Induced Endothelial Cell Cycle Arrest Prevents Arteriovenous Malformations in Hereditary Hemorrhagic Telangiectasia.

BACKGROUND: Distinct endothelial cell cycle states (early G1 versus late G1) provide different "windows of opportunity" to enable the differential expression of genes that regulate venous versus arterial specification, respectively. Endothelial cell cycle control and arteriovenous identities are disrupted in vascular malformations including arteriovenous shunts, the hallmark of hereditary hemorrhagic telangiectasia (HHT). To date, the mechanistic link between endothelial cell cycle regulation and the development of arteriovenous malformations (AVMs) in HHT is not known. METHODS: We used BMP (bone morphogenetic protein) 9/10 blocking antibodies and endothelial-specific deletion of activin A receptor like type 1 (Alk1) to induce HHT in Fucci (fluorescent ubiquitination-based cell cycle indicator) 2 mice to assess endothelial cell cycle states in AVMs. We also assessed the therapeutic potential of inducing endothelial cell cycle G1 state in HHT to prevent AVMs by repurposing the Food and Drug Administration-approved CDK (cyclin-dependent kinase) 4/6 inhibitor (CDK4/6i) palbociclib. RESULTS: We found that endothelial cell cycle state and associated gene expressions are dysregulated during the pathogenesis of vascular malformations in HHT. We also showed that palbociclib treatment prevented AVM development induced by BMP9/10 inhibition and Alk1 genetic deletion. Mechanistically, endothelial cell late G1 state induced by palbociclib modulates the expression of genes regulating arteriovenous identity, endothelial cell migration, metabolism, and VEGF-A (vascular endothelial growth factor A) and BMP9 signaling that collectively contribute to the prevention of vascular malformations. CONCLUSIONS: This study provides new insights into molecular mechanisms leading to HHT by defining how endothelial cell cycle is dysregulated in AVMs because of BMP9/10 and Alk1 signaling deficiencies, and how restoration of endothelial cell cycle control may be used to treat AVMs in patients with HHT.

Obesogenic diet disrupts tissue-specific mitochondrial gene signatures in the artery and capillary endothelium.

Endothelial cells (ECs) adapt to the unique needs of their resident tissue and metabolic perturbations, such as obesity. We sought to understand how obesity affects EC metabolic phenotypes, specifically mitochondrial gene expression. We investigated the mesenteric and adipose endothelium because these vascular beds have distinct roles in lipid homeostasis. Initially, we performed bulk RNA sequencing on ECs from mouse adipose and mesenteric vasculatures after a normal chow (NC) diet or high-fat diet (HFD) and found higher mitochondrial gene expression in adipose ECs compared with mesenteric ECs in both NC and HFD mice. Next, we performed single-cell RNA sequencing and categorized ECs as arterial, capillary, venous, or lymphatic. We found mitochondrial genes to be enriched in adipose compared with mesentery under NC conditions in artery and capillary ECs. After HFD, these genes were decreased in adipose ECs, becoming like mesenteric ECs. Transcription factor analysis revealed that peroxisome proliferator-activated receptor-γ (PPAR-γ) had high specificity in NC adipose artery and capillary ECs. These findings were recapitulated in single-nuclei RNA-sequencing data from human visceral adipose. The sum of these findings suggests that mesenteric and adipose arterial ECs metabolize lipids differently, and the transcriptional phenotype of the vascular beds converges in obesity due to downregulation of PPAR-γ in adipose artery and capillary ECs.NEW & NOTEWORTHY Using bulk and single-cell RNA sequencing on endothelial cells from adipose and mesentery, we found that an obesogenic diet induces a reduction in adipose endothelial oxidative phosphorylation gene expression, resulting in a phenotypic convergence of mesenteric and adipose endothelial cells. Furthermore, we found evidence that PPAR-γ drives this phenotypic shift. Mining of human data sets segregated based on body mass index supported these findings. These data point to novel mechanisms by which obesity induces endothelial dysfunction.

Clonal Hematopoiesis in Clinical and Experimental Heart Failure With Preserved Ejection Fraction.

