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We have found that genetic ablation of the Sost gene (protein: sclerostin) prevents aortic valve stenosis in aged, Western diet mice. This is a new role for sclerostin in the cardiovascular system. To the knowledge of the authors, this is one of the first studies directly manipulating sclerostin in a cardiovascular disease model and the first to specifically study the aortic valve. We also provide a potential new role for Hox genes in cardiovascular disease, noting pan- Hox upregulation in the aortic roots of sclerostin genetic knockouts. The role of Hox genes in postnatal cardiovascular health and disease is another burgeoning field of study to which this article contributes.

This article uses in vivo and ex vivo analysis to track the development of osteoporosis and left heart cardiovascular disease in an aged, high-cholesterol diet, mouse ovariectomy model. Mice develop early left ventricle hypertrophy without concurrent fibrosis or aortic valve stenosis. These findings allow for a new model of the study of left ventricle hypertrophy in postmenopausal osteoporosis that more closely mimics the natural progression of disease in female patients.

Despite numerous promising therapeutic targets, there are no proven medical treatments for calcific aortic stenosis (AS). Multiple stakeholders need to come together and several scientific, operational, and trial design challenges must be addressed to capitalize on the recent and emerging mechanistic insights into this prevalent heart valve disease. This review briefly discusses the pathobiology and most promising pharmacologic targets, screening, diagnosis and progression of AS, identification of subgroups that should be targeted in clinical trials, and the need to elicit the patient voice earlier rather than later in clinical trial design and implementation. Potential trial end points and tools for assessment and approaches to implementation and design of clinical trials are reviewed. The efficiencies and advantages offered by a clinical trial network and platform trial approach are highlighted. The objective is to provide practical guidance that will facilitate a series of trials to identify effective medical therapies for AS resulting in expansion of therapeutic options to complement mechanical solutions for late-stage disease.

Inflammation caused by infiltrating macrophages and T cells promotes plaque growth in atherosclerosis. Cadherin-11 (CDH11) is a cell-cell adhesion protein implicated in several fibrotic and inflammatory diseases. Much of the research on CDH11 concerns its role in fibroblasts, although its expression in immune cells has been noted as well. The objective of this study was to assess the effect of CDH11 on the atherosclerotic immune response. In vivo studies of atherosclerosis indicated an increase in Cdh11 in plaque tissue. However, global loss of Cdh11 resulted in increased atherosclerosis and inflammation. It also altered the immune response in circulating leukocytes, decreasing myeloid cell populations and increasing T-cell populations, suggesting possible impaired myeloid migration. Bone marrow transplants from Cdh11-deficient mice resulted in similar immune cell profiles. In vitro examination of Cdh11(−/−) macrophages revealed reduced migration, despite upregulation of a number of genes related to locomotion. Flow cytometry revealed an increase in CD3(+) and CD4(+) helper T-cell populations in the blood of both the global Cdh11 loss and the bone marrow transplant animals, possibly resulting from increased expression by Cdh11(−/−) macrophages of major histocompatibility complex class II molecule genes, which bind to CD4(+) T cells for coordinated activation. CDH11 fundamentally alters the immune response in atherosclerosis, resulting in part from impaired macrophage migration and altered macrophage-induced T-cell activation. NEW & NOTEWORTHY Cadherin-11 is well known to contribute to inflammatory and fibrotic disease. Here, we examined its role in atherosclerosis progression, which is predominantly an inflammatory process. We found that while cadherin-11 is associated with plaque progression, global loss of cadherin-11 exacerbated the disease phenotype. Moreover, loss of cadherin-11 in bone marrow-derived immune cells resulted in impaired macrophage migration and an unexplained increase in circulating helper T cells, presumably due to altered macrophage function without cadherin-11.

Cancer cells characteristically consume glucose through Warburg metabolism1, a process that forms the basis of tumour imaging by positron emission tomography (PET). Tumour-infiltrating immune cells also rely on glucose, and impaired immune cell metabolism in the tumour microenvironment (TME) contributes to immune evasion by tumour cells2–4. However, whether the metabolism of immune cells is dysregulated in the TME by cell-intrinsic programs or by competition with cancer cells for limited nutrients remains unclear. Here we used PET tracers to measure the access to and uptake of glucose and glutamine by specific cell subsets in the TME. Notably, myeloid cells had the greatest capacity to take up intratumoral glucose, followed by T cells and cancer cells, across a range of cancer models. By contrast, cancer cells showed the highest uptake of glutamine. This distinct nutrient partitioning was programmed in a cell-intrinsic manner through mTORC1 signalling and the expression of genes related to the metabolism of glucose and glutamine. Inhibiting glutamine uptake enhanced glucose uptake across tumour-resident cell types, showing that glutamine metabolism suppresses glucose uptake without glucose being a limiting factor in the TME. Thus, cell-intrinsic programs drive the preferential acquisition of glucose and glutamine by immune and cancer cells, respectively. Cell-selective partitioning of these nutrients could be exploited to develop therapies and imaging strategies to enhance or monitor the metabolic programs and activities of specific cell populations in the TME. Positron emission tomography measurements of nutrient uptake in cells of the tumour microenvironment reveal cell-intrinsic partitioning in which glucose uptake is higher in myeloid cells, whereas glutamine is preferentially acquired by cancer cells.

