Inhibition of endothelial histone deacetylase 2 shifts endothelial-mesenchymal transitions in cerebral arteriovenous malformation models.

Cerebral arteriovenous malformations (AVMs) are the most common vascular malformations worldwide and the leading cause of hemorrhagic strokes that may result in crippling neurological deficits. Here, using newly generated mouse models, we uncovered that cerebral endothelial cells (ECs) acquired mesenchymal markers and caused vascular malformations. Interestingly, we found that limiting endothelial histone deacetylase 2 (HDAC2) prevented cerebral ECs from undergoing mesenchymal differentiation and reduced cerebral AVMs. We found that endothelial expression of HDAC2 and enhancer of zeste homolog 1 (EZH1) was altered in cerebral AVMs. These alterations changed the abundance of H4K8ac and H3K27me in the genes regulating endothelial and mesenchymal differentiation, which caused the ECs to acquire mesenchymal characteristics and form AVMs. Together, this investigation demonstrated that the induction of HDAC2 altered specific histone modifications, which resulted in mesenchymal characteristics in the ECs and cerebral AVMs. The results provided insight into the epigenetic impact on AVMs.

CDK1 inhibition reduces osteogenesis in endothelial cells in vascular calcification.

Vascular calcification is a severe complication of cardiovascular diseases. Previous studies demonstrated that endothelial lineage cells transitioned into osteoblast-like cells and contributed to vascular calcification. Here, we found that inhibition of cyclin-dependent kinase (CDK) prevented endothelial lineage cells from transitioning to osteoblast-like cells and reduced vascular calcification. We identified a robust induction of CDK1 in endothelial cells (ECs) in calcified arteries and showed that EC-specific gene deletion of CDK1 decreased the calcification. We found that limiting CDK1 induced E-twenty-six specific sequence variant 2 (ETV2), which was responsible for blocking endothelial lineage cells from undergoing osteoblast differentiation. We also found that inhibition of CDK1 reduced vascular calcification in a diabetic mouse model. Together, the results highlight the importance of CDK1 suppression and suggest CDK1 inhibition as a potential option for treating vascular calcification.

Intrahepatic Transcriptomics Differentiate Advanced Fibrosis and Clinical Outcomes in Adults With Fontan Circulation.

BACKGROUND: The molecular mechanisms underlying Fontan-associated liver disease (FALD) remain largely unknown. OBJECTIVES: This study aimed to assess intrahepatic transcriptomic differences among patients with FALD according to the degree of liver fibrosis and clinical outcomes. METHODS: This retrospective cohort study included adults with the Fontan circulation. Baseline clinical, laboratory, imaging, and hemodynamic data as well as a composite clinical outcome (CCO) were extracted from medical records. Patients were classified into early or advanced fibrosis. RNA was isolated from formalin-fixed paraffin-embedded liver biopsy samples; RNA libraries were constructed with the use of an rRNA depletion method and sequenced on an Illumina Novaseq 6000. Differential gene expression and gene ontology analyses were performed with the use of DESeq2 and Metascape. RESULTS: A total of 106 patients (48% male, median age 31 years [IQR: 11.3 years]) were included. Those with advanced fibrosis had higher B-type natriuretic peptide levels and Fontan, mean pulmonary artery, and capillary wedge pressures. The CCO was present in 23 patients (22%) and was not predicted by advanced liver fibrosis, right ventricular morphology, presence of aortopulmonary collaterals, or Fontan pressures on multivariable analysis. Samples with advanced fibrosis had 228 upregulated genes compared with early fibrosis. Samples with the CCO had 894 upregulated genes compared with those without the CCO. A total of 136 upregulated genes were identified in both comparisons and were enriched in cellular response to cytokine stimulus or oxidative stress, VEGFA-VEGFR2 signaling pathway, TGF-ß signaling pathway, and vasculature development. CONCLUSIONS: Patients with FALD and advanced fibrosis or the CCO exhibited upregulated genes related to inflammation, congestion, and angiogenesis.

Two-step regulation by matrix Gla protein in brown adipose cell differentiation.

