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.

Pulsed Electrolysis Promotes CO2 Reduction to Ethanol on Heterostructured Cu2 O/Ag Catalysts.

The electrochemical conversion of carbon dioxide (CO2 ) into ethanol with high added value has attracted increasing attention. Here, an efficient catalyst with abundant Cu2 O/Ag interfaces for ethanol production under pulsed CO2 electrolysis is reported, which is composed of Cu2 O hollow nanospheres loaded with Ag nanoparticles (named as se-Cu2 O/Ag). The CO2 -to-ethanol Faradaic efficiency is prominently improved to 46.3% at a partial current density up to 417 mA cm-2 under pulsed electrolysis conditions in a neutral flow cell, notably outperforming conventional Cu catalysts during static electrolysis. In situ spectroscopy reveals the stabilized Cu+ species of se-Cu2 O/Ag during pulsed electrolysis and the enhanced adsorbed CO intermediate (* CO)coverage on the heterostructured catalyst. Density functional theory (DFT) calculations further confirm that the Cu2 O/Ag heterostructure stabilizes the * CO intermediate and promotes the coupling of * CO and adsorbed CH intermediate (* CH). Meanwhile, the stable Cu+ species under pulsed electrolysis favor the hydrogenation of adsorbed HCCOH intermediate (* HCCOH) to adsorbed HCCHOH intermediate (* HCCHOH) on the pathway to ethanol. The synergistic effect between the enhanced generation of * CO on Cu2 O/Ag and regenerated Cu+ species under pulsed electrolysis steers the reaction pathway toward ethanol. This work provides some insights into selective ethanol production from CO2 electroreduction via combined catalyst design and non-steady state electrolysis.

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.

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.

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.

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.

Enhanced CO Affinity on Cu Facilitates CO2 Electroreduction toward Multi-Carbon Products.

Electrochemical CO2 reduction reaction (CO2 RR) is a promising strategy for waste CO2 utilization and intermittent electricity storage. Herein, it is reported that bimetallic Cu/Pd catalysts with enhanced *CO affinity show a promoted CO2 RR performance for multi-carbon (C2+) production under industry-relevant high current density. Especially, bimetallic Cu/Pd-1% catalyst shows an outstanding CO2 -to-C2+ conversion with 66.2% in Faradaic efficiency (FE) and 463.2 mA cm-2 in partial current density. An increment in the FE ratios of C2+ products to CO  for Cu/Pd-1% catalyst further illuminates a preferable C2+ production. In situ Raman spectra reveal that the atop-bounded CO is dominated by low-frequency band CO on Cu/Pd-1% that leads to C2+ products on bimetallic catalysts, in contrast to the majority of high-frequency band CO on Cu that favors the formation of CO. Density function theory calculation confirms that bimetallic Cu/Pd catalyst enhances the *CO adsorption and reduces the Gibbs free energy of the CC coupling process, thereby favoring the formation of C2+ products.

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.

Identification and function of miRNA-mRNA interaction pairs during lateral root development of hemi-parasitic Santalum album L. seedlings.

Sandalwood (Santalum album L.) is a hemi-parasitic tree species famous for its santalol and santalene, which are extracted from its heartwood and roots. The ability to understand root functionality within its branched root system would benefit the regulation of sandalwood growth and enhance the commercial value of sandalwood. Phenotypic and anatomical evidence in this study indicated that seed germination stage 4 (SG4) seemed pivotal for lateral root (LR) morphogenesis. Small RNA (sRNA) high-throughput sequencing of root tissues at three sub-stages of SG4 (lateral root primordia initiation (LRPI), lateral root primordia development (LRPD), and lateral root primordia emergence (LRPE)) was performed to identify microRNAs (miRNAs) associated with LR development. A total of 135 miRNAs, including 70 differentially expressed miRNAs (DEMs), were screened. Ten DEMs were selected to investigate transcript abundance in different organs or developmental stages. Among 100 negative DEM-mRNA interaction pairs, four targets (Sa-miR166m_2, 408d, 858a, and novel_Sa-miR8) were selected for studying cleavage sites by 5' RLM-RACE validation. The expression mode of the four miRNA-mRNA pairs was investigated after indole-3-acetic acid (IAA) treatment. IAA enhanced the abundance of homeobox-leucine-zipper protein 32 (HOX32), laccase 12 (LAC12), myeloblastosis86 (MYB86), and pectin methylesterase inhibitor6 (PMEI6) target transcripts by reducing the expression of Sa-miR166m_2, 408d, 858a, and novel_Sa-miR8 in the first 10 min. A schematic model of miRNA-regulated LR development is proposed for this hemi-parasitic species. This novel genetic information for improving sandalwood root growth and development may allow for the cultivation of fast-growing and high-yielding plantations.