BACKGROUND: Clonal hematopoiesis (CH), which results from an array of nonmalignant driver gene mutations, can lead to altered immune cell function and chronic disease, and has been associated with worse outcomes in patients with heart failure (HF) with reduced ejection fraction. However, the role of CH in the prognosis of HF with preserved ejection fraction (HFpEF) has been understudied. This study aimed to characterize CH in patients with HFpEF and elucidate its causal role in a murine model. METHODS: Using a panel of 20 candidate CH driver genes and a variant allele fraction cutoff of 0.5%, ultradeep error-corrected sequencing identified CH in a cohort of 81 patients with HFpEF (mean age, 71±6 years; ejection fraction, 63±5%) and 36 controls without a diagnosis of HFpEF (mean age, 74±7 years; ejection fraction, 61.5±8%). CH was also evaluated in a replication cohort of 59 individuals with HFpEF. RESULTS: Compared with controls, there was an enrichment of TET2-mediated CH in the HFpEF patient cohort (12% versus 0%, respectively; P=0.02). In the HFpEF cohort, patients with CH exhibited exacerbated diastolic dysfunction in terms of E/e' (14.9 versus 11.7, respectively; P=0.0096) and E/A (1.69 versus 0.89, respectively; P=0.0206) compared with those without CH. The association of CH with exacerbated diastolic dysfunction was corroborated in a validation cohort of individuals with HFpEF. In accordance, patients with HFpEF, an age ≥70 years, and CH exhibited worse prognosis in terms of 5-year cardiovascular-related hospitalization rate (hazard ratio, 5.06; P=0.042) compared with patients with HFpEF and an age ≥70 years without CH. To investigate the causal role of CH in HFpEF, nonconditioned mice underwent adoptive transfer with Tet2-wild-type or Tet2-deficient bone marrow and were subsequently subjected to a high-fat diet/L-NAME (Nω-nitro-l-arginine methyl ester) combination treatment to induce features of HFpEF. This model of Tet2-CH exacerbated cardiac hypertrophy by heart weight/tibia length and cardiomyocyte size, diastolic dysfunction by E/e' and left ventricular end-diastolic pressure, and cardiac fibrosis compared with the Tet2-wild-type condition. CONCLUSIONS: CH is associated with worse heart function and prognosis in patients with HFpEF, and a murine experimental model of Tet2-mediated CH displays greater features of HFpEF.

Isolation of Highly Purified and Viable Retinal Endothelial Cells.

The neonatal mouse retinal vascularization model has been widely used in the vascular biology field to investigate mechanisms of angiogenesis and arterial-venous fate specification during blood vessel formation and maturation. Recent advances in next-generation sequencing can further elucidate mechanisms of blood vessel formation and remodeling in this, as well as other, vascular development models. However, an optimized method for isolating retinal endothelial cells that limits tissue digestion-induced cell damage is required for next-generation sequencing applications. In this study, we established a method for isolating neonatal retinal endothelial cells that optimizes cell viability and purity. The CD31+/CD45- endothelial cell population was fluorescence-activated cell sorting (FACS)-isolated from digested postnatal retinas, found to be highly enriched for endothelial cell gene expression, and exhibited no change in viability for 60 min post-FACS. Thus, this method for retinal endothelial cell isolation is compatible with next-generation sequencing applications. Combining this isolation method with next-generation sequencing will enable further delineation of mechanisms underlying vascular development and maturation.

Adapter Protein RapGEF1 Is Required for ERK1/2 Signaling in Response to Elevated Phosphate in Vascular Smooth Muscle Cells.

The sodium-dependent phosphate transporter, SLC20A1, is required for elevated inorganic phosphate (Pi) induced vascular smooth muscle cell (VSMC) matrix mineralization and phenotype transdifferentiation. Recently, elevated Pi was shown to induce ERK1/2 phosphorylation through SLC20A1 by Pi uptake-independent functions in VSMCs, suggesting a cell signaling response to elevated Pi. Previous studies identified Rap1 guanine nucleotide exchange factor (RapGEF1) as an SLC20A1-interacting protein and RapGEF1 promotes ERK1/2 phosphorylation through Rap1 activation. In this study, we tested the hypothesis that RapGEF1 is a critical component of the SLC20A1-mediated Pi-induced ERK1/2 phosphorylation pathway. Co-localization of SLC20A1 and RapGEF1, knockdown of RapGEF1 with siRNA, and small molecule inhibitors of Rap1, B-Raf, and Mek1/2 were investigated. SLC20A1 and RapGEF1 were co-localized in peri-membranous structures in VSMCs. Knockdown of RapGEF1 and small molecule inhibitors against Rap1, B-Raf, and Mek1/2 eliminated elevated Pi-induced ERK1/2 phosphorylation. Knockdown of RapGEF1 inhibited SM22α mRNA expression and blocked elevated Pi-induced downregulation of SM22α mRNA. Together, these data suggest that RapGEF1 is required for SLC20A1-mediated elevated Pi signaling through a Rap1/B-Raf/Mek1/2 cell signaling pathway, thereby promoting ERK1/2 phosphorylation and inhibiting SM22α gene expression in VSMCs.

