is lacking. We previously developed a novel self-organizing cellularized collagen hydrogel model that is adaptable, tunable, reproducible, and capable of mimicking the multitude of stimuli that cells experience. Our model produced self-assembled toroids of cells that were formed by 24 h. Data we present here show toroids initially form as early as 3 h after seeding. Additionally, toroids formed when cells were seeded on various collagen subtypes and were sensitive to the composition of the hydrogel. Moreover, we found differences in remodeling in toroid gels compared to gels with cells embedded in them using both a collagen binding peptide and rheology. Using scanning electron microscopy, we observed toroids forming a crater-like structure compared to whole gel contractions in mixed in gels. Finally, when multiple cells were mixed prior to seeding, heterogeneous toroids formed with some containing clusters of cells.

Alpha-Calcitonin Gene Related Peptide: New Therapeutic Strategies for the Treatment and Prevention of Cardiovascular Disease and Migraine.

Alpha-calcitonin gene-related peptide (α-CGRP) is a vasodilator neuropeptide of the calcitonin gene family. Pharmacological and gene knock-out studies have established a significant role of α-CGRP in normal and pathophysiological states, particularly in cardiovascular disease and migraines. α-CGRP knock-out mice with transverse aortic constriction (TAC)-induced pressure-overload heart failure have higher mortality rates and exhibit higher levels of cardiac fibrosis, inflammation, oxidative stress, and cell death compared to the wild-type TAC-mice. However, administration of α-CGRP, either in its native- or modified-form, improves cardiac function at the pathophysiological level, and significantly protects the heart from the adverse effects of heart failure and hypertension. Similar cardioprotective effects of the peptide were demonstrated in pressure-overload heart failure mice when α-CGRP was delivered using an alginate microcapsules-based drug delivery system. In contrast to cardiovascular disease, an elevated level of α-CGRP causes migraine-related headaches, thus the use of α-CGRP antagonists that block the interaction of the peptide to its receptor are beneficial in reducing chronic and episodic migraine headaches. Currently, several α-CGRP antagonists are being used as migraine treatments or in clinical trials for migraine pain management. Overall, agonists and antagonists of α-CGRP are clinically relevant to treat and prevent cardiovascular disease and migraine pain, respectively. This review focuses on the pharmacological and therapeutic significance of α-CGRP-agonists and -antagonists in various diseases, particularly in cardiac diseases and migraine pain.

Collagen Fibrillogenesis in the Mitral Valve: It's a Matter of Compliance.

Collagen fibers are essential structural components of mitral valve leaflets, their tension apparatus (chordae tendineae), and the associated papillary muscles. Excess or lack of collagen fibers in the extracellular matrix (ECM) in any of these structures can adversely affect mitral valve function. The organization of collagen fibers provides a sophisticated framework that allows for unidirectional blood flow during the precise opening and closing of this vital heart valve. Although numerous ECM molecules are essential for the differentiation, growth, and homeostasis of the mitral valve (e.g., elastic fibers, glycoproteins, and glycans), collagen fibers are key to mitral valve integrity. Besides the inert structural components of the tissues, collagen fibers are dynamic structures that drive outside-to-inside cell signaling, which informs valvular interstitial cells (VICs) present within the tissue environment. Diversity of collagen family members and the closely related collagen-like triple helix-containing proteins found in the mitral valve, will be discussed in addition to how defects in these proteins may lead to valve disease.

A Novel Alginate-Based Delivery System for the Prevention and Treatment of Pressure-Overload Induced Heart Failure.

