Temporal Progression of Aortic Valve Pathogenesis in a Mouse Model of Osteogenesis Imperfecta.

Organization of extracellular matrix (ECM) components, including collagens, proteoglycans, and elastin, is essential for maintaining the structure and function of heart valves throughout life. Mutations in ECM genes cause connective tissue disorders, including Osteogenesis Imperfecta (OI), and progressive debilitating heart valve dysfunction is common in these patients. Despite this, effective treatment options are limited to end-stage interventions. Mice with a homozygous frameshift mutation in col1a2 serve as a murine model of OI (oim/oim), and therefore, they were used in this study to examine the pathobiology of aortic valve (AoV) disease in this patient population at structural, functional, and molecular levels. Temporal echocardiography of oim/oim mice revealed AoV dysfunction by the late stages of disease in 12-month-old mice. However, structural and proteomic changes were apparent much earlier, at 3 months of age, and were associated with disturbances in ECM homeostasis primarily related to collagen and proteoglycan abnormalities and disorganization. Together, findings from this study provide insights into the underpinnings of late onset AoV dysfunction in connective tissue disease patients that can be used for the development of mechanistic-based therapies administered early to halt progression, thereby avoiding late-stage surgical intervention.

Exploring molecular profiles of calcification in aortic vascular smooth muscle cells and aortic valvular interstitial cells.

Cardiovascular calcification can occur in vascular and valvular structures and is commonly associated with calcium deposition and tissue mineralization leading to stiffness and dysfunction. Patients with chronic kidney disease and associated hyperphosphatemia have an elevated risk for coronary artery calcification (CAC) and calcific aortic valve disease (CAVD). However, there is mounting evidence to suggest that the susceptibility and pathobiology of calcification in these two cardiovascular structures may be different, yet clinically they are similarly treated. To better understand diversity in molecular and cellular processes that underlie hyperphosphatemia-induced calcification in vascular and valvular structures, we exposed aortic vascular smooth muscle cells (AVSMCs) and aortic valve interstitial cells (AVICs) to high (2.5 mM) phosphate (Ph) conditions in vitro, and examined cell-specific responses. To further identify hyperphosphatemic-specific responses, parallel studies were performed using osteogenic media (OM) as an alternative calcific stimulus. Consistent with clinical observations made by others, we show that AVSMCs are more susceptible to calcification than AVICs. In addition, bulk RNA-sequencing reveals that AVSMCs and AVICs activate robust ossification-programs in response to high phosphate or OM treatments, however, the signaling pathways, cellular processes and osteogenic-associated markers involved are cell- and treatment-specific. For example, compared to VSMCs, VIC-mediated calcification involves biological processes related to osteo-chondro differentiation and down regulation of 'actin cytoskeleton'-related genes, that are not observed in VSMCs. Furthermore, hyperphosphatemic-induced calcification in AVICs and AVSMCs is independent of P13K signaling, which plays a role in OM-treated cells. Together, this study provides a wealth of information suggesting that the pathogenesis of cardiovascular calcifications is significantly more diverse than previously appreciated.

Dissociation of pulse wave velocity and aortic wall stiffness in diabetic db/db mice: The influence of blood pressure.

Introduction: Vascular stiffness is a predictor of cardiovascular disease and pulse wave velocity (PWV) is the current standard for measuring in vivo vascular stiffness. Mean arterial pressure is the largest confounding variable to PWV; therefore, in this study we aimed to test the hypothesis that increased aortic PWV in type 2 diabetic mice is driven by increased blood pressure rather than vascular biomechanics. Methods and Results: Using a combination of in vivo PWV and ex vivo pressure myography, our data demonstrate no difference in ex vivo passive mechanics, including outer diameter, inner diameter, compliance (Db/db: 0.0094 ± 0.0018 mm2/mmHg vs. db/db: 0.0080 ± 0.0008 mm2/mmHg, p > 0.05 at 100 mmHg), and incremental modulus (Db/db: 801.52 ± 135.87 kPa vs. db/db: 838.12 ± 44.90 kPa, p > 0.05 at 100 mmHg), in normal versus diabetic 16 week old mice. We further report no difference in basal or active aorta biomechanics in normal versus diabetic 16 week old mice. Finally, we show here that the increase in diabetic in vivo aortic pulse wave velocity at baseline was completely abolished when measured at equivalent pharmacologically-modulated blood pressures, indicating that the elevated PWV was attributed to the concomitant increase in blood pressure at baseline, and therefore "stiffness." Conclusions: Together, these animal model data suggest an intimate regulation of blood pressure during collection of pulse wave velocity when determining in vivo vascular stiffness. These data further indicate caution should be exerted when interpreting elevated PWV as the pure marker of vascular stiffness.