Phosphate uptake-independent signaling functions of the type III sodium-dependent phosphate transporter, PiT-1, in vascular smooth muscle cells.

Vascular calcification (VC) is prevalent in chronic kidney disease and elevated serum inorganic phosphate (Pi) is a recognized risk factor. The type III sodium-dependent phosphate transporter, PiT-1, is required for elevated Pi-induced osteochondrogenic differentiation and matrix mineralization in vascular smooth muscle cells (VSMCs). However, the molecular mechanism(s) by which PiT-1 promotes these processes is unclear. In the present study, we confirmed that the Pi concentration required to induce osteochondrogenic differentiation and matrix mineralization of mouse VSMCs was well above that required for maximal Pi uptake, suggesting a signaling function of PiT-1 that was independent of Pi transport. Elevated Pi-induced signaling via ERK1/2 phosphorylation was abrogated in PiT-1 deficient VSMCs, but could be rescued by wild-type (WT) and a Pi transport-deficient PiT-1 mutant. Furthermore, both WT and transport-deficient PiT-1 mutants promoted osteochondrogenic differentiation as measured by decreased SM22α and increased osteopontin mRNA expression. Finally, compared to vector alone, expression of transport-deficient PiT-1 mutants promoted VSMC matrix mineralization, but not to the extent observed with PiT-1 WT. These data suggest that both Pi uptake-dependent and -independent functions of PiT-1 are important for VSMC processes mediating vascular calcification.

Sodium-dependent phosphate cotransporters and phosphate-induced calcification of vascular smooth muscle cells: redundant roles for PiT-1 and PiT-2.

OBJECTIVE: Elevated serum phosphate has emerged as a major risk factor for vascular calcification. The sodium-dependent phosphate cotransporter, PiT-1, was previously shown to be required for phosphate-induced osteogenic differentiation and calcification of cultured human vascular smooth muscle cells (VSMCs), but its importance in vascular calcification in vivo and the potential role of its homologue, PiT-2, have not been determined. We investigated the in vivo requirement for PiT-1 in vascular calcification using a mouse model of chronic kidney disease and the potential compensatory role of PiT-2 using in vitro knockdown and overexpression strategies. APPROACH AND RESULTS: Mice with targeted deletion of PiT-1 in VSMCs were generated (PiT-1(Δsm)). PiT-1 mRNA levels were undetectable, whereas PiT-2 mRNA levels were increased 2-fold in the vascular aortic media of PiT-1(Δsm) compared with PiT-1(flox/flox) control. When arterial medial calcification was induced in PiT-1(Δsm) and PiT-1(flox/flox) by chronic kidney disease followed by dietary phosphate loading, the degree of aortic calcification was not different between genotypes, suggesting compensation by PiT-2. Consistent with this possibility, VSMCs isolated from PiT-1(Δsm) mice had no PiT-1 mRNA expression, increased PiT-2 mRNA levels, and no difference in sodium-dependent phosphate uptake or phosphate-induced matrix calcification compared with PiT-1(flox/flox) VSMCs. Knockdown of PiT-2 decreased phosphate uptake and phosphate-induced calcification of PiT-1(Δsm) VSMCs. Furthermore, overexpression of PiT-2 restored these parameters in human PiT-1-deficient VSMCs. CONCLUSIONS: PiT-2 can mediate phosphate uptake and calcification of VSMCs in the absence of PiT-1. Mechanistically, PiT-1 and PiT-2 seem to serve redundant roles in phosphate-induced calcification of VSMCs.

Disruption of the Uty epigenetic regulator locus in hematopoietic cells phenocopies the profibrotic attributes of Y chromosome loss in heart failure.

