Continuous single-cell imaging of blood generation from haemogenic endothelium.

Despite decades of research, the identity of the cells generating the first haematopoietic cells in mammalian embryos is unknown. Indeed, whether blood cells arise from mesodermal cells, mesenchymal progenitors, bipotent endothelial-haematopoietic precursors or haemogenic endothelial cells remains controversial. Proximity of endothelial and blood cells at sites of embryonic haematopoiesis, as well as their similar gene expression, led to the hypothesis of the endothelium generating blood. However, owing to lacking technology it has been impossible to observe blood cell emergence continuously at the single-cell level, and the postulated existence of haemogenic endothelial cells remains disputed. Here, using new imaging and cell-tracking methods, we show that embryonic endothelial cells can be haemogenic. By continuous long-term single-cell observation of mouse mesodermal cells generating endothelial cell and blood colonies, it was possible to detect haemogenic endothelial cells giving rise to blood cells. Living endothelial and haematopoietic cells were identified by simultaneous detection of morphology and multiple molecular and functional markers. Detachment of nascent blood cells from endothelium is not directly linked to asymmetric cell division, and haemogenic endothelial cells are specified from cells already expressing endothelial markers. These results improve our understanding of the developmental origin of mammalian blood and the potential generation of haematopoietic stem cells from embryonic stem cells.

Deep learning on resting electrocardiogram to identify impaired heart rate recovery.

Background and Objective: Postexercise heart rate recovery (HRR) is an important indicator of cardiac autonomic function and abnormal HRR is associated with adverse outcomes. We hypothesized that deep learning on resting electrocardiogram (ECG) tracings may identify individuals with impaired HRR. Methods: We trained a deep learning model (convolutional neural network) to infer HRR based on resting ECG waveforms (HRRpred) among UK Biobank participants who had undergone exercise testing. We examined the association of HRRpred with incident cardiovascular disease using Cox models, and investigated the genetic architecture of HRRpred in genome-wide association analysis. Results: Among 56,793 individuals (mean age 57 years, 51% women), the HRRpred model was moderately correlated with actual HRR (r = 0.48, 95% confidence interval [CI] 0.47-0.48). Over a median follow-up of 10 years, we observed 2060 incident diabetes mellitus (DM) events, 862 heart failure events, and 2065 deaths. Higher HRRpred was associated with lower risk of DM (hazard ratio [HR] 0.79 per 1 standard deviation change, 95% CI 0.76-0.83), heart failure (HR 0.89, 95% CI 0.83-0.95), and death (HR 0.83, 95% CI 0.79-0.86). After accounting for resting heart rate, the association of HRRpred with incident DM and all-cause mortality were similar. Genetic determinants of HRRpred included known heart rate, cardiac conduction system, cardiomyopathy, and metabolic trait loci. Conclusion: Deep learning-derived estimates of HRR using resting ECG independently associated with future clinical outcomes, including new-onset DM and all-cause mortality. Inferring postexercise heart rate response from a resting ECG may have potential clinical implications and impact on preventive strategies warrants future study.

Cohort design and natural language processing to reduce bias in electronic health records research.

Electronic health record (EHR) datasets are statistically powerful but are subject to ascertainment bias and missingness. Using the Mass General Brigham multi-institutional EHR, we approximated a community-based cohort by sampling patients receiving longitudinal primary care between 2001-2018 (Community Care Cohort Project [C3PO], n = 520,868). We utilized natural language processing (NLP) to recover vital signs from unstructured notes. We assessed the validity of C3PO by deploying established risk models for myocardial infarction/stroke and atrial fibrillation. We then compared C3PO to Convenience Samples including all individuals from the same EHR with complete data, but without a longitudinal primary care requirement. NLP reduced the missingness of vital signs by 31%. NLP-recovered vital signs were highly correlated with values derived from structured fields (Pearson r range 0.95-0.99). Atrial fibrillation and myocardial infarction/stroke incidence were lower and risk models were better calibrated in C3PO as opposed to the Convenience Samples (calibration error range for myocardial infarction/stroke: 0.012-0.030 in C3PO vs. 0.028-0.046 in Convenience Samples; calibration error for atrial fibrillation 0.028 in C3PO vs. 0.036 in Convenience Samples). Sampling patients receiving regular primary care and using NLP to recover missing data may reduce bias and maximize generalizability of EHR research.

The Rho-GTPase cdc42 regulates neural progenitor fate at the apical surface.