Background: α-CGRP (alpha-calcitonin gene related peptide) is a cardioprotective neuropeptide. Our recent study demonstrated that the administration of native α-CGRP, using osmotic mini-pumps, protected against transverse aortic constriction (TAC) pressure-induced heart failure in mice. However, the short half-life of peptides and the non-applicability of osmotic pumps in humans limits the use of α-CGRP as a therapeutic agent for heart failure (HF). Here, we sought to comprehensively study a novel α-CGRP delivery system using alginate microcapsules to determine its bioavailability in vivo and to test for cardioprotective effects in HF mice. Methods: Native α-CGRP filled alginate microcapsules (200 µm diameter) were prepared using an electrospray method. The prepared alginate-α-CGRP microcapsules were incubated with rat cardiac H9c2 cells, mouse cardiac HL-1 cells, and human umbilical vein endothelial cells (HUVECs), and the cytotoxicity of the alginate-α-CGRP microcapsules was measured by a trypan-blue cell viability assay and a calcium dye fluorescent based assay. The efficacy of the alginate-α-CGRP microcapsules was tested in a TAC-pressure overload mouse model of heart failure. Male C57BL6 mice were divided into four groups: sham, sham-alginate-α-CGRP, TAC-only, and TAC-alginate-α-CGRP, and the TAC procedure was performed in the TAC-only and TAC-alginate-α-CGRP groups of mice to induce pressure-overload heart failure. After 2 or 15 days post-TAC, alginate-α-CGRP microcapsules (containing an α-CGRP dose of 6 mg/kg/mouse) were administered subcutaneously on alternate days, for 28 days, and echocardiography was performed weekly. After 28 days of peptide delivery, the mice were sacrificed and their hearts were collected for histological and biochemical analyses. Results: Our in vitro cell culture assays showed that alginate-α-CGRP microcapsules did not affect the viability of the cell lines tested. The alginate-α-CGRP microcapsules released their peptides for an extended period of time. Our echocardiography, biochemical, and histology data from HF mice demonstrated that the administration of alginate-α-CGRP microcapsules significantly improved all cardiac parameters examined in TAC-mice. When compared to sham mice, TAC significantly decreased cardiac functions (as determined by fraction shortening and ejection fraction) and markedly increased heart and lung weight, left ventricle (LV) cardiac cell size, cardiac apoptosis, and oxidative stress. In contrast, the administration of alginate-α-CGRP microcapsules significantly attenuated the increased heart and lung weight, LV cardiac cell size, apoptosis, and oxidative stress in TAC mice. Conclusion: Our results demonstrate that the encapsulation of α-CGRP in an alginate polymer is an effective strategy to improve peptide bioavailability in plasma and increase the duration of the therapeutic effect of the peptide throughout the treatment period. Furthermore, alginate mediates α-CGRP delivery, either prior to the onset or after the initiation of the symptom progression of pressure-overload, improves cardiac function, and protects hearts against pressure-induced HF.

Alpha-calcitonin gene-related peptide prevents pressure-overload induced heart failure: role of apoptosis and oxidative stress.

Alpha-calcitonin gene-related peptide (α-CGRP) is a 37-amino acid neuropeptide that plays an important protective role in modulating cardiovascular diseases. Deletion of the α-CGRP gene increases the vulnerability of the heart to pressure-induced heart failure and the administration of a modified α-CGRP agonist decreases this vulnerability. Systemic administration of α-CGRP decreases blood pressure in normotensive and hypertensive animals and humans. Here we examined the protective effect of long-term administration of native α-CGRP against pressure-overload heart failure and the likely mechanism(s) of its action. Transverse aortic constriction (TAC) was performed to induce pressure-overload heart failure in mice. We found that TAC significantly decreased left ventricular (LV) fractional shortening, ejection fraction, and α-CGRP content, and increased hypertrophy, dilation, and fibrosis compared to sham mice. Administration of α-CGRP-filled mini-osmotic pumps (4 mg/kg bwt/day) in TAC mice preserved cardiac function and LV α-CGRP levels, and reduced LV hypertrophy, dilation, and fibrosis to levels comparable to sham mice. Additionally, TAC pressure-overload significantly increased LV apoptosis and oxidative stress compared to the sham mice but these increases were prevented by α-CGRP administration. α-CGRP administration in TAC animals decreased LV AMPK phosphorylation levels and the expression of sirt1, both of which are regulatory markers of oxidative stress and energy metabolism. These results demonstrate that native α-CGRP is protective against pressure-overload induced heart failure. The mechanism of this cardio-protection is likely through the prevention of apoptosis and oxidative stress, possibly mediated by sirt1 and AMPK. Thus, α-CGRP is a potential therapeutic agent in preventing the progression to heart failure, and the cardio-protective action of α-CGRP is likely the result of a direct cellular effect; however, a partial vasodilatory blood pressure-dependent mechanism of α-CGRP cannot be excluded.