Heart failure affects millions of people worldwide, with men exhibiting a higher incidence than women. Our previous work has shown that mosaic loss of the Y chromosome (LOY) in leukocytes is causally associated with an increased risk for heart failure. Here, we show that LOY macrophages from the failing hearts of humans with dilated cardiomyopathy exhibit widespread changes in gene expression that correlate with cardiac fibroblast activation. Moreover, we identify the ubiquitously transcribed t et ratricopeptide Y-linked (Uty) gene in leukocytes as a causal locus for an accelerated progression of heart failure in male mice with LOY. We demonstrate that Uty disruption leads to epigenetic alterations in both monocytes and macrophages, increasing the propensity of differentiation into profibrotic macrophages. Treatment with a transforming growth factor-ß-neutralizing antibody prevented the cardiac pathology associated with Uty deficiency in leukocytes. These findings shed light on the mechanisms that contribute to the higher incidence of heart failure in men.

Stress produces aversion and potentiates cocaine reward by releasing endogenous dynorphins in the ventral striatum to locally stimulate serotonin reuptake.

Activation of the dynorphin/κ-opioid receptor (KOR) system by repeated stress exposure or agonist treatment produces place aversion, social avoidance, and reinstatement of extinguished cocaine place preference behaviors by stimulation of p38α MAPK, which subsequently causes the translocation of the serotonin transporter (SERT, SLC6A4) to the synaptic terminals of serotonergic neurons. In the present study we extend those findings by showing that stress-induced potentiation of cocaine conditioned place preference occurred by a similar mechanism. In addition, SERT knock-out mice did not show KOR-mediated aversion, and selective reexpression of SERT by lentiviral injection into the dorsal raphe restored the prodepressive effects of KOR activation. Kinetic analysis of several neurotransporters demonstrated that repeated swim stress exposure selectively increased the V(max) but not K(m) of SERT without affecting dopamine transport or the high-capacity, low-affinity transporters. Although the serotonergic neurons in the dorsal raphe project throughout the forebrain, a significant stress-induced increase in cell-surface SERT expression was only evident in the ventral striatum, and not in the dorsal striatum, hippocampus, prefrontal cortex, amygdala, or dorsal raphe. Stereotaxic microinjections of the long-lasting KOR antagonist norbinaltorphimine demonstrated that local KOR activation in the nucleus accumbens, but not dorsal raphe, mediated this stress-induced increase in ventral striatal surface SERT expression. Together, these results support the hypothesis that stress-induced activation of the dynorphin/KOR system produces a transient increase in serotonin transport locally in the ventral striatum that may underlie some of the adverse consequences of stress exposure, including the potentiation of the rewarding effects of cocaine.

Fibroblast growth factor 23 is not associated with and does not induce arterial calcification.

Elevated fibroblast growth factor 23 (FGF23) is associated with cardiovascular disease in patients with chronic kidney disease. As a potential mediating mechanism, FGF23 induces left ventricular hypertrophy; however, its role in arterial calcification is less clear. In order to study this, we quantified coronary artery and thoracic aorta calcium by computed tomography in 1501 patients from the Chronic Renal Insufficiency Cohort (CRIC) study within a median of 376 days (interquartile range 331-420 days) of baseline. Baseline plasma FGF23 was not associated with the prevalence or severity of coronary artery calcium after multivariable adjustment. In contrast, higher serum phosphate levels were associated with prevalence and severity of coronary artery calcium, even after adjustment for FGF23. Neither FGF23 nor serum phosphate were consistently associated with thoracic aorta calcium. We could not detect mRNA expression of FGF23 or its coreceptor, klotho, in human or mouse vascular smooth muscle cells, or normal or calcified mouse aorta. Whereas elevated phosphate concentrations induced calcification in vitro, FGF23 had no effect on phosphate uptake or phosphate-induced calcification regardless of phosphate concentration or even in the presence of soluble klotho. Thus, in contrast to serum phosphate, FGF23 is not associated with arterial calcification and does not promote calcification experimentally. Hence, phosphate and FGF23 promote cardiovascular disease through distinct mechanisms.

Obesity accelerates endothelial-to-mesenchymal transition in adipose tissues of mice and humans.