Developing small-diameter vascular grafts with human amniotic membrane: long-term evaluation of transplantation outcomes in a small animal model.

While current clinical utilization of large vascular grafts for vascular transplantation is encouraging, tissue engineering of small grafts still faces numerous challenges. This study aims to investigate the feasibility of constructing a small vascular graft from decellularized amniotic membranes (DAMs). DAMs were rolled around a catheter and each of the resulting grafts was crosslinked with (a) 0.1% glutaraldehyde; (b) 1-ethyl-3-(3-dimethylaminopropyl) crbodiimidehydro-chloride (20 mM)-N-hydroxy-succinimide (10 mM); (c) 0.5% genipin; and (d) no-crosslinking, respectively. Our results demonstrated the feasibility of using a rolling technique followed by lyophilization to transform DAM into a vessel-like structure. The genipin-crosslinked DAM graft showed an improved integrated structure, prolonged stability, proper mechanical property, and superior biocompatibility. After transplantation in rat abdominal aorta, the genipin-crosslinked DAM graft remained patent up to 16 months, with both endothelial and smooth muscle cell regeneration, which suggests that the genipin-crosslinked DAM graft has great potential to beimplementedas a small tissue engineered graft for futurevasculartransplantation.

Platelet tissue factor pathway inhibitor-α dampens cardiac thrombosis and associated fibrosis in mice.

BACKGROUND: Tissue factor pathway inhibitor (TFPI) is the primary inhibitor of events initiating the blood coagulation pathway. Tfpi-/- mice die during embryonic development. The absence of protease-activated receptor (PAR) 4, the major thrombin receptor on mouse platelets, rescues Tfpi-/-mice to adulthood. Among the 3 TFPI isoforms in mice, TFPIα is the only isoform within platelets (pltTFPIα) and the only isoform that inhibits prothrombinase, the enzymatic complex that converts prothrombin to thrombin. OBJECTIVES: To determine biological functions of pltTFPIα. METHODS: Tfpi-/-/Par4-/- mice were irradiated and transplanted with bone marrow from mice lacking or containing pltTFPIα. Thus, PAR4 expression was restored in the recipient mice, which differed selectively by the presence or absence of pltTFPIα and lacked other forms of TFPI. RESULTS: Recipient mice lacking pltTFPIα had reduced survival over the 200-day posttransplant period. Necropsy revealed radiation injury associated with large intraventricular platelet-rich thrombi, whereas other organs were not affected. Thrombi were associated with fibrotic presentations, including increased collagen deposition, periostin-positive activated fibroblasts, myofibroblasts, and macrophage infiltrates. Recipient mice containing pltTFPIα showed evidence of radiation injury but lacked heart pathology. CONCLUSIONS: Tfpi-/-/Par4-/- mice develop severe cardiac fibrosis following irradiation and transplantation with bone marrow lacking pltTFPIα. This pathology is markedly reduced when the mice are transplanted with bone marrow containing pltTFPIα. Thus, in this model system pltTFPIα has an important physiological role in dampening pathological responses mediated by activated platelets within the heart tissue.

Differential requirement for DICER1 activity during the development of mitral and tricuspid valves.