The coordinated generation of mechanical forces by cardiac myocytes is required for proper heart function. Myofibrils are the functional contractile units of force production within individual cardiac myocytes. At the molecular level, myosin motors form cross-bridges with actin filaments and use ATP to convert chemical energy into mechanical forces. The energetic efficiency of the cross-bridge cycle is influenced by the viscous damping of myofibril contraction. The viscoelastic response of myofibrils is an emergent property of their individual mechanical components. Previous studies have implicated titin-actin interactions, cell-ECM adhesion, and microtubules as regulators of the viscoelastic response of myofibrils. Here we probed the viscoelastic response of myofibrils using laser-assisted dissection. As a proof-of-concept, we found actomyosin contractility was required to endow myofibrils with their viscoelastic response, with blebbistatin treatment resulting in decreased myofibril tension and viscous damping. Focal adhesion kinase (FAK) is a key regulator of cell-ECM adhesion, microtubule stability, and myofibril assembly. We found inhibition of FAK signaling altered the viscoelastic properties of myofibrils. Specifically, inhibition of FAK resulted in increased viscous damping of myofibril retraction following laser ablation. This damping was not associated with acute changes in the electrophysiological properties of cardiac myocytes. These results implicate FAK as a regulator of mechanical properties of myofibrils.

Heart failure (HF) is a chronic, complex condition with increasing incidence worldwide, necessitating the development of novel therapeutic strategies. This has led to the current clinical strategies, which only treat symptoms of HF without addressing the underlying causes. Multiple animal models have been developed in an attempt to recreate the chronic HF phenotype that arises following a variety of myocardial injuries. Although significant strides have been made in HF research, an understanding of more specific mechanisms will require distinguishing models that resemble HF with preserved ejection fraction (HFpEF) from those with reduced ejection fraction (HFrEF). Therefore, current mouse models of HF need to be re-assessed to determine which of them most closely recapitulate the specific etiology of HF being studied. This will allow for the development of therapies targeted specifically at HFpEF or HFrEF. This review will summarize the commonly used mouse models of HF and discuss which aspect of human HF each model replicates, focusing on whether HFpEF or HFrEF is induced, to allow better investigation into pathophysiological mechanisms and treatment strategies.

Pressure overload of the heart is characterized by concentric hypertrophy and interstitial fibrosis. Cardiac fibroblasts (CFs) in the ventricular wall become activated during injury and synthesize and compact the extracellular matrix, which causes interstitial fibrosis and stiffening of the ventricular heart walls. Talin1 (Tln1) and Talin2 (Tln2) are mechanosensitive proteins that participate in focal adhesion transmission of signals from the extracellular environment to the actin cytoskeleton of CFs. The aim of the present study was to determine whether the removal of Tln1 and Tln2 from CFs would reduce interstitial fibrosis and cardiac hypertrophy. Twelve-week-old male and female Tln2-null (Tln2(−/−)) and Tln2-null, CF-specific Tln1 knockout (Tln2(−/−);Tln1(CF−/−)) mice were given angiotensin-II (ANG II) (1.5 mg/kg/day) or saline through osmotic pumps for 8 wk. Cardiomyocyte area and measures of heart thickness were increased in the male ANG II-infused Tln2(−/−);Tln1(CF−/−) mice, whereas there was no increase in interstitial fibrosis. Systolic blood pressure was increased in the female Tln2(−/−);Tln1(CF−/−) mice after ANG II infusion compared with the Tln2(−/−) mice. However, there was no increase in cardiac hypertrophy in the Tln2(−/−);Tln1(CF−/−) mice, which was seen in the Tln2(−/−) mice. Collectively, these data indicate that in male mice, the absence of Tln1 and Tln2 in CFs leads to cardiomyocyte hypertrophy in response to ANG II, whereas it results in a hypertrophy-resistant phenotype in female mice. These findings have important implications for the role of mechanosensitive proteins in CFs and their impact on cardiomyocyte function in the pathogenesis of hypertension and cardiac hypertrophy. NEW & NOTEWORTHY The role of talins has been previously studied in cardiomyocytes; however, these mechanotransductive proteins that are members of the focal adhesion complex have not been examined in cardiac fibroblasts previously. We hypothesized that loss of talins in cardiac fibroblasts would reduce interstitial fibrosis in the heart with a pressure overload model. However, we found that although loss of talins did not alter fibrosis, it did result in cardiomyocyte and ventricular hypertrophy.