Protective Role of α-Calcitonin Gene-Related Peptide in Cardiovascular Diseases.

α-Calcitonin gene-related peptide (α-CGRP) is a regulatory neuropeptide of 37 amino acids. It is widely distributed in the central and peripheral nervous system, predominantly in cell bodies of the dorsal root ganglion (DRG). It is the most potent vasodilator known to date and has inotropic and chronotropic effects. Using pharmacological and genetic approaches, our laboratory and other research groups established the protective role of α-CGRP in various cardiovascular diseases such as heart failure, experimental hypertension, myocardial infarction, and myocardial ischemia/reperfusion injury (I/R injury). α-CGRP acts as a depressor to attenuate the rise in blood pressure in three different models of experimental hypertension: (1) DOC-salt, (2) subtotal nephrectomy-salt, and (3) L-NAME-induced hypertension during pregnancy. Subcutaneous administration of α-CGRP lowers the blood pressure in hypertensive and normotensive humans and rodents. Recent studies also demonstrated that an α-CGRP analog, acylated α-CGRP, with extended half-life (~7 h) reduces blood pressure in Ang-II-induced hypertensive mouse, and protects against abdominal aortic constriction (AAC)-induced heart failure. Together, these studies suggest that α-CGRP, native or a modified form, may be a potential therapeutic agent to treat patients suffering from cardiac diseases.

Perfusion Tissue Culture Initiates Differential Remodeling of Internal Thoracic Arteries, Radial Arteries, and Saphenous Veins.

Adaptive remodeling processes are essential to the maintenance and viability of coronary artery bypass grafts where clinical outcomes depend strongly on the tissue source. In this investigation, we utilized an ex vivo perfusion bioreactor to culture porcine analogs of common human bypass grafts: the internal thoracic artery (ITA), the radial artery (RA), and the great saphenous vein (GSV), and then evaluated samples acutely (6 h) and chronically (7 days) under in situ or coronary-like perfusion conditions. Although morphologically similar, primary cells harvested from the ITA illustrated lower intimal and medial, but not adventitial, cell proliferation rates than those from the RA or GSV. Basal gene expression levels were similar in all vessels, with only COL3A1, SERPINE1, FN1, and TGFB1 being differentially expressed prior to culture; however, over half of all genes were affected nominally by the culturing process. When exposed to coronary-like conditions, RAs and GSVs experienced pathological remodeling not present in ITAs or when vessels were studied in situ. Many of the remodeling genes perturbed at 6 h were restored after 7 days (COL3A1, FN1, MMP2, and TIMP1) while others (SERPINE1, TGFB1, and VCAM1) were not. The findings elucidate the potential mechanisms of graft failure and highlight strategies to encourage healthy ex vivo pregraft conditioning.

Removing vessel constriction on the embryonic heart results in changes in valve gene expression, morphology, and hemodynamics.