Introduction: Vascular dysfunction and chronic inflammation are characteristics of obesity-induced adipose tissue dysfunction. Proinflammatory cytokines can drive an endothelial-to-mesenchymal transition (EndoMT), where endothelial cells undergo a phenotypic switch to mesenchymal-like cells that are pro-inflammatory and pro-fibrotic. In this study, we sought to determine whether obesity can promote EndoMT in adipose tissue. Methods: Mice in which endothelial cells are lineage-traced with eYFP were fed a high-fat/high-sucrose (HF/HS) or Control diet for 13, 26, and 52 weeks, and EndoMT was assessed in adipose tissue depots as percentage of CD45-CD31-Acta2+ mesenchymal-like cells that were eYFP +. EndoMT was also assessed in human adipose endothelial cells through cell culture assays and by the analysis of single cell RNA sequencing datasets obtained from the visceral adipose tissues of obese individuals. Results: Quantification by flow cytometry showed that mice fed a HF/HS diet display a time-dependent increase in EndoMT over Control diet in subcutaneous adipose tissue (+3.0%, +2.6-fold at 13 weeks; +10.6%, +3.2-fold at 26 weeks; +11.8%, +2.9-fold at 52 weeks) and visceral adipose tissue (+5.5%, +2.3-fold at 13 weeks; +20.7%, +4.3-fold at 26 weeks; +25.7%, +4.8-fold at 52 weeks). Transcriptomic analysis revealed that EndoMT cells in visceral adipose tissue have enriched expression of genes associated with inflammatory and TGFß signaling pathways. Human adipose-derived microvascular endothelial cells cultured with TGF-ß1, IFN-γ, and TNF-α exhibited a similar upregulation of EndoMT markers and induction of inflammatory response pathways. Analysis of single cell RNA sequencing datasets from visceral adipose tissue of obese patients revealed a nascent EndoMT sub-cluster of endothelial cells with reduced PECAM1 and increased ACTA2 expression, which was also enriched for inflammatory signaling genes and other genes associated with EndoMT. Discussion: These experimental and clinical findings show that chronic obesity can accelerate EndoMT in adipose tissue. We speculate that EndoMT is a feature of adipose tissue dysfunction that contributes to local inflammation and the systemic metabolic effects of obesity..

SLC20a1/PiT-1 is required for chorioallantoic placental morphogenesis.

The placenta mediates the transport of nutrients, such as inorganic phosphate (Pi), between the maternal and fetal circulatory systems. The placenta itself also requires high levels of nutrient uptake as it develops to provide critical support for fetal development. This study aimed to determine placental Pi transport mechanisms using in vitro and in vivo models. We observed that Pi (P33) uptake in BeWo cells is sodium dependent and that SLC20A1/Slc20a1 is the most highly expressed placental sodium-dependent transporter in mouse (microarray), human cell line (RT-PCR) and term placenta (RNA-seq), supporting that normal growth and maintenance of the mouse and human placenta requires SLC20A1/Slc20a1. Slc20a1 wild-type (Slc20a1+/+) and knockout (Slc20a1-/-) mice were produced through timed intercrosses and displayed yolk sac angiogenesis failure as expected at E10.5. E9.5 tissues were analyzed to test whether placental morphogenesis requires Slc20a1. At E9.5, the developing placenta was reduced in size in Slc20a1-/-. Multiple structural abnormalities were also observed in the Slc20a1-/-chorioallantois. We determined that monocarboxylate transporter 1 protein (MCT1+) cells were reduced in developing Slc20a1-/-placenta, confirming that Slc20a1 loss reduced trophoblast syncytiotrophoblast 1 (SynT-I) coverage. Next, we examined the cell type-specific Slc20a1 expression and SynT molecular pathways in silico and identified Notch/Wnt as a pathway of interest that regulates trophoblast differentiation. We further observed that specific trophoblast lineages express Notch/Wnt genes that associate with endothelial cell tip-and-stalk cell markers. In conclusion, our findings support that Slc20a1 mediates the symport of Pi into SynT cells, providing critical support for their differentiation and angiogenic mimicry function at the developing maternal-fetal interface.

Connexin 37 sequestering of activated-ERK in the cytoplasm promotes p27-mediated endothelial cell cycle arrest.

Connexin37-mediated regulation of cell cycle modulators and, consequently, growth arrest lack mechanistic understanding. We previously showed that arterial shear stress up-regulates Cx37 in endothelial cells and activates a Notch/Cx37/p27 signaling axis to promote G1 cell cycle arrest, and this is required to enable arterial gene expression. However, how induced expression of a gap junction protein, Cx37, up-regulates cyclin-dependent kinase inhibitor p27 to enable endothelial growth suppression and arterial specification is unclear. Herein, we fill this knowledge gap by expressing wild-type and regulatory domain mutants of Cx37 in cultured endothelial cells expressing the Fucci cell cycle reporter. We determined that both the channel-forming and cytoplasmic tail domains of Cx37 are required for p27 up-regulation and late G1 arrest. Mechanistically, the cytoplasmic tail domain of Cx37 interacts with, and sequesters, activated ERK in the cytoplasm. This then stabilizes pERK nuclear target Foxo3a, which up-regulates p27 transcription. Consistent with previous studies, we found this Cx37/pERK/Foxo3a/p27 signaling axis functions downstream of arterial shear stress to promote endothelial late G1 state and enable up-regulation of arterial genes.