The mechanisms regulating endothelial cell response to hemodynamic forces required for heart valve development, especially valve remodeling, remain elusive. Tie1, an endothelial specific receptor tyrosine kinase, is up-regulated by oscillating shear stress and is required for lymphatic valve development. In this study, we demonstrate that valvular endothelial Tie1 is differentially expressed in a dynamic pattern predicted by disturbed flow during valve remodeling. Following valvular endocardial specific deletion of Tie1 in mice, we observed enlarged aortic valve leaflets, decreased valve stiffness and valvular insufficiency. Valve abnormalities were only detected in late gestation and early postnatal mutant animals and worsened with age. The mutant mice developed perturbed extracellular matrix (ECM) deposition and remodeling characterized by increased glycosaminoglycan and decreased collagen content, as well as increased valve interstitial cell expression of Sox9, a transcription factor essential for normal ECM maturation during heart valve development. This study provides the first evidence that Tie1 is involved in modulation of late valve remodeling and suggests that an important Tie1-Sox9 signaling axis exists through which disturbed flows are converted by endocardial cells to paracrine Sox9 signals to modulate normal matrix remodeling of the aortic valve.

Calcific aortic valve disease (CAVD) is highly prevalent and has no pharmaceutical treatment. Surgical replacement of the aortic valve has proved effective in advanced disease but is costly, time limited, and in many cases not optimal for elderly patients. This has driven an increasing interest in noninvasive therapies for patients with CAVD. Adaptive immune cell signaling in the aortic valve has shown potential as a target for such a therapy. Up to 15% of cells in the healthy aortic valve are hematopoietic in origin, and these cells, which include macrophages, T lymphocytes, and B lymphocytes, are increased further in calcified specimens. Additionally, cytokine signaling has been shown to play a causative role in aortic valve calcification both in vitro and in vivo. This review summarizes the physiological presence of hematopoietic cells in the valve, innate and adaptive immune cell infiltration in disease states, and the cytokine signaling pathways that play a significant role in CAVD pathophysiology and may prove to be pharmaceutical targets for this disease in the near future.

Visual Abstract, Highlights • Celecoxib use is associated with diagnosis of aortic stenosis in analysis of electronic medical records. • Celecoxib treatment increases dystrophic calcification of aortic valve interstitial cells in vitro. • Dimethyl celecoxib, which binds CDH11, prevents TGF-β1–mediated calcification of aortic valve interstitial cells in vitro., Summary Calcific aortic valve disease is a progressive fibrocalcific process that can only be treated with valve replacement. Cadherin-11 has recently been identified as a potential therapeutic target for calcific aortic valve disease. The already approved drug celecoxib, a cyclooxygenase-2 inhibitor, binds cadherin-11, and was investigated as a therapeutic against calcific aortic valve disease. Unexpectedly, celecoxib treatment led to hallmarks of myofibroblast activation and calcific nodule formation in vitro. Retrospective electronic medical record analysis of celecoxib, ibuprofen, and naproxen revealed a unique association of celecoxib use and aortic stenosis.

BACKGROUND: Myocardial infarction (MI) induces an intense injury response which ultimately generates a collagen-dominated scar. While required to prevent ventricular rupture, the fibrotic process is often sustained in a manner detrimental to optimal recovery. Cardiac myofibroblasts are the cells tasked with depositing and remodeling collagen and are a prime target to limit the fibrotic process post-MI. Serotonin 2B receptor (5-HT(2B)) signaling has been shown to be harmful in a variety of cardiopulmonary pathologies and could play an important role in mediating scar formation after MI. METHODS: We employed two pharmacologic antagonists to explore the effect of 5-HT(2B) inhibition on outcomes post-MI and characterized the histological and microstructural changes involved in tissue remodeling. Inducible, 5-HT(2B) ablation driven by Tcf21(MCM) and Postn(MCM) were used to evaluate resident cardiac fibroblast- and myofibroblast-specific contributions of 5-HT(2B,) respectively. RNA sequencing was used to motivate subsequent in vitro analyses to explore cardiac fibroblast phenotype. RESULTS: 5-HT(2B) antagonism preserved cardiac structure and function by facilitating a less fibrotic scar, indicated by decreased scar thickness and decreased border zone area. 5-HT(2B) antagonism resulted in collagen fiber redistribution to thinner collagen fibers which were more anisotropic, enhancing left ventricular contractility, while fibrotic tissue stiffness was decreased, limiting the hypertrophic response of uninjured cardiomyocytes. Using a tamoxifen-inducible Cre, we ablated 5-HT(2B) from Tcf21-lineage resident cardiac fibroblasts and saw similar improvements to the pharmacologic approach. Tamoxifen-inducible Cre-mediated ablation of 5-HT(2B) after onset of injury in Postn-lineage myofibroblasts also improved cardiac outcomes. RNA sequencing and subsequent in vitro analyses corroborate a decrease in fibroblast proliferation, migration, and remodeling capabilities through alterations in Dnajb4 expression and Src phosphorylation. CONCLUSIONS: Together, our findings illustrate that 5-HT(2B) expression in either cardiac fibroblasts or activated myofibroblasts directly contributes to excessive scar formation, resulting in adverse remodeling and impaired cardiac function after MI.