Mitral and tricuspid valves are essential for unidirectional blood flow in the heart. They are derived from similar cell sources, and yet congenital dysplasia affecting both valves is clinically rare, suggesting the presence of differential regulatory mechanisms underlying their development. Here, we specifically inactivated Dicer1 in the endocardium during cardiogenesis and found that Dicer1 deletion caused congenital mitral valve stenosis and regurgitation, whereas it had no impact on other valves. We showed that hyperplastic mitral valves were caused by abnormal condensation and extracellular matrix (ECM) remodeling. Our single-cell RNA sequencing analysis revealed impaired maturation of mesenchymal cells and abnormal expression of ECM genes in mutant mitral valves. Furthermore, expression of a set of miRNAs that target ECM genes was significantly lower in tricuspid valves compared to mitral valves, consistent with the idea that the miRNAs are differentially required for mitral and tricuspid valve development. We thus reveal miRNA-mediated gene regulation as a novel molecular mechanism that differentially regulates mitral and tricuspid valve development, thereby enhancing our understanding of the non-association of inborn mitral and tricuspid dysplasia observed clinically.

A Systematic Review of Ebstein's Anomaly with Left Ventricular Noncompaction.

Traditional definitions of Ebstein's anomaly (EA) and left ventricular noncompaction (LVNC), two rare congenital heart defects (CHDs), confine disease to either the right or left heart, respectively. Around 15-29% of patients with EA, which has a prevalence of 1 in 20,000 live births, commonly manifest with LVNC. While individual EA or LVNC literature is extensive, relatively little discussion is devoted to the joint appearance of EA and LVNC (EA/LVNC), which poses a higher risk of poor clinical outcomes. We queried PubMed, Medline, and Web of Science for all peer-reviewed publications from inception to February 2022 that discuss EA/LVNC and found 58 unique articles written in English. Here, we summarize and extrapolate commonalities in clinical and genetic understanding of EA/LVNC to date. We additionally postulate involvement of shared developmental pathways that may lead to this combined disease. Anatomical variation in EA/LVNC encompasses characteristics of both CHDs, including tricuspid valve displacement, right heart dilatation, and left ventricular trabeculation, and dictates clinical presentation in both age and severity. Disease treatment is non-specific, ranging from symptomatic management to invasive surgery. Apart from a few variant associations, mainly in sarcomeric genes MYH7 and TPM1, the genetic etiology and pathogenesis of EA/LVNC remain largely unknown.

RBFOX2 is required for establishing RNA regulatory networks essential for heart development.

Human genetic studies identified a strong association between loss of function mutations in RBFOX2 and hypoplastic left heart syndrome (HLHS). There are currently no Rbfox2 mouse models that recapitulate HLHS. Therefore, it is still unknown how RBFOX2 as an RNA binding protein contributes to heart development. To address this, we conditionally deleted Rbfox2 in embryonic mouse hearts and found profound defects in cardiac chamber and yolk sac vasculature formation. Importantly, our Rbfox2 conditional knockout mouse model recapitulated several molecular and phenotypic features of HLHS. To determine the molecular drivers of these cardiac defects, we performed RNA-sequencing in Rbfox2 mutant hearts and identified dysregulated alternative splicing (AS) networks that affect cell adhesion to extracellular matrix (ECM) mediated by Rho GTPases. We identified two Rho GTPase cycling genes as targets of RBFOX2. Modulating AS of these two genes using antisense oligos led to cell cycle and cell-ECM adhesion defects. Consistently, Rbfox2 mutant hearts displayed cell cycle defects and inability to undergo endocardial-mesenchymal transition, processes dependent on cell-ECM adhesion and that are seen in HLHS. Overall, our work not only revealed that loss of Rbfox2 leads to heart development defects resembling HLHS, but also identified RBFOX2-regulated AS networks that influence cell-ECM communication vital for heart development.

Pulmonary Vein Stenosis: Moving From Past Pessimism to Future Optimism.