OBJECTIVE: Bone morphogenetic protein (BMP) signaling is intricately involved in adipose tissue development. BMP7 together with BMP4 have been implicated in brown adipocyte differentiation but their roles during development remains poorly specified. Matrix Gla protein (MGP) inhibits BMP4 and BMP7 and is expressed in endothelial and progenitor cells. The objective was to determine the role of MGP in brown adipose tissue (BAT) development. METHODS: The approach included global and cell-specific Mgp gene deletion in combination with RNA analysis, immunostaining, thermogenic activity, and in vitro studies. RESULTS: The results revealed that MGP directs brown adipogenesis at two essential steps. Endothelial-derived MGP limits triggering of white adipogenic differentiation in the perivascular region, whereas MGP derived from adipose cells supports the transition of CD142-expressing progenitor cells to brown adipogenic maturity. Both steps were important to optimize the thermogenic function of BAT. Furthermore, MGP derived from both sources impacted vascular growth. Reduction of MGP in either endothelial or adipose cells expanded the endothelial cell population, suggesting that MGP is a factor in overall plasticity of adipose tissue. CONCLUSION: MGP displays a dual and cell-specific function in BAT, essentially creating a "cellular shuttle" that coordinates brown adipogenic differentiation with vascular growth during development.

Aurora Kinase A Regulates Cell Transitions in Glucocorticoid-Induced Bone Loss.

Glucocorticoid-induced bone loss is a severe and toxic effect of long-term therapy with glucocorticoids, which are currently prescribed for millions of people worldwide. Previous studies have uncovered that glucocorticoids reciprocally converted osteoblast lineage cells into endothelial-like cells to cause bone loss and showed that the modulations of Foxc2 and Osterix were the causative factors that drove this harmful transition of osteoblast lineage cells. Here, we find that the inhibition of aurora kinase A halts this transition and prevents glucocorticoid-induced bone loss. We find that aurora A interacts with the glucocorticoid receptor and show that this interaction is required for glucocorticoids to modulate Foxc2 and Osterix. Together, we identify a new potential approach to counteracting unwanted transitions of osteoblast lineage cells in glucocorticoid treatment and may provide a novel strategy for ameliorating glucocorticoid-induced bone loss.

Role of Matrix Gla Protein in Transforming Growth Factor-ß Signaling and Nonalcoholic Steatohepatitis in Mice.

BACKGROUND & AIMS: Nonalcoholic steatohepatitis (NASH) is a complex disease involving both genetic and environmental factors in its onset and progression. We analyzed NASH phenotypes in a genetically diverse cohort of mice, the Hybrid Mouse Diversity Panel, to identify genes contributing to disease susceptibility. METHODS: A "systems genetics" approach, involving integration of genetic, transcriptomic, and phenotypic data, was used to identify candidate genes and pathways in a mouse model of NASH. The causal role of Matrix Gla Protein (MGP) was validated using heterozygous MGP knockout (Mgp+/-) mice. The mechanistic role of MGP in transforming growth factor-beta (TGF-ß) signaling was examined in the LX-2 stellate cell line by using a loss of function approach. RESULTS: Local cis-acting regulation of MGP was correlated with fibrosis, suggesting a causal role in NASH, and this was validated using loss of function experiments in 2 models of diet-induced NASH. Using single-cell RNA sequencing, Mgp was found to be primarily expressed in hepatic stellate cells and dendritic cells in mice. Knockdown of MGP expression in stellate LX-2 cells led to a blunted response to TGF-ß stimulation. This was associated with reduced regulatory SMAD phosphorylation and TGF-ß receptor ALK1 expression as well as increased expression of inhibitory SMAD6. Hepatic MGP expression was found to be significantly correlated with the severity of fibrosis in livers of patients with NASH, suggesting relevance to human disease. CONCLUSIONS: MGP regulates liver fibrosis and TGF-ß signaling in hepatic stellate cells and contributes to NASH pathogenesis.

GSK3ß Inhibition Ameliorates Atherosclerotic Calcification.

