Cell-programmed nutrient partitioning in the tumour microenvironment.

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.

Guidelines on the use of sex and gender in cardiovascular research.

In cardiovascular research, sex and gender have not typically been considered in research design and reporting until recently. This has resulted in clinical research findings from which not only all women, but also gender-diverse individuals have been excluded. The resulting dearth of data has led to a lack of sex- and gender-specific clinical guidelines and raises serious questions about evidence-based care. Basic research has also excluded considerations of sex. Including sex and/or gender as research variables not only has the potential to improve the health of society overall now, but it also provides a foundation of knowledge on which to build future advances. The goal of this guidelines article is to provide advice on best practices to include sex and gender considerations in study design, as well as data collection, analysis, and interpretation to optimally establish rigor and reproducibility needed to inform clinical decision-making and improve outcomes. In cardiovascular physiology, incorporating sex and gender is a necessary component when optimally designing and executing research plans. The guidelines serve as the first guidance on how to include sex and gender in cardiovascular research. We provide here a beginning path toward achieving this goal and improve the ability of the research community to interpret results through a sex and gender lens to enable comparison across studies and laboratories, resulting in better health for all.

Circulating prostate cancer cells have differential resistance to fluid shear stress-induced cell death.

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.

Targeting 5-HT2B Receptor Signaling Prevents Border Zone Expansion and Improves Microstructural Remodeling After Myocardial Infarction.

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-HT2B) 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-HT2B inhibition on outcomes after MI and characterized the histological and microstructural changes involved in tissue remodeling. Inducible 5-HT2B ablation driven by Tcf21MCM and PostnMCM was used to evaluate resident cardiac fibroblast- and myofibroblast-specific contributions of 5-HT2B, respectively. RNA sequencing was used to motivate subsequent in vitro analyses to explore cardiac fibroblast phenotype. RESULTS: 5-HT2B antagonism preserved cardiac structure and function by facilitating a less fibrotic scar, indicated by decreased scar thickness and decreased border zone area. 5-HT2B 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-HT2B from Tcf21-lineage resident cardiac fibroblasts and saw similar improvements to the pharmacological approach. Tamoxifen-inducible Cre-mediated ablation of 5-HT2B 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-HT2B expression in either cardiac fibroblasts or activated myofibroblasts directly contributes to excessive scar formation, resulting in adverse remodeling and impaired cardiac function after MI.

Evaluation of early bilateral ovariectomy in mice as a model of left heart disease.

Postmenopausal women tend to have worse cardiovascular outcomes in a manner that is associated with osteoporosis severity. In this study, we performed the first evaluation of the left ventricle and aortic valve phenotype of ovariectomized mice aged on Western diet to 1 yr. Disease was monitored in vivo using echocardiography and dual X-ray absorptiometry imaging and ex vivo using quantitative histological and immunostaining analysis. Mice had decreased bone mineral density in response to ovariectomy and increased fat mass in response to Western diet. Ovariectomized mice had a significantly increased left ventricle mass compared with control animals, absent of fibrosis. There was a slight increase in aortic valve peak velocity but no change in mean pressure gradient across the valve in the ovariectomy group. There was no evidence of leaflet hypertrophy, fibrosis, or calcification. This model of ovariectomy may present a novel method of studying left ventricle hypertrophy in female populations but does not have a phenotype for the study of aortic stenosis. This is particularly useful as it does not require genetic manipulation or drug treatment and more faithfully mimics aging, high-cholesterol diet, and postmenopausal osteoporosis that many female patients experience potentially resulting in a more translatable disease model.NEW & NOTEWORTHY 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.

Sclerostin ablation prevents aortic valve stenosis in mice.