Pulmonary Vein Stenosis (PVS) is a rare disease with a prevalence of around 1. 7 cases per 100,000 children under 2 years old. Treatment options for this disease have not provided great results and pathophysiology of this condition is still poorly understood. Here, we will review the history of PVS including diagnostic tools and treatments, the current management approach, and what the future holds for this devastating disease.

Tgfß1-Cthrc1 Signaling Plays an Important Role in the Short-Term Reparative Response to Heart Valve Endothelial Injury.

OBJECTIVE: Aortic valve disease is a common worldwide health burden with limited treatment options. Studies have shown that the valve endothelium is critical for structure-function relationships, and disease is associated with its dysfunction, damage, or injury. Therefore, therapeutic targets to maintain a healthy endothelium or repair damaged endothelial cells could hold promise. In this current study, we utilize a surgical mouse model of heart valve endothelial cell injury to study the short-term response at molecular and cellular levels. The goal is to determine if the native heart valve exhibits a reparative response to injury and identify the mechanisms underlying this process. Approach and Results: Mild aortic valve endothelial injury and abrogated function was evoked by inserting a guidewire down the carotid artery of young (3 months) and aging (16-18 months) wild-type mice. Short-term cellular responses were examined at 6 hours, 48 hours, and 4 weeks following injury, whereas molecular profiles were determined after 48 hours by RNA-sequencing. Within 48 hours following endothelial injury, young wild-type mice restore endothelial barrier function in association with increased cell proliferation, and upregulation of transforming growth factor beta 1 (Tgfß1) and the glycoprotein, collagen triple helix repeat containing 1 (Cthrc1). Interestingly, this beneficial response to injury was not observed in aging mice with known underlying endothelial dysfunction. CONCLUSIONS: Data from this study suggests that the healthy valve has the capacity to respond to mild endothelial injury, which in short term has beneficial effects on restoring endothelial barrier function through acute activation of the Tgfß1-Cthrc1 signaling axis and cell proliferation.

Molecular and Mechanical Mechanisms of Calcification Pathology Induced by Bicuspid Aortic Valve Abnormalities.

Bicuspid aortic valve (BAV) is a congenital defect affecting 1-2% of the general population that is distinguished from the normal tricuspid aortic valve (TAV) by the existence of two, rather than three, functional leaflets (or cusps). BAV presents in different morphologic phenotypes based on the configuration of cusp fusion. The most common phenotypes are Type 1 (containing one raphe), where fusion between right coronary and left coronary cusps (BAV R/L) is the most common configuration followed by fusion between right coronary and non-coronary cusps (BAV R/NC). While anatomically different, BAV R/L and BAV R/NC configurations are both associated with abnormal hemodynamic and biomechanical environments. The natural history of BAV has shown that it is not necessarily the primary structural malformation that enforces the need for treatment in young adults, but the secondary onset of premature calcification in ~50% of BAV patients, that can lead to aortic stenosis. While an underlying genetic basis is a major pathogenic contributor of the structural malformation, recent studies have implemented computational models, cardiac imaging studies, and bench-top methods to reveal BAV-associated hemodynamic and biomechanical alterations that likely contribute to secondary complications. Contributions to the field, however, lack support for a direct link between the external valvular environment and calcific aortic valve disease in the setting of BAV R/L and R/NC BAV. Here we review the literature of BAV hemodynamics and biomechanics and discuss its previously proposed contribution to calcification. We also offer means to improve upon previous studies in order to further characterize BAV and its secondary complications.

Genetic and Developmental Contributors to Aortic Stenosis.