Background: Myocardial infarction (MI) induces an intense injury response that ultimately generates a collagen-dominated scar. Although required to prevent ventricular rupture, the fibrotic process is often sustained in a manner detrimental to optimal recovery. Cardiac myofibroblasts are the cells tasked with depositing and remodeling collagen and are a prime target to limit the fibrotic process after MI. Serotonin 2B receptor (5-HT 2B ) signaling has been shown to be harmful in a variety of cardiopulmonary pathologies and could play an important role in mediating scar formation after MI. Methods: We used 2 pharmacological antagonists to explore the effect of 5-HT 2B inhibition on outcomes after MI and characterized the histological and microstructural changes involved in tissue remodeling. Inducible 5-HT 2B ablation driven by Tcf21 MCM and Postn MCM was used to evaluate resident cardiac fibroblast- and myofibroblast-specific contributions of 5-HT 2B , respectively. RNA sequencing was used to motivate subsequent in vitro analyses to explore cardiac fibroblast phenotype. Results: 5-HT 2B antagonism preserved cardiac structure and function by facilitating a less fibrotic scar, indicated by decreased scar thickness and decreased border zone area. 5-HT 2B antagonism resulted in collagen fiber redistribution to thinner collagen fibers that were more anisotropic, enhancing left ventricular contractility, whereas fibrotic tissue stiffness was decreased, limiting the hypertrophic response of uninjured cardiomyocytes. Using a tamoxifen-inducible Cre, we ablated 5-HT 2B from Tcf21 -lineage resident cardiac fibroblasts and saw similar improvements to the pharmacological approach. Tamoxifen-inducible Cre-mediated ablation of 5-HT 2B after onset of injury in Postn -lineage myofibroblasts also improved cardiac outcomes. RNA sequencing and subsequent in vitro analyses corroborate a decrease in fibroblast proliferation, migration, and remodeling capabilities through alterations in Dnajb4 expression and Src phosphorylation. Conclusions: Together, our findings illustrate that 5-HT 2B expression in either cardiac fibroblasts or activated myofibroblasts directly contributes to excessive scar formation, resulting in adverse remodeling and impaired cardiac function after MI.

Cardiopulmonary disease is driven by a plethora of factors, some well characterized and others still undiscovered. The 5-HT2B serotonin receptor (5-HT2BR) is one such factor that has gained attention and contributed to the understanding of disease development. 5-HT2BR is present throughout the cardiopulmonary system, most notably in cardiac valves, lung tissue, and the myocardium. Persistent activation of the receptor contributes to disease onset, initially reported following administration of anti-obesity compounds whose metabolites were potent 5-HT2BR agonists. These patients had increased incidences of valve disease as well as pulmonary arterial hypertension. The 5-HT2BR is also integral to normal cardiac development, and there is evidence describing the impact of 5-HT2BR activation inducing cardiac hypertrophy. The body of research highlights the importance of serotonergic signaling, specifically through 5-HT2BR, in the development of cardiopulmonary disease and merits consideration into investigating the therapeutic potential manipulating 5-HT2BR signaling.

Calcific aortic valve disease (CAVD) is a deadly disease that is rising in prevalence due to population aging. While the disease is complex and poorly understood, one well-documented driver of valvulopathy is serotonin agonism. Both serotonin overexpression, as seen with carcinoid tumors and drug-related agonism, such as with Fenfluramine use, are linked with various diseases of the valves. Thus, the objective of this study was to determine if genetic ablation or pharmacological antagonism of the 5-HT2Bserotonin receptor (gene:Htr2b) could improve the hemodynamic and histological progression of calcific aortic valve disease.Htr2bmutant mice were crossed withNotch1+/-mice, an established small animal model of CAVD, to determine if genetic ablation affects CAVD progression. To assess the effect of pharmacological inhibition on CAVD progression,Notch1+/-mice were treated with the 5-HT2Breceptor antagonist SB204741. Mice were analyzed using echocardiography, histology, immunofluorescence, and real-time quantitative polymerase chain reaction.Htr2bmutant mice showed lower aortic valve peak velocity and mean pressure gradient–classical hemodynamic indicators of aortic valve stenosis–without concurrent left ventricle change. 5-HT2Breceptor antagonism, however, did not affect hemodynamic progression. Leaflet thickness, collagen density, and CAVD-associated transcriptional markers were not significantly different in any group. This study reveals that genetic ablation ofHtr2battenuates hemodynamic development of CAVD in theNotch1+/-mice, but pharmacological antagonism may require high doses or long-term treatment to slow progression.

Calcific aortic valve disease (CAVD) is a deadly disease that is rising in prevalence due to population aging. While the disease is complex and poorly understood, one well-documented driver of valvulopathy is serotonin agonism. Both serotonin overexpression, as seen with carcinoid tumors and drug-related agonism, such as with Fenfluramine use, are linked with various diseases of the valves. Thus, the objective of this study was to determine if genetic ablation or pharmacological antagonism of the 5-HT2Bserotonin receptor (gene:Htr2b) could improve the hemodynamic and histological progression of calcific aortic valve disease.Htr2bmutant mice were crossed withNotch1+/-mice, an established small animal model of CAVD, to determine if genetic ablation affects CAVD progression. To assess the effect of pharmacological inhibition on CAVD progression,Notch1+/-mice were treated with the 5-HT2Breceptor antagonist SB204741. Mice were analyzed using echocardiography, histology, immunofluorescence, and real-time quantitative polymerase chain reaction.Htr2bmutant mice showed lower aortic valve peak velocity and mean pressure gradient – classical hemodynamic indicators of aortic valve stenosis – without concurrent left ventricle change. 5-HT2Breceptor antagonism, however, did not affect hemodynamic progression. Leaflet thickness, collagen density, and CAVD-associated transcriptional markers were not significantly different in any group. This study reveals that genetic ablation ofHtr2battenuates hemodynamic development of CAVD in theNotch1+/-mice, but pharmacological antagonism may require high doses or long-term treatment to slow progression.