Connexin 43-mediated neurovascular interactions regulate neurogenesis in the adult brain subventricular zone.

The subventricular zone (SVZ) is the largest neural stem cell (NSC) niche in the adult brain; herein, the blood-brain barrier is leaky, allowing direct interactions between NSCs and endothelial cells (ECs). Mechanisms by which direct NSC-EC interactions in the adult SVZ control NSC behavior are unclear. We found that Cx43 is highly expressed by SVZ NSCs and ECs, and its deletion in either leads to increased NSC proliferation and neuroblast generation, suggesting that Cx43-mediated NSC-EC interactions maintain NSC quiescence. This is further supported by single-cell RNA sequencing and in vitro studies showing that ECs control NSC proliferation by regulating expression of genes associated with NSC quiescence and/or activation in a Cx43-dependent manner. Cx43 mediates these effects in a channel-independent manner involving its cytoplasmic tail and ERK activation. Such insights inform adult NSC regulation and maintenance aimed at stem cell therapies for neurodegenerative disorders.

Importance of clonal hematopoiesis in heart failure.

Heart failure is prevalent in the elderly population. Inflammatory processes can contribute to the progression of heart failure by altering the balance of tissue healing and pathological remodeling during the injury response. New findings show that aging can alter immune cell phenotypes through the process of clonal hematopoiesis. This condition results from acquired somatic DNA mutations in specific driver genes that give rise to clonal expansions of mutant hematopoietic cells with overactive inflammatory properties. Recent clinical and experimental studies have shown that clonal hematopoiesis is prevalent in heart failure patients and associated with poor prognosis. In this review, we summarize current evidence that associates clonal hematopoiesis with the progression of heart failure. We further describe the mechanistic links between clonal hematopoiesis and the pro-inflammatory responses that can contribute to pathological outcomes in the heart. Finally, we provide perspectives on future research directions in the area of clonal hematopoiesis and heart failure.

Endothelial cell cycle state determines propensity for arterial-venous fate.

During blood vessel development, endothelial cells become specified toward arterial or venous fates to generate a circulatory network that provides nutrients and oxygen to, and removes metabolic waste from, all tissues. Arterial-venous specification occurs in conjunction with suppression of endothelial cell cycle progression; however, the mechanistic role of cell cycle state is unknown. Herein, using Cdh5-CreERT2;R26FUCCI2aR reporter mice, we find that venous endothelial cells are enriched for the FUCCI-Negative state (early G1) and BMP signaling, while arterial endothelial cells are enriched for the FUCCI-Red state (late G1) and TGF-ß signaling. Furthermore, early G1 state is essential for BMP4-induced venous gene expression, whereas late G1 state is essential for TGF-ß1-induced arterial gene expression. Pharmacologically induced cell cycle arrest prevents arterial-venous specification defects in mice with endothelial hyperproliferation. Collectively, our results show that distinct endothelial cell cycle states provide distinct windows of opportunity for the molecular induction of arterial vs. venous fate.

Hematopoietic loss of Y chromosome leads to cardiac fibrosis and heart failure mortality.

Hematopoietic mosaic loss of Y chromosome (mLOY) is associated with increased risk of mortality and age-related diseases in men, but the causal and mechanistic relationships have yet to be established. Here, we show that male mice reconstituted with bone marrow cells lacking the Y chromosome display increased mortality and age-related profibrotic pathologies including reduced cardiac function. Cardiac macrophages lacking the Y chromosome exhibited polarization toward a more fibrotic phenotype, and treatment with a transforming growth factor ß1-neutralizing antibody ameliorated cardiac dysfunction in mLOY mice. A prospective study revealed that mLOY in blood is associated with an increased risk for cardiovascular disease and heart failure-associated mortality. Together, these results indicate that hematopoietic mLOY causally contributes to fibrosis, cardiac dysfunction, and mortality in men.

Directed Differentiation of Hemogenic Endothelial Cells from Human Pluripotent Stem Cells.