The objective of this study was to test the hypothesis that targeting sclerostin would accelerate the progression of aortic valve stenosis. Sclerostin (mouse gene, Sost) is a secreted glycoprotein that acts as a potent regulator of bone remodeling. Antibody therapy targeting sclerostin is approved for osteoporosis but results from a stage III clinical trial showed multiple off-target cardiovascular effects. Wild-type (WT, Sost+/+) and Sost-gene knockout-expression (Null, Sost-/-) mice were generated and maintained to 12 mo of age on a high-cholesterol diet to induce aortic valve stenosis. Mice were examined by echocardiography, histology, and RNAseq. Immortalized valve interstitial cells were developed from each genotype for in vitro studies. Null mice developed a bone overgrowth phenotype, similar to patients with sclerosteosis. Surprisingly, however, WT mice developed hemodynamic signs of aortic valve stenosis, whereas Null mice were unchanged. WT mice had thicker aortic valve leaflets and higher amounts of α-smooth muscle actin, a marker myofibroblast activation and dystrophic calcification, with very little evidence of Runx2 expression, a marker of osteogenic calcification. RNAseq analysis of aortic roots indicated the HOX family of transcription factors was significantly upregulated in Null mice, and valve interstitial cells from Null animals were enriched with Hoxa1, Hoxb2, and Hoxd3 subtypes with downregulated Hoxa7. In addition, Null valve interstitial cells were shown to be less contractile than their WT counterparts. Contrary to our hypothesis, sclerostin targeting prevented hallmarks of aortic valve stenosis and indicates that targeted antibody treatments for osteoporosis may be beneficial for these patients regarding aortic stenosis.NEW & NOTEWORTHY 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.

Loss of talin in cardiac fibroblasts results in augmented ventricular cardiomyocyte hypertrophy in response to pressure overload.

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-/-;Tln1CF-/-) 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-/-;Tln1CF-/- mice, whereas there was no increase in interstitial fibrosis. Systolic blood pressure was increased in the female Tln2-/-;Tln1CF-/- mice after ANG II infusion compared with the Tln2-/- mice. However, there was no increase in cardiac hypertrophy in the Tln2-/-;Tln1CF-/- 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.

Impaired macrophage trafficking and increased helper T-cell recruitment with loss of cadherin-11 in atherosclerotic immune response.

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.

Mouse Models of Heart Failure with Preserved or Reduced Ejection Fraction.

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.

Macrophages Promote Aortic Valve Cell Calcification and Alter STAT3 Splicing.

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 STAT3ß 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.

Loss of flow responsive Tie1 results in ImpairedAortic valve remodeling.

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.

Bone Marrow-Derived Proangiogenic Cells Mediate Pulmonary Arteriole Stiffening via Serotonin 2B Receptor Dependent Mechanism.

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-HT2B (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-HT2B 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-HT2B 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-HT2B 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-HT2B signaling-dependent manner.

Adaptive immune cells in calcific aortic valve disease.

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.

Cadherin-11 as a regulator of valve myofibroblast mechanobiology.

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)-ß1 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-ß1 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-ß1 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.

Mechanobiology of myofibroblast adhesion in fibrotic cardiac disease.

Fibrotic cardiac disease, a leading cause of death worldwide, manifests as substantial loss of function following maladaptive tissue remodeling. Fibrosis can affect both the heart valves and the myocardium and is characterized by the activation of fibroblasts and accumulation of extracellular matrix. Valvular interstitial cells and cardiac fibroblasts, the cell types responsible for maintenance of cardiac extracellular matrix, are sensitive to changing mechanical environments, and their ability to sense and respond to mechanical forces determines both normal development and the progression of disease. Recent studies have uncovered specific adhesion proteins and mechano-sensitive signaling pathways that contribute to the progression of fibrosis. Integrins form adhesions with the extracellular matrix, and respond to changes in substrate stiffness and extracellular matrix composition. Cadherins mechanically link neighboring cells and are likely to contribute to fibrotic disease propagation. Finally, transition to the active myofibroblast phenotype leads to maladaptive tissue remodeling and enhanced mechanotransductive signaling, forming a positive feedback loop that contributes to heart failure. This Commentary summarizes recent findings on the role of mechanotransduction through integrins and cadherins to perpetuate mechanically induced differentiation and fibrosis in the context of cardiac disease.