Cancer cells characteristically consume glucose through Warburg metabolism

ObjectiveTo evaluate how common echocardiographic metrics of aortic stenosis (AS) influence the proportion of patients who may be categorised as having severe stenosis and therefore considered for valve replacement.MethodsRetrospective analysis was performed of all echocardiograms with aortic valve area (AVA) ≤1.2 cm2 and peak jet velocity (Vmax) ≥3 m/s from 1 December 2014 through 30 October 2017 at a single academic medical centre. Echocardiographic indices collected include AVA, Vmax, left ventricular ejection fraction, stroke volume and annotated aortic stenosis severity.ResultsAmong 807 patients with AVA ≤1.2 cm2 and Vmax ≥3 m/s (44.0% female, median age 74 years (IQR: 66–81)), 45.6% had Vmax ≥4 m/s, while 75.8% had AVA ≤1 cm2. 40.0% of patients had concordant indices (Vmax ≥4 m/s and AVA ≤1 cm2), and 35.8% had discordant indices (Vmax 2) of severe AS. Compared with those with concordant indices, patients with discordant indices were more commonly female (54.0% vs 44.3%, pConclusionsPatients with discordant indices, who are disproportionately female, are commonly described in clinical echocardiography reports as having less than severe AS. Given the potential benefit of AVR in patients with AVA ≤1 cm2 regardless of Vmax, this could have important clinical implications.

Circulating tumor cells (CTCs) are exposed to fluid shear stress (FSS) of greater than 1000 dyn/cm2 (100 Pa) in circulation. Normally, CTCs that are exposed to FSS of this magnitude die. However, some CTCs develop resistance to this FSS, allowing them to colonize distant organs. We explored how prostate CTCs can resist cell death in response to forces of this magnitude. The DU145, PC3 and LNCaP human prostate cancer cell lines were used to represent cells of different metastatic origins. The cell lines were briefly treated with an average FSS of 3950 dyn/cm2 (395 Pa) using a 30 G needle and a syringe pump. DU145 cells had no change in cell viability, PC3 cells had some cell death and LNCaP cells exhibited significant cell death. These cell death responses correlated with increased cell membrane damage, less efficient membrane repair and increased stiffness. Additionally, FSS treatment prevented the LNCaP FSS-sensitive cell line from forming a growing tumor in vivo. This suggests that these properties play a role in FSS resistance and could represent potential targets for disrupting blood-borne metastasis., Summary: Prostate cancer cells have different sensitivities to fluid forces that alter their resistance to elevated blood flow-level fluid shear stress.

Objective: Macrophages have been described in calcific aortic valve disease, but it is unclear if they promote or counteract calcification. We aimed to determine how macrophages are involved in calcification using the Notch1 +/− model of calcific aortic valve disease. Approach and Results: Macrophages in wild-type and Notch1 +/− murine aortic valves were characterized by flow cytometry. Macrophages in Notch1 +/− aortic valves had increased expression of MHCII (major histocompatibility complex II). We then used bone marrow transplants to test if differences in Notch1 +/− macrophages drive disease. Notch1 +/− mice had increased valve thickness, macrophage infiltration, and proinflammatory macrophage maturation regardless of transplanted bone marrow genotype. In vitro approaches confirm that Notch1 +/− aortic valve cells promote macrophage invasion as quantified by migration index and proinflammatory phenotypes as quantified by Ly6C and CCR2 positivity independent of macrophage genotype. Finally, we found that macrophage interaction with aortic valve cells promotes osteogenic, but not dystrophic, calcification and decreases abundance of the STAT3β isoform. Conclusions: This study reveals that Notch1 +/− aortic valve disease involves increased macrophage recruitment and maturation driven by altered aortic valve cell secretion, and that increased macrophage recruitment promotes osteogenic calcification and alters STAT3 splicing. Further investigation of STAT3 and macrophage-driven inflammation as therapeutic targets in calcific aortic valve disease is warranted.