BACKGROUND: The formation of healthy heart valves throughout embryonic development is dependent on both genetic and epigenetic factors. Hemodynamic stimuli are important epigenetic regulators of valvulogenesis, but the resultant molecular pathways that control valve development are poorly understood. Here we describe how the heart and valves recover from the removal of a partial constriction (banding) of the OFT/ventricle junction (OVJ) that temporarily alters blood flow velocity through the embryonic chicken heart (HH stage 16/17). Recovery is described in terms of 24- and 48-hr gene expression, morphology, and OVJ hemodynamics. RESULTS: Collectively, these studies show that after 24 hr of recovery, important epithelial-mesenchymal transformation (EMT) genes TGFßRIII and Cadherin 11 (CDH11) transcript levels normalize return to control levels, in contrast to Periostin and TGFß,3 which remain altered. In addition, after 48 hr of recovery, TGFß3 and CDH11 transcript levels remain normalized, whereas TGFßRIII and Periostin are down-regulated. Analyses of OFT cushion volumes in the hearts show significant changes, as does the ratio of cushion to cell volume at 24 hr post band removal (PBR). Morphologically, the hearts show visible alteration following band removal when compared to their control age-matched counterparts. CONCLUSIONS: Although some aspects of the genetic/cellular profiles affected by altered hemodynamics seem to be reversed, not all gene expression and cardiac growth normalize following 48 hr of band removal. Developmental Dynamics 247:531-541, 2018. © 2017 Wiley Periodicals, Inc.

Functional Tissue Engineering: A Prevascularized Cardiac Muscle Construct for Validating Human Mesenchymal Stem Cells Engraftment Potential In Vitro.

The influence of somatic stem cells in the stimulation of mammalian cardiac muscle regeneration is still in its early stages, and so far, it has been difficult to determine the efficacy of the procedures that have been employed. The outstanding question remains whether stem cells derived from the bone marrow or some other location within or outside of the heart can populate a region of myocardial damage and transform into tissue-specific differentiated progenies, and also exhibit functional synchronization. Consequently, this necessitates the development of an appropriate in vitro three-dimensional (3D) model of cardiomyogenesis and prompts the development of a 3D cardiac muscle construct for tissue engineering purposes, especially using the somatic stem cell, human mesenchymal stem cells (hMSCs). To this end, we have created an in vitro 3D functional prevascularized cardiac muscle construct using embryonic cardiac myocytes (eCMs) and hMSCs. First, to generate the prevascularized scaffold, human cardiac microvascular endothelial cells (hCMVECs) and hMSCs were cocultured onto a 3D collagen cell carrier (CCC) for 7 days under vasculogenic culture conditions; hCMVECs/hMSCs underwent maturation, differentiation, and morphogenesis characteristic of microvessels, and formed dense vascular networks. Next, the eCMs and hMSCs were cocultured onto this generated prevascularized CCCs for further 7 or 14 days in myogenic culture conditions. Finally, the vascular and cardiac phenotypic inductions were characterized at the morphological, immunological, biochemical, molecular, and functional levels. Expression and functional analyses of the differentiated progenies revealed neo-cardiomyogenesis and neo-vasculogenesis. In this milieu, for instance, not only were hMSCs able to couple electromechanically with developing eCMs but were also able to contribute to the developing vasculature as mural cells, respectively. Hence, our unique 3D coculture system provides us a reproducible and quintessential in vitro 3D model of cardiomyogenesis and a functioning prevascularized 3D cardiac graft that can be utilized for personalized medicine.

A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System.

The new model presented here can be used to understand the influence of hemodynamics on specific cardiac developmental processes, at the cellular and molecular level. To alter intracardiac hemodynamics, fertilized chicken eggs are incubated in a humidified chamber to obtain embryos of the desired stage (HH17). Once this developmental stage is achieved, the embryo is maintained ex ovo and hemodynamics in the embryonic heart are altered by partially constricting the outflow tract (OFT) with a surgical suture at the junction of the OFT and ventricle (OVJ). Control embryos are also cultured ex ovo but are not subjected to the surgical intervention. Banded and control embryos are then incubated in a humidified incubator for the desired period of time, after which 2D ultrasound is employed to analyze the change in blood flow velocity at the OVJ as a result of OFT banding. Once embryos are maintained ex ovo, it is important to ensure adequate hydration in the incubation chamber so as to prevent drying and eventually embryo death. Using this new banded model, it is now possible to perform analyses of changes in the expression of key players involved in valve development and to understand the role of hemodynamics on cellular responses in vivo, which could not be achieved previously.