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.

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.

Red Cell Distribution Width, Unlike Neutrophil Lymphocyte Ratio Is Unable to Accurately Gauge Enhanced Systemic Inflammation Associated With Panoramic Imaged Carotid Plaque.

INTRODUCTION: We have previously shown that panoramic X-rays (PXs) demonstrating calcified carotid artery atheromas (CCAA) are associated with increased systemic inflammation demonstrating increased neutrophil lymphocyte ratios (NLRs), a validated risk indicator of fatal myocardial infarctions arising from coronary artery atherosclerosis. Using this same cohort of patients (with minor adjustments because of missing data), we sought to determine if a like association existed between PXs evidencing CCAA and elevated red blood cell distribution width (RDW) given conflicting data as its reliability relative to NLR as a biologic marker of system inflammation. We hypothesized that CCAAs on PXs would simultaneously be associated with both increased NLR and RDW. MATERIALS AND METHODS: Investigators implemented a cross-sectional study design. Study sample consisted of patient medical records and PXs of white men ≥ 55 years. Two groups (N = 50 each) were constituted, one with atheromas (CCAA+) and without atheromas (CCAA-). The predictor variable was CCAA+ and outcome variables were NLR and RDW. Bootstrapping analysis was employed to analyze the differences in mean NLRs and RDWs between groups since the data was not normally distributed. Statistical significance determined to be ≤ 0.05 for all tests. The Medical Center's Institutional Review Board approved the research protocol. RESULTS: A study group of 50 CCAA+ men (mean age 71; range 58-89 years) demonstrated a mean NLR of 2.98 ± 1.38 and an RDW of 13.21 ± 0.85. A control group of 50 CCAA- males (mean age 70 range; 55-91 years) evidenced a mean NLR of 2.38 ± 0.77 and an RDW of 13.16 ± 0.77. Bootstrapping comparison of NLR values evidenced significant (P = 0.008) difference (95% confidence interval of difference of mean: - 0.4272, 0.4384; observed effect size: 0.579) between groups; however, there was no significant difference in RDW values between the groups. Furthermore, logistic regression modeling demonstrated that for a one unit increase in NLR the odds of being CCAA+ (vs. CCAA-) increases by a factor of 1.659. CONCLUSION: The existence of CCAA seen on PXs of elderly white men is associated with significantly (P = 0.008) elevated NLR values but is not associated with increases in RDW.

Elevated White Blood Cell Count Resultant Atherogenesis is Associated With Panoramic-Imaged Carotid Plaque.

PURPOSE: Atherosclerotic plaques develop as a result of a low-grade, chronic, systemic inflammatory response to the injury of endothelial cells arising from lipid deposition within the intima. Increased white blood cell count (WBCC) is both a validated "biologic marker" of the extent of this inflammatory process and a key participant in the development of subsequent atherosclerotic ischemic heart disease manifesting as myocardial infarction. We sought to determine if calcified carotid artery plaque (CCAP) on a panoramic image (PI), also a validated risk indicator of future myocardial infarction, is associated with increased WBCC. PATIENTS AND METHODS: We retrospectively evaluated the PI and medical records of White male military veterans aged 55 years and older treated by a VA dental service. Established were 2 cohorts of patients, 50 having plaques (CCAP+) and 50 without plaques (CCAP-). Predictor variable was CCAP+; outcome variable was WBCC. Bootstrapping analysis determined the differences in mean WBCCs between groups. Statistical significance set at ≤ 0.05. RESULTS: The study group, (mean age 74; range 59 to 91 years) demonstrated a mean WBCC of 8,062 per mm3. The control group, (mean age 72 range; 57 to 94) evidenced a mean WBCC of 7,058 per mm3. Bootstrapping analysis of WBCC values demonstrated a significant (P = .012) difference (95% confidence interval of difference of mean, -806, 742; observed effect size, 1004) between groups. CONCLUSIONS: The presence of CCAP demonstrated on PIs of older Caucasian men is associated with elevated WBCC. Concomitant presence of CCAP on PI and increased WBCC (≥7,800 per mm3) amplifies need for medical consultation before intravenous anesthesia and maxillofacial surgical procedures.