INTRODUCTION: Induced pluripotent stem cell (iPSC)-derived endothelial cells (ECs) have the potential for therapeutic application in several cardiovascular diseases. Mechanical strain is known to regulate EC behavior and stem cell differentiation and may play a role in directing EC differentiation of iPSCs. H19, a long non-coding RNA (lncRNA), is known to affect ECs in several mechanically relevant pathologies and may play a role in this process as well. Therefore, we investigated expression changes of H19 resulting from mechanical stimulation during EC differentiation, as well as functional effects on EC tube formation. METHODS: iPSCs were subjected to 5% cyclic mechanical strain during EC differentiation. RT-PCR and flow cytometry were used to assess changes in mesoderm differentiation and gene expression in the final ECs as a result of strain. Functional outcomes of mechanically differentiated ECs were assessed with a tube formation assay and changes in H19. H19 was also overexpressed in human umbilical vein endothelial cells (HUVECs) to assess its role in non-H19-expressing ECs. RESULTS: Mechanical strain promoted mesoderm differentiation, marked by increased expression of brachyury 24 h after initiation of differentiation. Strain also increased expression of H19, CD31, VE-cadherin, and VEGFR2 in differentiated ECs. Strain-differentiated ECs formed tube networks with higher junction and endpoint density than statically-differentiated ECs. Overexpression of H19 in HUVECs resulted in similar patterns of tube formation. CONCLUSIONS: H19 expression is increased by mechanical strain and promotes tube branching in iPSC-derived ECs.

Acute kidney injury (AKI) portends a poor clinical prognosis and increases the risk for the development of chronic kidney disease (CKD). Currently, there are no therapies to treat AKI or prevent its progression to CKD. Wnt/β-catenin is a critical regulator of kidney development that is up-regulated after injury. Most of the literature support a beneficial role for Wnt/β-catenin in AKI, but suggest that this pathway promotes the progression of tubulointerstitial fibrosis, the hallmark of CKD progression. We review the role of Wnt/β-catenin in renal injury with a focus on its potential as a therapeutic target in AKI and in AKI to CKD transition.

Cadherin-11 (CDH11) is upregulated in a variety of fibrotic diseases, including arthritis and calcific aortic valve disease. Our recent work has identified CDH11 as a potential therapeutic target and shown that treatment with a CDH11 functional blocking antibody can prevent hallmarks of calcific aortic valve disease in mice. The present study investigated the role of CDH11 in regulating the mechanobiological behavior of valvular interstitial cells believed to cause calcification. Aortic valve interstitial cells were harvested from Cdh11+/+, Cdh11+/−, and Cdh11−/− immortomice. Cells were subjected to inflammatory cytokines transforming growth factor (TGF)-β1 and IL-6 to characterize the molecular mechanisms by which CDH11 regulates their mechanobiological changes. Histology was performed on aortic valves from Cdh11+/+, Cdh11+/−, and Cdh11−/− mice to identify key responses to CDH11 deletion in vivo. We showed that CDH11 influences cell behavior through its regulation of contractility and its ability to bind substrates via focal adhesions. We also show that transforming growth factor-β1 overrides the normal relationship between CDH11 and smooth muscle α-actin to exacerbate the myofibroblast disease phenotype. This phenotypic switch is potentiated through the IL-6 signaling axis and could act as a paracrine mechanism of myofibroblast activation in neighboring aortic valve interstitial cells in a positive feedback loop. These data suggest CDH11 is an important mediator of the myofibroblast phenotype and identify several mechanisms by which it modulates cell behavior. NEW & NOTEWORTHY Cadherin-11 influences valvular interstitial cell contractility by regulating focal adhesions and inflammatory cytokine secretion. Transforming growth factor-β1 overrides the normal balance between cadherin-11 and smooth muscle α-actin expression to promote a myofibroblast phenotype. Cadherin-11 is necessary for IL-6 and chitinase-3-like protein 1 secretion, and IL-6 promotes contractility. Targeting cadherin-11 could therapeutically influence valvular interstitial cell phenotypes in a multifaceted manner.

BackgroundOver one million Americans experience myocardial infarction (MI) every year, and the resulting scar and subsequent cardiac fibrosis contribute to heart failure and death. A specialized cell-cell adhesion protein, cadherin-11 (CDH11), contributes to inflammation and fibrosis in rheumatoid arthritis, pulmonary fibrosis, and aortic valve calcification but has not yet been studied in the context of cardiac remodeling after MI. We hypothesized that targeting CDH11 function after MI would reduce inflammation-driven fibrotic remodeling and infarct expansion to improve functional outcomes in mice.MethodsMI was induced by ligation of the left anterior descending artery in transgenic mice with reduced or ablated CDH11, wild type mice receiving bone marrow transplants from Cdh11 transgenic animals, and wild type mice treated with a functional blocking antibody against CDH11 (SYN0012). Cardiac function was measured by echocardiography, expression of cell populations was quantified by flow cytometry, and tissue remodeling by altered histological assessment and transcription of inflammatory and pro-angiogenic genes by qPCR. Co-culture was used to assess interactions between cardiac fibroblasts and macrophages.ResultsMI increased transcription of Cdh11 in non-cardiomyocyte cells. Mice with deletion of Cdh11 and wild type mice receiving bone marrow transplants from Cdh11 transgenic animals had improved cardiac function and dimensions after MI. Animals given SYN0012 had improved cardiac function, reduced tissue remodeling, and altered transcription of inflammatory and proangiogenic genes. Targeting CDH11 also reduced the number of bone marrow-derived myeloid cells and increased pro-angiogenic cells in the heart three days after MI, consistent with a decrease in transcription and expression of IL-6 in the infarct region. Cardiac fibroblast and macrophage interactions led to an increase in IL-6 secretion that was reduced with SYN0012 treatment in vitro.ConclusionsOur findings suggest that CDH11-expressing cells contribute to inflammation-driven fibrotic remodeling after MI, and that targeting CDH11 with a blocking antibody improves cardiac function after MI. This improvement is likely mediated by altered recruitment of bone marrow-derived cells, thereby limiting the macrophage-induced expression of IL-6 by fibroblasts and promoting vascularization.