Endothelial-mesenchymal transition (EndMT) drives endothelium to contribute to atherosclerotic calcification. In a previous study, we showed that glycogen synthase kinase-3ß (GSK3ß) inhibition induced ß-catenin and reduced mothers against DPP homolog 1 (SMAD1) in order to redirect osteoblast-like cells towards endothelial lineage, thereby reducing vascular calcification in Matrix Gla Protein (Mgp) deficiency and diabetic Ins2Akita/wt mice. Here, we report that GSK3ß inhibition or endothelial-specific deletion of GSK3ß reduces atherosclerotic calcification. We also find that alterations in ß-catenin and SMAD1 induced by GSK3ß inhibition in the aortas of Apoe-/- mice are similar to Mgp-/- mice. Together, our results suggest that GSK3ß inhibition reduces vascular calcification in atherosclerotic lesions through a similar mechanism to that in Mgp-/- mice.

Cell Transitions Contribute to Glucocorticoid-Induced Bone Loss.

Glucocorticoid-induced bone loss is a toxic effect of long-term therapy with glucocorticoids resulting in a significant increase in the risk of fracture. Here, we find that glucocorticoids reciprocally convert osteoblast-lineage cells into endothelial-like cells. This is confirmed by lineage tracing showing the induction of endothelial markers in osteoblast-lineage cells following glucocorticoid treatment. Functional studies show that osteoblast-lineage cells isolated from glucocorticoid-treated mice lose their capacity for bone formation but simultaneously improve vascular repair. We find that the glucocorticoid receptor directly targets Foxc2 and Osterix, and the modulations of Foxc2 and Osterix drive the transition of osteoblast-lineage cells to endothelial-like cells. Together, the results suggest that glucocorticoids suppress osteogenic capacity and cause bone loss at least in part through previously unrecognized osteoblast-endothelial transitions.

COVID-19 Infection May Drive EC-like Myofibroblasts towards Myofibroblasts to Contribute to Pulmonary Fibrosis.

COVID-19 has an extensive impact on Homo sapiens globally. Patients with COVID-19 are at an increased risk of developing pulmonary fibrosis. A previous study identified that myofibroblasts could be derived from pulmonary endothelial lineage cells as an important cell source that contributes to pulmonary fibrosis. Here, we analyzed publicly available data and showed that COVID-19 infection drove endothelial lineage cells towards myofibroblasts in pulmonary fibrosis of patients with COVID-19. We also discovered a similar differentiation trajectory in mouse lungs after viral infection. The results suggest that COVID-19 infection leads to the development of pulmonary fibrosis partly through the activation of endothelial cell (EC)-like myofibroblasts.

Intrahepatic transcriptomics differentiate advanced fibrosis and clinical outcomes in adults with the Fontan circulation.

Background: The molecular mechanisms underlying Fontan associated liver disease (FALD) remain largely unknown. We aimed to assess intrahepatic transcriptomic differences among patients with FALD according to the degree of liver fibrosis and clinical outcomes. Methods: This retrospective cohort study included adults with the Fontan circulation at the Ahmanson/UCLA Adult Congenital Heart Disease Center. Clinical, laboratory, imaging and hemodynamic data prior to the liver biopsy were extracted from medical records. Patients were classified into early (F1-F2) or advanced fibrosis (F3-F4). RNA was isolated from formalin-fixed paraffin embedded liver biopsy samples; RNA libraries were constructed using rRNA depletion method and sequencing was performed on Illumina Novaseq 6000. Differential gene expression and gene ontology analyses were carried out using DESeq2 and Metascape. Medical records were comprehensively reviewed for a composite clinical outcome which included decompensated cirrhosis, hepatocellular carcinoma, liver transplantation, protein-losing enteropathy, chronic kidney disease stage 4 or higher, or death. Results: Patients with advanced fibrosis had higher serum BNP levels and Fontan, mean pulmonary artery and capillary wedge pressures. The composite clinical outcome was present in 23 patients (22%) and was predicted by age at Fontan, right ventricular morphology and presence of aortopulmonary collaterals on multivariable analysis. Samples with advanced fibrosis had 228 up-regulated genes compared to early fibrosis. Samples with the composite clinical outcome had 894 up-regulated genes compared to those without it. A total of 136 up-regulated genes were identified in both comparisons and these genes were enriched in cellular response to cytokine stimulus, response to oxidative stress, VEGFA-VEGFR2 signaling pathway, TGF-beta signaling pathway, and vasculature development. Conclusions: Patients with FALD and advanced liver fibrosis or the composite clinical outcome exhibit up-regulated genes including pathways related to inflammation, congestion, and angiogenesis. This adds further insight into FALD pathophysiology.