Matrigel Mattress: A Method for the Generation of Single Contracting Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

RATIONALE: The lack of measurable single-cell contractility of human-induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs) currently limits the utility of hiPSC-CMs for evaluating contractile performance for both basic research and drug discovery. OBJECTIVE: To develop a culture method that rapidly generates contracting single hiPSC-CMs and allows quantification of cell shortening with standard equipment used for studying adult CMs. METHODS AND RESULTS: Single hiPSC-CMs were cultured for 5 to 7 days on a 0.4- to 0.8-mm thick mattress of undiluted Matrigel (mattress hiPSC-CMs) and compared with hiPSC-CMs maintained on a control substrate (<0.1-mm thick 1:60 diluted Matrigel, control hiPSC-CMs). Compared with control hiPSC-CMs, mattress hiPSC-CMs had more rod-shape morphology and significantly increased sarcomere length. Contractile parameters of mattress hiPSC-CMs measured with video-based edge detection were comparable with those of freshly isolated adult rabbit ventricular CMs. Morphological and contractile properties of mattress hiPSC-CMs were consistent across cryopreserved hiPSC-CMs generated independently at another institution. Unlike control hiPSC-CMs, mattress hiPSC-CMs display robust contractile responses to positive inotropic agents, such as myofilament calcium sensitizers. Mattress hiPSC-CMs exhibit molecular changes that include increased expression of the maturation marker cardiac troponin I and significantly increased action potential upstroke velocity because of a 2-fold increase in sodium current (INa). CONCLUSIONS: The Matrigel mattress method enables the rapid generation of robustly contracting hiPSC-CMs and enhances maturation. This new method allows quantification of contractile performance at the single-cell level, which should be valuable to disease modeling, drug discovery, and preclinical cardiotoxicity testing.

Notch1 Mutation Leads to Valvular Calcification Through Enhanced Myofibroblast Mechanotransduction.

OBJECTIVE: Calcific aortic valve disease (CAVD) is a significant cardiovascular disorder, and controversy exists as to whether it is primarily a dystrophic or osteogenic process in vivo. In this study, we sought to clarify the mechanism of CAVD by assessing a genetic mutation, Notch1 heterozygosity, which leads to CAVD with 100% penetrance in humans. APPROACH AND RESULTS: Murine immortalized Notch1(+/-) aortic valve interstitial cells (AVICs) were isolated and expanded in vitro. Molecular signaling of wild-type and Notch1(+/-) AVICs were compared to identify changes in pathways that have been linked to CAVD-transforming growth factor-ß1/bone morphogenetic protein, mitogen-activated protein kinase, and phosphoinositide 3-kinase/protein kinase B-and assessed for calcification potential. Additionally, AVIC mechanobiology was studied in a physiologically relevant, dynamic mechanical environment (10% cyclic strain) to investigate differences in responses between the cell types. We found that Notch1(+/-) AVICs resembled a myofibroblast-like phenotype expressing higher amounts of cadherin-11, a known mediator of dystrophic calcification, and decreased Runx2, a known osteogenic marker. We determined that cadherin-11 expression is regulated by Akt activity, and inhibition of Akt phosphorylation significantly reduced cadherin-11 expression. Moreover, in the presence of cyclic strain, Notch1(+/-) AVICs exhibited significantly upregulated phosphorylation of Akt at Ser473 and smooth muscle α-actin expression, indicative of a fully activated myofibroblast. Finally, these Notch1-mediated alterations led to enhanced dystrophic calcific nodule formation. CONCLUSIONS: This study presents novel insights in our understanding of Notch1-mediated CAVD by demonstrating that the mutation leads to AVICs that are fully activated myofibroblasts, resulting in dystrophic, but not osteogenic, calcification.

Identification of a common Wnt-associated genetic signature across multiple cell types in pulmonary arterial hypertension.

Understanding differences in gene expression that increase risk for pulmonary arterial hypertension (PAH) is essential to understanding the molecular basis for disease. Previous studies on patient samples were limited by end-stage disease effects or by use of nonadherent cells, which are not ideal to model vascular cells in vivo. These studies addressed the hypothesis that pathological processes associated with PAH may be identified via a genetic signature common across multiple cell types. Expression array experiments were initially conducted to analyze cell types at different stages of vascular differentiation (mesenchymal stromal and endothelial) derived from PAH patient-specific induced pluripotent stem (iPS) cells. Molecular pathways that were altered in the PAH cell lines were then compared with those in fibroblasts from 21 patients, including those with idiopathic and heritable PAH. Wnt was identified as a target pathway and was validated in vitro using primary patient mesenchymal and endothelial cells. Taken together, our data suggest that the molecular lesions that cause PAH are present in all cell types evaluated, regardless of origin, and that stimulation of the Wnt signaling pathway was a common molecular defect in both heritable and idiopathic PAH.