Stem cell persistence into adulthood requires self-renewal from early developmental stages. In the developing mouse brain, only apical progenitors located at the ventricle are self-renewing, whereas basal progenitors gradually deplete. However, nothing is known about the mechanisms regulating the fundamental difference between these progenitors. Here we show that the conditional deletion of the small Rho-GTPase cdc42 at different stages of neurogenesis in mouse telencephalon results in an immediate increase in basal mitoses. Whereas cdc42-deficient progenitors have normal cell cycle length, orientation of cell division and basement membrane contact, the apical location of the Par complex and adherens junctions are gradually lost, leading to an increasing failure of apically directed interkinetic nuclear migration. These cells then undergo mitoses at basal positions and acquire the fate of basal progenitors. Thus, cdc42 has a crucial role at the apical pole of progenitors, thereby regulating the position of mitoses and cell fate.

Inhibition of the Keap1-Nrf2 protein-protein interaction protects retinal cells and ameliorates retinal ischemia-reperfusion injury.

The Nrf2-Keap1 pathway regulates transcription of a wide array of antioxidant and cytoprotective genes and offers critical protection against oxidative stress. This pathway has demonstrated benefit for a variety of retinal conditions. Retinal ischemia plays a pivotal role in many vision threatening diseases. Retinal vascular endothelial cells are an important participant in ischemic injury. In this setting, Nrf2 provides a protective pathway via amelioration of oxidative stress and inflammation. In this study, we investigated a potent small molecule inhibitor of the Nrf2-Keap1 protein-protein interaction (PPI), CPUY192018, for its therapeutic potential in retinal cells and retinal ischemia-reperfusion injury. In human retinal endothelial cells (HREC), treatment with CPUY192018 increased Nrf2 protein levels and nuclear translocation, stimulated Nrf2-ARE-induced transcriptional capacity, and induced Nrf2 target gene expression. Furthermore, CPUY192018 protected HREC against oxidative stress and inflammatory activation. CPUY192018 also activated Nrf2 and suppressed inflammatory response in macrophages. In the retinal ischemia-reperfusion (I/R) model, administration of CPUY192018 induced Nrf2 target gene activation in the retina. Both systemic and topical treatment with CPUY192018 rescued visual function after ischemia-reperfusion injury. Taken together, these findings indicate that small molecule Keap1-Nrf2 PPI inhibitors can activate the Nrf2 pathway in the retina and provide protection against retinal ischemic and inflammatory injury, suggesting Keap1-Nrf2 PPI inhibition in the treatment of retinal conditions.

Loss of the transcription factor RBPJ induces disease-promoting properties in brain pericytes.

Sufficient vascular supply is indispensable for brain development and function, whereas dysfunctional blood vessels are associated with human diseases such as vascular malformations, stroke or neurodegeneration. Pericytes are capillary-associated mesenchymal cells that limit vascular permeability and protect the brain by preserving blood-brain barrier integrity. Loss of pericytes has been linked to neurodegenerative changes in genetically modified mice. Here, we report that postnatal inactivation of the Rbpj gene, encoding the transcription factor RBPJ, leads to alteration of cell identity markers in brain pericytes, increases local TGFß signalling, and triggers profound changes in endothelial behaviour. These changes, which are not mimicked by pericyte ablation, imperil vascular stability and induce the acquisition of pathological landmarks associated with cerebral cavernous malformations. In adult mice, loss of Rbpj results in bigger stroke lesions upon ischemic insult. We propose that brain pericytes can acquire deleterious properties that actively enhance vascular lesion formation and promote pathogenic processes.

MAPK phosphatase-1 represents a novel anti-inflammatory target of glucocorticoids in the human endothelium.

Glucocorticoids are well-established anti-inflammatory drugs thought to mainly act by inhibition of proinflammatory transcription factors like NF-kappaB. In recent years, however, transcription factor-independent mechanisms of glucocorticoid action have been proposed, namely the influence on MAPK pathways. Here we identify MAPK phosphatase-1 (MKP-1) as a pivotal mediator of the anti-inflammatory action of glucocorticoids in the human endothelium. We applied dexamethasone (Dex) to TNF-alpha-activated human endothelial cells and used the adhesion molecule E-selectin as inflammatory read-out parameter. Dex is known to reduce the expression of E-selectin, which is largely regulated by NF-kappaB. Here, we communicate that Dex at low concentrations (1-100 nM) markedly attenuates E-selectin expression without affecting NF-kappaB. Importantly, Dex is able to increase the expression of MKP-1, which causes an inactivation of TNF-alpha-induced p38 MAPK and mediates inhibition of E-selectin expression. In endothelial MKP-1(-/-) cells differentiated from MKP-1(-/-) embryonic stem cells and in MKP-1-silenced human endothelial cells, Dex did not inhibit TNF-alpha-evoked E-selectin expression. Thus, our findings introduce MKP-1 as a novel and crucial mediator of the anti-inflammatory action of glucocorticoids at low concentrations in the human endothelium and highlight MKP-1 as an important and promising anti-inflammatory drug target.