GSK3ß Inhibition Reduced Vascular Calcification in Ins2Akita/+ Mice.

Endothelial-mesenchymal transition (EndMT) drives the endothelium to contribute to vascular calcification in diabetes mellitus. In our previous study, we showed that glycogen synthase kinase-3ß (GSK3ß) inhibition induces ß-catenin and reduces mothers against DPP homolog 1 (SMAD1) to direct osteoblast-like cells toward endothelial lineage, thereby reducing vascular calcification in Matrix Gla Protein (Mgp) deficiency. Here, we report that GSK3ß inhibition reduces vascular calcification in diabetic Ins2Akita/wt mice. Cell lineage tracing reveals that GSK3ß inhibition redirects endothelial cell (EC)-derived osteoblast-like cells back to endothelial lineage in the diabetic endothelium of Ins2Akita/wt mice. We also find that the alterations in ß-catenin and SMAD1 by GSK3ß inhibition in the aortic endothelium of diabetic Ins2Akita/wt mice are similar to Mgp-/- mice. Together, our results suggest that GSK3ß inhibition reduces vascular calcification in diabetic arteries through a similar mechanism to that in Mgp-/- mice.

Regulating the cell shift of endothelial cell-like myofibroblasts in pulmonary fibrosis.

Pulmonary fibrosis is a common and severe fibrotic lung disease with high morbidity and mortality. Recent studies have reported a large number of unwanted myofibroblasts appearing in pulmonary fibrosis, and shown that the sustained activation of myofibroblasts is essential for unremitting interstitial fibrogenesis. However, the origin of these myofibroblasts remains poorly understood. Here, we create new mouse models of pulmonary fibrosis and identify a previously unknown population of endothelial cell (EC)-like myofibroblasts in normal lung tissue. We show that these EC-like myofibroblasts significantly contribute myofibroblasts to pulmonary fibrosis, which is confirmed by single-cell RNA sequencing of human pulmonary fibrosis. Using the transcriptional profiles, we identified a small molecule that redirects the differentiation of EC-like myofibroblasts and reduces pulmonary fibrosis in our mouse models. Our study reveals the mechanistic underpinnings of the differentiation of EC-like myofibroblasts in pulmonary fibrosis and may provide new strategies for therapeutic interventions.

Single-Cell RNA-Seq Identifies Dynamic Cardiac Transition Program from ADCs Induced by Leukemia Inhibitory Factor.

Adipose-derived cells (ADCs) from white adipose tissue are promising stem cell candidates because of their large regenerative reserves and the potential for cardiac regeneration. However, given the heterogeneity of ADC and its unsolved mechanisms of cardiac acquisition, ADC-cardiac transition efficiency remains low. In this study, we explored the heterogeneity of ADCs and the cellular kinetics of 39,432 single-cell transcriptomes along the leukemia inhibitory factor (LIF)-induced ADC-cardiac transition. We identified distinct ADC subpopulations that reacted differentially to LIF when entering the cardiomyogenic program, further demonstrating that ADC-myogenesis is time-dependent and initiates from transient changes in nuclear factor erythroid 2-related factor 2 (Nrf2) signaling. At later stages, pseudotime analysis of ADCs navigated a trajectory with 2 branches corresponding to activated myofibroblast or cardiomyocyte-like cells. Our findings offer a high-resolution dissection of ADC heterogeneity and cell fate during ADC-cardiac transition, thus providing new insights into potential cardiac stem cells.

Progenitor cells from brown adipose tissue undergo neurogenic differentiation.