Aortic stenosis (AS) remains one of the most common forms of valve disease, with significant impact on patient survival. The disease is characterized by left ventricular outflow obstruction and encompasses a series of stenotic lesions starting from the left ventricular outflow tract to the descending aorta. Obstructions may be subvalvar, valvar, or supravalvar and can be present at birth (congenital) or acquired later in life. Bicuspid aortic valve, whereby the aortic valve forms with two instead of three cusps, is the most common cause of AS in younger patients due to primary anatomic narrowing of the valve. In addition, the secondary onset of premature calcification, likely induced by altered hemodynamics, further obstructs left ventricular outflow in bicuspid aortic valve patients. In adults, degenerative AS involves progressive calcification of an anatomically normal, tricuspid aortic valve and is attributed to lifelong exposure to multifactoral risk factors and physiological wear-and-tear that negatively impacts valve structure-function relationships. AS continues to be the most frequent valvular disease that requires intervention, and aortic valve replacement is the standard treatment for patients with severe or symptomatic AS. While the positive impacts of surgical interventions are well documented, the financial burden, the potential need for repeated procedures, and operative risks are substantial. In addition, the clinical management of asymptomatic patients remains controversial. Therefore, there is a critical need to develop alternative approaches to prevent the progression of left ventricular outflow obstruction, especially in valvar lesions. This review summarizes our current understandings of AS cause; beginning with developmental origins of congenital valve disease, and leading into the multifactorial nature of AS in the adult population.

Nitric oxide prevents aortic valve calcification by S-nitrosylation of USP9X to activate NOTCH signaling.

Calcific aortic valve disease (CAVD) is an increasingly prevalent condition, and endothelial dysfunction is implicated in its etiology. We previously identified nitric oxide (NO) as a calcification inhibitor by its activation of NOTCH1, which is genetically linked to human CAVD. Here, we show NO rescues calcification by an S-nitrosylation-mediated mechanism in porcine aortic valve interstitial cells and single-cell RNA-seq demonstrated NO regulates the NOTCH pathway. An unbiased proteomic approach to identify S-nitrosylated proteins in valve cells found enrichment of the ubiquitin-proteasome pathway and implicated S-nitrosylation of USP9X (ubiquitin specific peptidase 9, X-linked) in NOTCH regulation during calcification. Furthermore, S-nitrosylated USP9X was shown to deubiquitinate and stabilize MIB1 for NOTCH1 activation. Consistent with this, genetic deletion of Usp9x in mice demonstrated CAVD and human calcified aortic valves displayed reduced S-nitrosylation of USP9X. These results demonstrate a previously unidentified mechanism by which S-nitrosylation-dependent regulation of a ubiquitin-associated pathway prevents CAVD.

KPT-330 Prevents Aortic Valve Calcification via a Novel C/EBPß Signaling Pathway.

[Figure: see text].

Biology and Biomechanics of the Heart Valve Extracellular Matrix.

Heart valves are dynamic structures that, in the average human, open and close over 100,000 times per day, and 3 × 109 times per lifetime to maintain unidirectional blood flow. Efficient, coordinated movement of the valve structures during the cardiac cycle is mediated by the intricate and sophisticated network of extracellular matrix (ECM) components that provide the necessary biomechanical properties to meet these mechanical demands. Organized in layers that accommodate passive functional movements of the valve leaflets, heart valve ECM is synthesized during embryonic development, and remodeled and maintained by resident cells throughout life. The failure of ECM organization compromises biomechanical function, and may lead to obstruction or leaking, which if left untreated can lead to heart failure. At present, effective treatment for heart valve dysfunction is limited and frequently ends with surgical repair or replacement, which comes with insuperable complications for many high-risk patients including aged and pediatric populations. Therefore, there is a critical need to fully appreciate the pathobiology of biomechanical valve failure in order to develop better, alternative therapies. To date, the majority of studies have focused on delineating valve disease mechanisms at the cellular level, namely the interstitial and endothelial lineages. However, less focus has been on the ECM, shown previously in other systems, to be a promising mechanism-inspired therapeutic target. Here, we highlight and review the biology and biomechanical contributions of key components of the heart valve ECM. Furthermore, we discuss how human diseases, including connective tissue disorders lead to aberrations in the abundance, organization and quality of these matrix proteins, resulting in instability of the valve infrastructure and gross functional impairment.

Constructing and evaluating caspase-activatable adeno-associated virus vector for gene delivery to the injured heart.