Spatiotemporal endothelial cell - pericyte association in tumors as shown by high resolution 4D intravital imaging.

Endothelial cells and pericytes are integral cellular components of the vasculature with distinct interactive functionalities. To study dynamic interactions between these two cells we created two transgenic animal lines. A truncated eNOS (endothelial nitric oxide synthase) construct was used as a GFP tag for endothelial cell evaluation and an inducible Cre-lox recombination, under control of the Pdgfrb (platelet derived growth factor receptor beta) promoter, was created for pericyte assessment. Also, eNOStag-GFP animals were crossed with the already established Cspg4-DsRed mice expressing DsRed fluorescent protein in pericytes. For intravital imaging we used tumors implanted in the dorsal skinfold of these transgenic animals. This setup allowed us to study time and space dependent complexities, such as distribution, morphology, motility, and association between both vascular cell types in all angiogenetic stages, without the need for additional labeling. Moreover, as fluorescence was still clearly detectable after fixation, it is possible to perform comparative histology following intravital evaluation. These transgenic mouse lines form an excellent model to capture collective and individual cellular and subcellular endothelial cell - pericyte dynamics and will help answer key questions on the cellular and molecular relationship between these two cells.

Pericytes regulate VEGF-induced endothelial sprouting through VEGFR1.

Pericytes adhere to the abluminal surface of endothelial tubules and are required for the formation of stable vascular networks. Defective endothelial cell-pericyte interactions are frequently observed in diseases characterized by compromised vascular integrity such as diabetic retinopathy. Many functional properties of pericytes and their exact role in the regulation of angiogenic blood vessel growth remain elusive. Here we show that pericytes promote endothelial sprouting in the postnatal retinal vasculature. Using genetic and pharmacological approaches, we show that the expression of vascular endothelial growth factor receptor 1 (VEGFR1) by pericytes spatially restricts VEGF signalling. Angiogenic defects caused by pericyte depletion are phenocopied by intraocular injection of VEGF-A or pericyte-specific inactivation of the murine gene encoding VEGFR1. Our findings establish that pericytes promote endothelial sprouting, which results in the loss of side branches and the enlargement of vessels when pericyte function is impaired or lost.

Dynamics of endothelial cell behavior in sprouting angiogenesis.

The vertebrate body contains an extensive blood vessel network that forms, with a few exceptions, by endothelial sprouting from the existing vasculature. This process, termed angiogenesis, involves complex and highly dynamic interactions between endothelial cells and their environment. Pro-angiogenic signals, such as VEGF, promote endothelial motility, filopodia extension and proliferation, and, together with Notch signaling, controls whether specific endothelial cells become lead tip cells or trailing stalk cells. Sprouts then convert into endothelial tubules and form connections with other vessels, which requires the local suppression of motility and the formation of new cell-cell junctions. We here review the dynamics of angiogenesis in the context of key molecules and pathways controlling tip cell selection, sprouting and the formation of new vessels.

Induction of pluripotency in human cord blood unrestricted somatic stem cells.

OBJECTIVE: Generation of induced pluripotent stem (iPS) cells from human cord blood (CB)-derived unrestricted somatic stem cells and evaluation of their molecular signature and differentiation potential in comparison to human embryonic stem cells. MATERIALS AND METHODS: Unrestricted somatic stem cells isolated from human CB were reprogrammed to iPS cells using retroviral expression of the transcription factors OCT4, SOX2, KLF4, and C-MYC. The reprogrammed cells were analyzed morphologically, by quantitative reverse transcription polymerase chain reaction, genome-wide microRNA and methylation profiling, and gene expression microarrays, as well as in their pluripotency potential by in vivo teratoma formation in severe combined immunodeficient mice and in vitro differentiation. RESULTS: CB iPS cells are very similar to human embryonic stem cells morphologically, at their molecular signature, and in their differentiation potential. CONCLUSIONS: Human CB-derived unrestricted somatic stem cells offer an attractive source of cells for generation of iPS cells. Our findings open novel perspectives to generate human leukocyte antigen-matched pluripotent stem cell banks based on existing CB banks. Besides the obvious relevance of a second-generation CB iPS cell bank for pharmacological and toxicological testing, its application for autologous or allogenic regenerative cell transplantation appears feasible.