Epigenetic dysregulation of long noncoding RNA H19 was recently found to be associated with calcific aortic valve disease (CAVD) in humans by repressing NOTCH1 transcription. This finding offers a possible epigenetic explanation for the abundance of cases of CAVD that are not explained by any clear genetic mutation. In this study, we examined the effect of age and sex on epigenetic dysregulation of H19 and subsequent aortic stenosis. Cohorts of littermate, wild‐type C57BL/6 mice were studied at developmental ages analogous to human middle age through advanced age. Cardiac and aortic valve function were assessed with M‐mode echocardiography and pulsed wave Doppler ultrasound, respectively. Bisulfite sequencing was used to determine methylation‐based epigenetic regulation of H19, and RT‐PCR was used to determine changes in gene expression profiles. Male mice were found to have higher peak systolic velocities than females, with several of the oldest mice showing signs of early aortic stenosis. The imprinting control region of H19 was not hypomethylated with age, and H19 expression was lower in the aortic valves of older mice than in the youngest group. These results suggest that age‐related upregulation of H19 is not observed in murine aortic valves and that other factors may initiate H19‐related CAVD in humans.

Summary: The mechanical properties of the actin cortex regulate shape changes during cell division, cell migration, and tissue morphogenesis. We show that modulation of myosin II (MII) filament composition allows tuning of surface tension at the cortex to maintain cell shape during cytokinesis. Our results reveal that MIIA generates cortex tension, while MIIB acts as a stabilizing motor and its inclusion in MII hetero-filaments reduces cortex tension. Tension generation by MIIA drives faster cleavage furrow ingression and bleb formation. We also show distinct roles for the motor and tail domains of MIIB in maintaining cytokinetic fidelity. Maintenance of cortical stability by the motor domain of MIIB safeguards against shape instability-induced chromosome missegregation, while its tail domain mediates cortical localization at the terminal stages of cytokinesis to mediate cell abscission. Because most non-muscle contractile systems are cortical, this tuning mechanism will likely be applicable to numerous processes driven by myosin-II contractility. : Taneja et al. describe distinct roles for the two myosin-II paralogs in regulating actin cortex mechanics during cell division. Myosin-IIA generates cortex tension, while myosin-IIB maintains cortical stability. Optimal levels of the two paralogs within hetero-filaments at the cortex are required for shape stability and cytokinetic fidelity during cell division. Keywords: myosin IIA, myosin IIB, actin cortex, binucleation, cortex tension, hydrostatic pressure, cytokinesis, cell division, bleb, spindle

Calcific aortic valve disease (CAVD) is a condition causing stiffening of the aortic valve, impeding cardiac function and resulting in significant morbidity worldwide. CAVD is thought to be driven by the persistent activation of the predominant cell type in the valve, aortic valve interstitial cells (AVICs), into myofibroblasts, resulting in subsequent calcification and stenosis of the valve. Although much of the research into CAVD focuses on AVICs, the aortic valve endothelial cells (AVECs) have been shown to regulate AVICs and maintain tissue homeostasis. Exposed to distinct flow patterns during the cardiac cycle, the AVECs lining either side of the valve demonstrate crucial differences which could contribute to the preferential formation of calcific nodules on the aorta-facing (fibrosa) side of the valve. Cadherin-11 (CDH11) is a cell–cell adhesion protein which has been previously associated with AVIC myofibroblast activation, nodule formation, and CAVD in mice. In this study, we investigated the role of CDH11 in AVECs and examined side-specific differences. The aorta-facing or fibrosa endothelial cells (fibAVECs) express higher levels of CDH11 than the ventricle-facing or ventricularis endothelial cells (venAVECs). This increase in expression corresponds with increased contraction of a free-floating collagen gel compared to venAVECs. Additionally, co-culture of fibAVECs with AVICs demonstrated decreased contraction compared to an AVIC + AVIC control, but increased contraction compared to the venAVECs co-culture. This aligns with the known preferential formation of calcific nodules on the fibrosa. These results together indicate a potential role for CDH11 expression by AVECs in regulating AVIC contraction and subsequent calcification.

Cardiac fibrosis represents an enormous health concern as it is prevalent in nearly every form of cardiovascular disease, the leading cause of death worldwide. Fibrosis is characterized by the activation of fibroblasts into myofibroblasts, a contractile cell type that secretes significant amounts of extracellular matrix components; however, the onset of this condition is also due to persistent inflammation and the cellular responses to a changing mechanical environment. In this review, we provide an overview of the pro-fibrotic, pro-inflammatory, and biomechanical mechanisms that lead to cardiac fibrosis in cardiovascular diseases. We then discuss cadherin-11, an intercellular adhesion protein present on both myofibroblasts and inflammatory cells, as a potential link for all three of the fibrotic mechanisms. Since experimentally blocking cadherin-11 dimerization prevents fibrotic diseases including cardiac fibrosis, understanding how this protein can be targeted for therapeutic use could lead to better treatments for patients with heart disease.