Adeno-associated virus (AAV) is a promising vector for gene therapy, but its broad tropism can be detrimental if the transgene being delivered is harmful when expressed ubiquitously in the body, i.e. in non-target tissues. Delivering the transgene of interest to target cells at levels high enough to be therapeutically effective while maintaining safety by minimizing delivery to off-target cells is a prevalent challenge in the field of gene therapy. We have developed a protease activatable vector (provector) platform based on AAV9 that can be injected systemically to deliver therapeutic transgenes site-specifically to diseased cells by responding to extracellular proteases present at the disease site. The provector platform consists of a peptide insertion into the virus capsid which disrupts the virus' ability to bind to cell surface receptors. This peptide contains a blocking motif (aspartic acid residues) flanked on either side by cleavage sequences that are recognized by certain proteases. Exposure to proteases cleaves the peptides off the capsid, activating or "switching ON" the provector. In response to the activation, the provectors regain their ability to bind and transduce cells. Here, we have designed a provector that is activated by cysteine aspartic proteases (caspases), which have roles in inflammation and apoptosis and thus are elevated at sites of diseases such as heart failure, neurodegenerative diseases, and ischemic stroke. This provector demonstrates a 200-fold reduction in transduction ability in the OFF state compared to AAV9, reducing the virus' ability to transduce off-target healthy tissue. Following exposure to and proteolysis by caspase-3, the provector shows a 95-fold increase in transduction compared to the OFF state. The switchable transduction behavior was found to be a direct result of the peptide insertion ablating the ability of the virus to bind to cells. In vivo studies were conducted to characterize the biodistribution, blood circulation time, neutralizing antibody formation, and targeted delivery ability of the caspase-activatable provector in a model of heart failure.

Effect of Left and Right Coronary Flow Waveforms on Aortic Sinus Hemodynamics and Leaflet Shear Stress: Correlation with Calcification Locations.

Coronary flow induces hemodynamic alterations in the aortic sinus region. The objectives of this study are to: (1) investigate the differences among sinus hemodynamics and leaflet wall shear stresses engendered by the left versus right versus non-coronary flow and (2) correlate respective wall shear stresses with leaflet calcification in patients. A left heart simulator flow loop with a tunable coronary circuit provided physiological coronary flow waveforms corresponding to the left coronary cusp case (LCC), right coronary cusp case (RCC), and non-coronary cusp case (NCC). High spatio-temporal resolution particle image velocimetry was conducted to quantify leaflet wall shear stress and sinus vorticity fields and to measure aortic leaflet tip kinematics. Thirty-one patients with severe calcific aortic valve disease were segmented from CT data for the calcific volumes in their respective left, right, and non-coronary cusps. Leaflet tip position during systole shows the RCC has a wider leaflet opening compared to LCC and NCC. Velocity and vorticity fields combined with leaflet position data show that sinus vorticity is diminished (peak ~ 43 s-1) in the LCC while RCC and NCC maintain high vorticity (~ 1200 and ~ 950 s-1 respectively). WSS magnitudes greater than 0.3 Pa show 20 and 81% greater occurrences in the LCC and RCC respectively compared to NCC. Significant differences [X2 (2, n = 31) = 7.31, p = 0.0258] between the calcification levels in each cusp of the patient population. Coronary flow differences between LCC, RCC, and NCC show significant impact on leaflet kinematics and sinus flow hemodynamics. Clinical data correlations of the coronary flow cases indicate the left coronary cusp has a higher likelihood of calcification compared to the right.

Smooth Muscle α-Actin Expression in Mitral Valve Interstitial Cells is Important for Mediating Extracellular Matrix Remodeling.