Altered Hemodynamics in the Embryonic Heart Affects Outflow Valve Development.

Cardiac valve structure and function are primarily determined during early development. Consequently, abnormally-formed heart valves are the most common type of congenital heart defects. Several adult valve diseases can be backtracked to abnormal valve development, making it imperative to completely understand the process and regulation of heart valve development. Epithelial-to-mesenchymal transition (EMT) plays an important role in the development of heart valves. Though hemodynamics is vital to valve development, its role in regulating EMT is still unknown. In this study, intracardiac hemodynamics were altered by constricting the outflow tract (OFT)/ventricle junction (OVJ) of HH16-17 (Hamilton and Hamburger (HH) Stage 16-17) chicken embryos, ex ovo for 24 h. The constriction created an increase in peak and time-averaged centerline velocity along the OFT without changes to volumetric flow or heart rate. Computational fluid dynamics was used to estimate the level of increased spatially-averaged wall shear stresses on the OFT cushion from AMIRA reconstructions. OFT constriction led to a significant decrease in OFT cushion volume and the number of invaded mesenchyme in the OFT cushion. qPCR analysis revealed altered mRNA expression of a representative panel of genes, vital to valve development, in the OFT cushions from banded hearts. This study indicates the importance of hemodynamics in valve development.

The impact of flow-induced forces on the morphogenesis of the outflow tract.

One percent of infants are born with congenital heart disease (CHD), which commonly involves outflow tract (OFT) defects. These infants often require complex surgeries, which are associated with long term adverse remodeling effects, and receive replacement valves with limited strength, biocompatibility, and growth capability. To address these problematic issues, researchers have carried out investigations in valve development and valve mechanics. A longstanding hypothesis is that flow-induced forces regulate fibrous valve development, however, the specific mechanisms behind this mechanotransduction remain unclear. The purpose of this study was to implement an in vitro system of outflow tract development to test the response of embryonic OFT tissues to fluid flow. A dynamic, three-dimensional bioreactor system was used to culture embryonic OFT tissue under different levels of flow as well as the absence of flow. In the absence of flow, OFT tissues took on a more primitive phenotype that is characteristic of early OFT cushion development where widely dispersed mesenchymal cells are surrounded by a sparse, disorganized extracellular matrix (ECM). Whereas OFT tissues subjected to physiologically matched flow formed compact mounds of cells, initated, fibrous ECM development, while prolonged supraphysiological flow resulted in abnormal tissue remodeling. This study indicates that both the timing and magnitude of flow alter cellular processes that determine if OFT precursor tissue undergoes normal or pathological development. Specifically, these experiments showed that flow-generated forces regulate the deposition and localization of fibrous ECM proteins, indicating that mechanosensitive signaling pathways are capable of driving pathological OFT development if flows are not ideal.

Impact of the controlled release of a connexin 43 peptide on corneal wound closure in an STZ model of type I diabetes.

The alpha-carboxy terminus 1 (αCT1) peptide is a synthetically produced mimetic modified from the DDLEI C-terminus sequence of connexin 43 (Cx43). Previous research using various wound healing models have found promising therapeutic effects when applying the drug, resulting in increased wound healing rates and reduced scarring. Previous data suggested a rapid metabolism rate in vitro, creating an interest in long term release. Using a streptozotocin (STZ) type I diabetic rat model with a surgically induced corneal injury, we delivered αCT1 both directly, in a pluronic gel solution, and in a sustained system, using polymeric alginate-poly-l-ornithine (A-PLO) microcapsules (MC). Fluorescent staining of wound area over a 5 day period indicated a significant increase in wound closure rates for both αCT1 and αCT1 MC treated groups, withαCT1 MC groups showing the most rapid wound closure overall. Analysis of inflammatory reaction to the treatment groups indicated significantly lower levels of both Interferon Inducible T-Cell Alpha Chemoattractant (ITAC) and Tumor Necrosis Factor Alpha (TNFα) markers using confocal quantification and ELISA assays. Additional analysis examining genes selected from the EMT pathway using RT-PCR and Western blotting suggested αCT1 modification of Transforming Growth Factor Beta 2 (TGFß2), Keratin 8 (Krt8), Estrogen Receptor 1 (Esr1), and Glucose Transporter 4 (Glut4) over a 14 day period. Combined, this data indicated a possible suppression of the inflammatory response by αCT1, leading to increased wound healing rates.