Calcific aortic valve disease (CAVD) accounts for ≈15 000 patient deaths per year in the United States, and intervention occurs only when severe stenosis requires surgical replacement of the valve. Recently, it was discovered that long noncoding RNA H19 expression silences NOTCH1 in cases of idiopathic CAVD,1 similar to the heritable form in patients with NOTCH1 mutations.2 We have previously reported that valves from idiopathic cases are enriched for the cell junction protein, cadherin-11 (CDH11),3 and that valve interstitial cells from Notch1 +/− mice overexpress CDH11.4 Thus, we speculate that CDH11 is downstream of NOTCH1 receptor dysfunction and may be a hallmark in both heritable and idiopathic CAVD.5 Here, we sought to determine whether targeting CDH11, genetically or pharmacologically, would prevent CAVD in Notch1 +/− mice. Notch1 +/+ and Notch1 +/− mice were given a high-fat/high-cholesterol diet starting at 10 weeks. At 4 months, 10 mg/kg SYN0012, a CDH11-blocking antibody, or IgG2a isotype control was administered by intraperitoneal injection once a week for 8 …

Rationale: Pulmonary arterial hypertension is a deadly disease of the pulmonary vasculature for which no disease-modifying therapies exist. Small-vessel stiffening and remodeling are fundamental pathological features of pulmonary arterial hypertension that occur early and drive further endovascular cell dysfunction. Bone marrow (BM)–derived proangiogenic cells (PACs), a specialized heterogeneous subpopulation of myeloid lineage cells, are thought to play an important role in pathogenesis. Objective: To determine whether BM-derived PACs directly contributed to experimental pulmonary hypertension (PH) by promoting small-vessel stiffening through 5-HT 2B (serotonin 2B receptor)–mediated signaling. Methods and Results: We performed BM transplants using transgenic donor animals expressing diphtheria toxin secondary to activation of an endothelial-specific tamoxifen-inducible Cre and induced experimental PH using hypoxia with SU5416 to enhance endovascular injury and ablated BM-derived PACs, after which we measured right ventricular systolic pressures in a closed-chest procedure. BM-derived PAC lineage tracing was accomplished by transplanting BM from transgenic donor animals with fluorescently labeled hematopoietic cells and treating mice with a 5-HT 2B antagonist. BM-derived PAC ablation both prevented and reversed experimental PH with SU5416-enhanced endovascular injury, reducing the number of muscularized pulmonary arterioles and normalizing arteriole stiffness as measured by atomic force microscopy. Similarly, treatment with a pharmacological antagonist of 5-HT 2B also prevented experimental PH, reducing the number and stiffness of muscularized pulmonary arterioles. PACs accelerated pulmonary microvascular endothelial cell injury response in vitro, and the presence of BM-derived PACs significantly correlated with stiffer pulmonary arterioles in pulmonary arterial hypertension patients and mice with experimental PH. RNA sequencing of BM-derived PACs showed that 5-HT 2B antagonism significantly altered biologic pathways regulating cell proliferation, locomotion and migration, and cytokine production and response to cytokine stimulus. Conclusions: Together, our findings illustrate that BM-derived PACs directly contribute to experimental PH with SU5416-enhanced endovascular injury by mediating small-vessel stiffening and remodeling in a 5-HT 2B signaling–dependent manner.

Like its systemic counterpart, pulmonary arterial hypertension (PAH) results in remodeling and fibrosis of the right ventricle as it attempts to adapt to the increased pressure overload. This eventually leads to contractile dysfunction, and RV failure is the primary cause of death in PAH patients. The G protein-coupled thromboxane/prostanoid (TP) receptor is expressed in vascular smooth muscle and immune cells, and is upregulated in cardiomyocytes following PAH. Activation of the cardiac TP receptor increases cardiomyocyte intracellular calcium and can lead to arrhythmias. We previously reported that oral treatment with the TP receptor antagonist ifetroban prevents RV fibrosis in a mouse pressure overload model of PAH. Here, we investigate the effects of TP receptor activation on calcium handling in RV cardiomyocytes and explore treatment of established RV remodeling with ifetroban, compared with prevention. Fixed pressure overload of the RV via pulmonary arterial banding (PAB) caused an increase in contractility and resting (end-diastolic) intracellular calcium in individual cardiomyocytes after 3 weeks; this occurred in conjunction with RV dilation, fibrosis, and stiffness. Surprisingly, total calcium content of the sarcoplasmic reticulum was increased following PAB. Antagonism with ifetroban decreased formation of fibrosis in a time-dependent manner. However, treatment with antagonist following establishment of RV fibrosis still prevented the cardiomyocyte increase in end-diastolic calcium. This suggests a multi-factorial contribution of the TP receptor in the RV response to PAH. Further studies continue to analyze changes in calcium-dependent signaling, as well as the contribution of the cardiomyocyte TP receptor to both fibrosis and calcium handling.