Multipotent cells derived from white adipose tissue have been shown to differentiate into multiple lineages including neurogenic lineages. However, the high innervation of brown adipose tissue by the sympathetic nervous system suggest it might be a better source of neural precursor cells. To investigate potential differences between white and brown progenitors, we cultured white and brown dedifferentiated fat (wDFAT and brDFAT) cells from mouse and human adipose tissue and compared marker expression of neural precursors, and neuronal and glial cells, using fluorescence-activated cell sorting, bright-field imaging, immunofluorescence, and RNA analysis by qPCR. The results showed that both wDFAT and brDFAT cells had the capacity to generate neuronal and glial-like cells under neurogenic conditions. However, the brDFAT cells exhibited enhanced propensity for neurogenic differentiation. The neurogenic cells were at least in part derived from Adiponectin-expressing cells. TdTomato-expressing cells derived from Adiponectin (Adipoq) Cre ERT2 -tdTomato flox/flox mice gave rise to individual cells and cell clusters with neurogenic characteristics. Moreover, human brDFAT cells demonstrated a similar ability to undergo neurogenic differentiation after treatment with neurogenic medium, as assessed by immunofluorescence and qPCR. Together, our results support that brDFAT cells have ability to undergo neurogenic differentiation.

Novel Structures of Type 1 Glyceraldehyde-3-phosphate Dehydrogenase from Escherichia coli Provide New Insights into the Mechanism of Generation of 1,3-Bisphosphoglyceric Acid.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a highly conserved enzyme involved in the ubiquitous process of glycolysis and presents a loop (residues 208-215 of Escherichia coli GAPDH) in two alternative conformations (I and II). It is uncertain what triggers this loop rearrangement, as well as which is the precise site from which phosphate attacks the thioacyl intermediate precursor of 1,3-bisphosphoglycerate (BPG). To clarify these uncertainties, we determined the crystal structures of complexes of wild-type GAPDH (WT) with NAD and phosphate or G3P, and of essentially inactive GAPDH mutants (C150S, H177A), trapping crystal structures for the thioacyl intermediate or for ternary complexes with NAD and either phosphate, BPG, or G3P. Analysis of these structures reported here lead us to propose that phosphate is located in the "new Pi site" attacks the thioester bond of the thioacyl intermediate to generate 1,3-bisphosphoglyceric acid (BPG). In the structure of the thioacyl intermediate, the mobile loop is in conformation II in subunits O, P, and R, while both conformations coexist in subunit Q. Moreover, only the Q subunit hosts bound NADH. In the R subunit, only the pyrophosphate part of NADH is well defined, and NADH is totally absent from the O and P subunits. Thus, the change in loop conformation appears to occur after NADH is produced, before NADH is released. In addition, two new D-glyceraldehyde-3-phosphate (G3P) binding forms are observed in WT.NAD.G3P and C150A+H177A.NAD.G3P. In summary, this paper improves our understanding of the GAPDH catalytic mechanism, particularly regarding BPG formation.

The Mechanobiology of Endothelial-to-Mesenchymal Transition in Cardiovascular Disease.

Endothelial cells (ECs) lining the cardiovascular system are subjected to a highly dynamic microenvironment resulting from pulsatile pressure and circulating blood flow. Endothelial cells are remarkably sensitive to these forces, which are transduced to activate signaling pathways to maintain endothelial homeostasis and respond to changes in the environment. Aberrations in these biomechanical stresses, however, can trigger changes in endothelial cell phenotype and function. One process involved in this cellular plasticity is endothelial-to-mesenchymal transition (EndMT). As a result of EndMT, ECs lose cell-cell adhesion, alter their cytoskeletal organization, and gain increased migratory and invasive capabilities. EndMT has long been known to occur during cardiovascular development, but there is now a growing body of evidence also implicating it in many cardiovascular diseases (CVD), often associated with alterations in the cellular mechanical environment. In this review, we highlight the emerging role of shear stress, cyclic strain, matrix stiffness, and composition associated with EndMT in CVD. We first provide an overview of EndMT and context for how ECs sense, transduce, and respond to certain mechanical stimuli. We then describe the biomechanical features of EndMT and the role of mechanically driven EndMT in CVD. Finally, we indicate areas of open investigation to further elucidate the complexity of EndMT in the cardiovascular system. Understanding the mechanistic underpinnings of the mechanobiology of EndMT in CVD can provide insight into new opportunities for identification of novel diagnostic markers and therapeutic interventions.

Contributions of the Endothelium to Vascular Calcification.