Microencapsulation of stem cells to study cellular interactions.

Microencapsulation is a technique used in both controlled delivery of materials over time as well as preservation of these materials while delivery is occurring. The range of materials able to be encapsulated is variable, from drugs to living cells. The latter is described here. Electrospray microencapsulation applies a high-voltage field, through which a polymeric material is extruded. A gelling bath, comprising a cross-linking material, is used to create a stable hydrogel containing secondary substances intended for delivery. Control of extrusion parameters, such as flow rate and voltage, allows for specification of diameter and pore sizes of the microcapsules.

A synthetic connexin 43 mimetic peptide augments corneal wound healing.

The ability to safely and quickly close wounds and lacerations is an area of need in regenerative medicine, with implications toward healing a wide range of tissues and wounds. Using an in vivo corneal injury model, our study applied a newly developed peptide capable of promotion of wound healing and epithelial regeneration. The alpha-carboxy terminus 1 (αCT1) peptide is a 25 amino acid peptide from the C-terminus of connexin 43 (Cx43), modified to promote cellular uptake. Previous studies applying αCT1 to excisional skin wounds in porcine models produced tissues having an overall reduced level of scar tissue and decreased healing time. Rapid metabolism of αCT1 in previous work led to the investigation of extended release on wound healing rate used in this study. Here we delivered αCT1 both directly, in a concentrated pluronic solution, and in a sustained system, using polymeric alginate-poly-l-ornithine (A-PLO) microcapsules. Cell toxicity analysis showed minimal cell-loss with microcapsule treatment. Measurement of wound healing using histology and fluorescence microscopy indicated significant reduction in healing time of αCT1 microcapsule treated rat corneas compared with controls (88% vs. 38%). RT-PCR analysis showed an initial up regulation followed by down regulation of the gene keratin-19 (Krt19). Zonula occludens 1 (ZO-1) showed an opposite down regulation followed by an up regulation whereas Cx43 showed a biphasic response. Inflammatory indexes demonstrated a reduction in the inflammation of corneas treated with αCT1 microcapsules when compared with pluronic gel vehicle. These results suggest αCT1, when applied in a sustained release system, acts as a beneficial wound healing treatment.

Zyxin regulates cell migration and differentiation in EMT during chicken AV valve morphogenesis.

During heart valve development, epithelial-mesenchymal transformation (EMT) is a key process for valve formation. EMT leads to the generation of mesenchymal cells that will eventually become the interstitial cells (fibroblasts) of the mature valve. During EMT, cell architecture and motility change markedly; significant changes are also observed in various signaling pathways. Here we systematically examined the expression, localization, and function of zyxin, a focal adhesion protein, in EMT during atrioventricular (AV) valve morphogenesis. Expression and localization studies showed that zyxin was expressed in the AV canal region during crucial stages of valve development. An in vitro 3D collagen gel culture system was used to determine zyxin function either after siRNA gene knockdown or after overexpression. Our studies revealed that zyxin overexpression inhibited endocardial cell migration and cell differentiation and also led to a decrease in the number of migrating mesenchymal cells. Moreover, correlative cytoskeletal changes were apparent in response to both overexpression and knockdown treatments. Thus, zyxin appears to play a role as a regulator of cell migration and differentiation during EMT in chicken AV valve formation.