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial cells, which line blood vessel lumens, from human pluripotent stem cells is crucial for a multitude of tissue engineering and regeneration applications. In vivo, primordial endothelial cells are derived from the mesodermal lineage and are specified toward specific subtypes, including arterial, venous, capillary, hemogenic, and lymphatic. Hemogenic endothelial cells are of particular interest because, during development, they give rise to hematopoietic stem and progenitor cells, which then generate all blood lineages throughout life. Thus, creating a system to generate hemogenic endothelial cells in vitro would provide an opportunity to study endothelial-to-hematopoietic transition, and may lead to ex vivo production of human blood products and reduced reliance on human donors. While several protocols exist for the derivation of progenitor and primordial endothelial cells, generation of well-characterized hemogenic endothelial cells from human stem cells has not been described. Here, a method for the derivation of hemogenic endothelial cells from human embryonic stem cells in approximately 1 week is presented: a differentiation protocol with primitive streak cells formed in response to GSK3ß inhibitor (CHIR99021), then mesoderm lineage induction mediated by bFGF, followed by primordial endothelial cell development promoted by BMP4 and VEGF-A, and finally hemogenic endothelial cell specification induced by retinoic acid. This protocol yields a well-defined population of hemogenic endothelial cells that can be used to further understand their molecular regulation and endothelial-to-hematopoietic transition, which has the potential to be applied to downstream therapeutic applications.

Isolation of Murine Retinal Endothelial Cells for Next-Generation Sequencing.

Recent improvements in next-generation sequencing have advanced researchers' knowledge of molecular and cellular biology, with several studies revealing novel paradigms in vascular biology. Applying these methods to models of vascular development requires the optimization of cell isolation techniques from embryonic and postnatal tissues. Cell yield, viability, and purity all need to be maximal to obtain accurate and reproducible results from next-generation sequencing approaches. The neonatal mouse retinal vascularization model is used by researchers to study mechanisms of vascular development. Researchers have used this model to investigate mechanisms of angiogenesis and arterial-venous fate specification during blood vessel formation and maturation. Applying next-generation sequencing techniques to study the retinal vascular development model requires optimization of a method for the isolation of retinal endothelial cells that maximizes cell yield, viability, and purity. This protocol describes a method for murine retinal tissue isolation, digestion, and purification using fluorescence-activated cell sorting (FACS). The results indicate that the FACS-purified CD31+/CD45- endothelial cell population is highly enriched for endothelial cell gene expression and exhibits no change in viability for 60 min post-FACS. Included are representative results of next-generation sequencing approaches on endothelial cells isolated using this method, including bulk RNA sequencing and single-cell RNA sequencing, demonstrating that this method for retinal endothelial cell isolation is compatible with next-generation sequencing applications. This method of retinal endothelial cell isolation will allow for advanced sequencing techniques to reveal novel mechanisms of vascular development.

The Cell Surface Receptors Ror1/2 Control Cardiac Myofibroblast Differentiation.

Background A hallmark of heart failure is cardiac fibrosis, which results from the injury-induced differentiation response of resident fibroblasts to myofibroblasts that deposit extracellular matrix. During myofibroblast differentiation, fibroblasts progress through polarization stages of early proinflammation, intermediate proliferation, and late maturation, but the regulators of this progression are poorly understood. Planar cell polarity receptors, receptor tyrosine kinase-like orphan receptor 1 and 2 (Ror1/2), can function to promote cell differentiation and transformation. In this study, we investigated the role of the Ror1/2 in a model of heart failure with emphasis on myofibroblast differentiation. Methods and Results The role of Ror1/2 during cardiac myofibroblast differentiation was studied in cell culture models of primary murine cardiac fibroblast activation and in knockout mouse models that underwent transverse aortic constriction surgery to induce cardiac injury by pressure overload. Expression of Ror1 and Ror2 were robustly and exclusively induced in fibroblasts in hearts after transverse aortic constriction surgery, and both were rapidly upregulated after early activation of primary murine cardiac fibroblasts in culture. Cultured fibroblasts isolated from Ror1/2 knockout mice displayed a proinflammatory phenotype indicative of impaired myofibroblast differentiation. Although the combined ablation of Ror1/2 in mice did not result in a detectable baseline phenotype, transverse aortic constriction surgery led to the death of all mice by day 6 that was associated with myocardial hyperinflammation and vascular leakage. Conclusions Together, these results show that Ror1/2 are essential for the progression of myofibroblast differentiation and for the adaptive remodeling of the heart in response to pressure overload.