Vascular calcification (VC) increases morbidity and mortality and constitutes a significant obstacle during percutaneous interventions and surgeries. On a cellular and molecular level, VC is a highly regulated process that involves abnormal cell transitions and osteogenic differentiation, re-purposing of signaling pathways normally used in bone, and even formation of osteoclast-like cells. Endothelial cells have been shown to contribute to VC through a variety of means. This includes direct contributions of osteoprogenitor cells generated through endothelial-mesenchymal transitions in activated endothelium, with subsequent migration into the vessel wall. The endothelium also secretes pro-osteogenic growth factors, such as bone morphogenetic proteins, inflammatory mediators and cytokines in conditions like hyperlipidemia, diabetes, and renal failure. High phosphate levels caused by renal disease have deleterious effects on the endothelium, and induction of tissue non-specific alkaline phosphatase adds to the calcific process. Furthermore, endothelial activation promotes proteolytic destruction of the internal elastic lamina that serves, among other things, as a stabilizer of the endothelium. Appropriate bone mineralization is highly dependent on active angiogenesis, but it is unclear whether the same relationship exists in VC. Through its location facing the vascular lumen, the endothelium is the first to encounter circulating factor and bone marrow-derived cells that might contribute to osteoclast-like versus osteoblast-like cells in the vascular wall. In the same way, the endothelium may be the easiest target to reach with treatments aimed at limiting calcification. This review provides a brief summary of the contributions of the endothelium to VC as we currently know them.

Panoramic imaged carotid atheromas are associated with increased neutrophil count: both validated, independent predictors of near-term myocardial infarction.

OBJECTIVES: Panoramic images (PXs) demonstrating calcified carotid artery atheromas (CCAAs) are associated with heightened risk of near-term myocardial infarction (MI). Elevated neutrophil counts (NC) within normal range 2,500-6,000 per mm3 are likewise associated with future MI signaling the role neutrophils play in the chronic inflammation process underlying coronary artery atherogenesis. We determined if CCAAs on PXs are associated with increased NC. METHODS: Investigators implemented a retrospective study of PXs and accompanying medical records of white males ≥ 65 years treated by a VA dental service. Two groups (N = 60 each) were constituted, one with atheromas (CCAA+) and one without (CCAA-). Predictor variable was CCAA + and outcome variable was NC. Bootstrapping analysis determined the difference in mean NCs between two groups, significance set at ≤0.05. RESULTS: The study group of (CCAA+) (mean age 75.9; range 69-91 years) demonstrated a mean NC of 4,843 per mm3 and control group (CCAA-) (mean age 75.3; range; 66-94) a mean NC of 4,108 per mm3. The difference between the groups was significant (p = 0.0008) (95% CI of difference of mean: -432, 431; observed effect size 736). CONCLUSIONS: CCAAs on PXs of elderly white males are associated with elevated NC; amplifying need for medical consultation prior to invasive dental procedures.

Shifting osteogenesis in vascular calcification.

Transitions between cell fates commonly occur in development and disease. However, reversing an unwanted cell transition in order to treat disease remains an unexplored area. Here, we report a successful process of guiding ill-fated transitions toward normalization in vascular calcification. Vascular calcification is a severe complication that increases the all-cause mortality of cardiovascular disease but lacks medical therapy. The vascular endothelium is a contributor of osteoprogenitor cells to vascular calcification through endothelial-mesenchymal transitions, in which endothelial cells (ECs) gain plasticity and the ability to differentiate into osteoblast-like cells. We created a high-throughput screening and identified SB216763, an inhibitor of glycogen synthase kinase 3 (GSK3), as an inducer of osteoblastic-endothelial transition. We demonstrated that SB216763 limited osteogenic differentiation in ECs at an early stage of vascular calcification. Lineage tracing showed that SB216763 redirected osteoblast-like cells to the endothelial lineage and reduced late-stage calcification. We also found that deletion of GSK3ß in osteoblasts recapitulated osteoblastic-endothelial transition and reduced vascular calcification. Overall, inhibition of GSK3ß promoted the transition of cells with osteoblastic characteristics to endothelial differentiation, thereby ameliorating vascular calcification.