BACKGROUND: Mitral valve prolapse (MVP) affects 3-6% of the total population including those with connective tissue disorders. Treatment is limited, and patients commonly require surgery which can be impermanent and insuperable. Abnormal prolapse of mitral valve leaflets into the left atria is caused by disturbances to the composition and organization of the extracellular matrix (ECM), that weaken biomechanics. This process, known as myxomatous degeneration is characterized by an abnormal accumulation of proteoglycans, in addition to collagen fiber disruption and elastic fiber fragmentation. The underlying mechanisms that promote myxomatous degeneration to the point of biomechanical failure are unknown, but previous histological studies of end-stage diseased tissue have reported abnormal α-smooth muscle actin (SMA) in a subset of heart valve interstitial cells (VICs); however, the contribution of these abnormal cells to MVP pathogenesis has not been extensively examined. METHODS: In vivo and in vitro approaches were used. Mice harboring a Fbn1C1039G mutation mimic human Marfan Syndrome and develop MVP. Using these mice, temporal and spatial changes in SMA expression relative to myxomatous degeneration were examined using histological techniques. In parallel in vitro experiments, SMA expression was downregulated in primary porcine mitral VICs directly using siRNA, and indirectly using the actin depolymerizing agent Latrunculin A. In addition, the regulation of SMA in VICs by mechanical stiffness was explored relative to ECM remodeling. RESULTS: We show, in mitral valves from Fbn1C1039G/+ mice, that abnormal increases in SMA expression in VICs are evident during early postnatal stages of disease, prior to significant myxomatous degeneration as indicated at later stages by increased proteoglycans and collagen type I (Col1a1). Furthermore, abnormal SMA expression continues to increase during the course of pathogenesis and is localized to the mid belly region of the mitral valve leaflets from 10 weeks. Using an in vitro approach, we demonstrate that reduced SMA function by direct siRNA or indirect Latrunculin A treatment attenuates proteoglycan and Col1a1 expression in porcine mitral VICs. While upstream, we provide insights to show that SMA is regulated by mechanical tension in VICs to promote changes in ECM homeostasis. CONCLUSIONS: Together, our data show that in VICs, SMA, an actin binding protein, is important for mediating ECM remodeling associated with phenotypes observed in myxomatous degeneration, and its expression is regulated by mechanical tension. These novel insights could inform the development of future non-surgical therapeutics to halt the progression of mitral valve degeneration thereby avoiding end-stage prolapse.

Disruption of foxc1 genes in zebrafish results in dosage-dependent phenotypes overlapping Axenfeld-Rieger syndrome.

The Forkhead Box C1 (FOXC1) gene encodes a forkhead/winged helix transcription factor involved in embryonic development. Mutations in this gene cause dysgenesis of the anterior segment of the eye, most commonly Axenfeld-Rieger syndrome (ARS), often with other systemic features. The developmental mechanisms and pathways regulated by FOXC1 remain largely unknown. There are two conserved orthologs of FOXC1 in zebrafish, foxc1a and foxc1b. To further examine the role of FOXC1 in vertebrates, we generated foxc1a and foxc1b single knockout zebrafish lines and bred them to obtain various allelic combinations. Three genotypes demonstrated visible phenotypes: foxc1a-/- single homozygous and foxc1-/- double knockout homozygous embryos presented with similar characteristics comprised of severe global vascular defects and early lethality, as well as microphthalmia, periocular edema and absence of the anterior chamber of the eye; additionally, fish with heterozygous loss of foxc1a combined with homozygosity for foxc1b (foxc1a+/-;foxc1b-/-) demonstrated craniofacial defects, heart anomalies and scoliosis. All other single and combined genotypes appeared normal. Analysis of foxc1 expression detected a significant increase in foxc1a levels in homozygous and heterozygous mutant eyes, suggesting a mechanism for foxc1a upregulation when its function is compromised; interestingly, the expression of another ARS-associated gene, pitx2, was responsive to the estimated level of wild-type Foxc1a, indicating a possible role for this protein in the regulation of pitx2 expression. Altogether, our results support a conserved role for foxc1 in the formation of many organs, consistent with the features observed in human patients, and highlight the importance of correct FOXC1/foxc1 dosage for vertebrate development.