Characterization of polymeric microcapsules containing a low molecular weight peptide for controlled release.

A need exists to prolong the release of rapidly metabolized peptides of a low molecular weight, while delivering this peptide without environmental interference. Previous studies have used bovine serum albumin (BSA) as a model peptide to study release characteristics from alginate microcapsules. BSA is 66 kDa in size, while the peptide of interest here, connexin-43 carboxyl-terminus mimetic peptide (αCT1), is only 3.4 kDa. Such a change in size results in a much different set of release parameters. Our overall goal is a sustained release over a 24+ h period. Prolonged application of the peptide to a wound site to investigate therapeutic effects is ideal. As a result, a diffusion method using alginate microcapsules, along with the addition of poly-l-lysine and poly-l-ornithine, has been explored. We first aimed to establish and characterize our parameters through a set of parametric tests. Variations in polymer coating, change in pH, and changes in loading ratio have previously been shown to effect release using model compounds. Here we test specific changes in these parameters to show effects on the release of αCT1. Additionally, the microcapsules were attached to several biomaterials and surgical implants by ultraviolet cross-linking to study the effectiveness of attachment and delivery. Analysis and measurements using phase contrast microscopy, scanning electron microscopy, and atomic force microscopy were used to characterize changes in microcapsule morphology.

Fluid flow forces and rhoA regulate fibrous development of the atrioventricular valves.

Fibrous development of the extracellular matrix (ECM) of cardiac valves is necessary for proper heart function. Pathological remodeling of valve ECM is observed in both pediatric and adult cardiac disorders. It is well established that intracardiac hemodynamics play a significant role in the morphogenesis of cardiovascular tissues. However, the mechanisms that transduce mechanical forces into morphogenetic processes are not well understood. Here, we report the development of a three-dimensional, in vitro culture system that allows for culture of embryonic valve tissue under specific pulsatile flow conditions. This system was used to investigate the role that fluid flow plays in fibrous ECM expression during valve formation and to test the underlying cellular mechanisms that regulate this mechanotransduction. When cultured under pulsatile flow, developing valve tissues upregulated fibrous ECM expression at both the transcript and protein levels in comparison to no-flow controls. Flow-cultured valve tissues also underwent morphological development, as cushions elongated into leaflet-like structures that were absent in no-flow controls. Furthermore, rhoA, a member of the cytoskeletal actin-regulating GTPase family of proteins, was upregulated and activated by flow culture. Inhibition of the downstream rhoA effector kinase, ROCK, blocked flow-driven fibrous ECM accumulation and tissue stiffening, while the addition of lysophosphatidic acid (LPA), a rhoA activator, stimulated fibrous ECM deposition and tissue stiffening. These results support a prominent role for the rhoA pathway in the mechanotransduction of hemodynamic forces during fibrous remodeling of developing valve tissue. Our results also point to a potential link between regulation of the actinomyosin cytoskeleton and fibrous ECM synthesis in cardiovascular tissues.

Self-organizing tissue-engineered constructs in collagen hydrogels.

A novel self-organizing behavior of cellularized gels composed of collagen type 1 that may have utility for tissue engineering is described. Depending on the starting geometry of the tissue culture well, toroidal rings of cells or hollow spheroids were prompted to form autonomously when cells were seeded onto the top of gels and the gels released from attachment to the culture well 12 to 24 h after seeding. Cells within toroids assumed distinct patterns of alignment not seen in control gels in which cells had been mixed in. In control gels, cells formed complex three-dimensional arrangements and assumed relatively higher levels of heterogeneity in expression of the fibronectin splice variant ED-A--a marker of epithelial mesenchymal transformation. The tissue-like constructs resulting from this novel self-organizing behavior may have uses in wound healing and regenerative medicine, as well as building blocks for the iterative assembly of synthetic biological structures.