Generating human artery and vein cells from pluripotent stem cells highlights the arterial tropism of Nipah and Hendra viruses.

Stem cell research endeavors to generate specific subtypes of classically defined "cell types." Here, we generate >90% pure human artery or vein endothelial cells from pluripotent stem cells within 3-4 days. We specified artery cells by inhibiting vein-specifying signals and vice versa. These cells modeled viral infection of human vasculature by Nipah and Hendra viruses, which are extraordinarily deadly (∼57%-59% fatality rate) and require biosafety-level-4 containment. Generating pure populations of artery and vein cells highlighted that Nipah and Hendra viruses preferentially infected arteries; arteries expressed higher levels of their viral-entry receptor. Virally infected artery cells fused into syncytia containing up to 23 nuclei, which rapidly died. Despite infecting arteries and occupying ∼6%-17% of their transcriptome, Nipah and Hendra largely eluded innate immune detection, minimally eliciting interferon signaling. We thus efficiently generate artery and vein cells, introduce stem-cell-based toolkits for biosafety-level-4 virology, and explore the arterial tropism and cellular effects of Nipah and Hendra viruses.

Origins and functional differences of blood endothelial cells.

The interests in blood endothelial cells arise from their therapeutic potential in vascular repair and regeneration. Our understanding of blood endothelial cells that exist in the circulation has been evolving significantly from the original concept of endothelial progenitor cells. Many studies have uncovered heterogeneities of blood endothelial subtypes where some cells express both endothelial and hematopoietic antigens, and others possess either mature or immature endothelial markers. Due to the lack of definitive cell marker identities, there have been momentums in the field to adopt a technical-oriented labeling system based on the cells' involvement in postnatal neovascularization and cell culture derivatives. Our review streamlines nomenclatures for blood endothelial subtypes and standardizes understanding of their functional differences. Broadly, we will discuss about myeloid angiogenic cells (MACs), endothelial colony-forming cells (ECFCs), blood outgrowth endothelial cells (BOECs) and circulating endothelial cells (CECs). The strategic location of blood endothelial cells confers them essential roles in supporting physiological processes. MACs exert angiogenic effects through paracrine mechanisms, while ECFCs are recruited to sites of vascular injury to participate directly in new vessel formation. BOECs are an in vitro derivative of ECFCs. CECs are shed into the bloodstream from damaged vessels, hence reflective of endothelial dysfunction. With clarity on the functional attributes of blood endothelial subtypes, we present recent advances in their applications in disease modelling, along with serving as biomarkers of vascular tissue homeostasis.

Endothelial-immune crosstalk contributes to vasculopathy in nonalcoholic fatty liver disease.

The top cause of mortality in patients with nonalcoholic fatty liver disease (NAFLD) is cardiovascular complications. However, mechanisms of NAFLD-associated vasculopathy remain understudied. Here, we show that blood outgrowth endothelial cells (BOECs) from NAFLD subjects exhibit global transcriptional upregulation of chemokines and human leukocyte antigens. In mouse models of diet-induced NAFLD, we confirm heightened endothelial expressions of CXCL12 in the aortas and the liver vasculatures, and increased retention of infiltrated leukocytes within the vessel walls. To elucidate endothelial-immune crosstalk, we performed immunoprofiling by single-cell analysis, uncovering T cell intensification in NAFLD patients. Functionally, treatment with a CXCL12-neutralizing antibody is effective at moderating the enhanced chemotactic effect of NAFLD BOECs in recruiting CD8+ T lymphocytes. Interference with the CXCL12-CXCR4 axis using a CXCR4 antagonist also averts the impact of immune cell transendothelial migration and restores endothelial barrier integrity. Clinically, we detect threefold more circulating damaged endothelial cells in NAFLD patients than in healthy controls. Our work provides insight into the modulation of interactions with effector immune cells to mitigate endothelial injury in NAFLD.

Left Atrial Phasic Function in Older Adults Is Associated with Fibrotic and Low-Grade Inflammatory Pathways.

INTRODUCTION: Concomitant risk factors challenge the mechanistic understanding of cardiac aging. We determined the degree to which the left atrial function could be distinguished by advanced cardiac magnetic resonance (CMR) imaging in older adults and assessed associations between the left atrial function and the plasma biomarkers related to biological aging and cardiovascular disease [serum monocyte chemoattractant protein-1 (MCP1), matrix metallopeptidase 9 (MMP-9), B-type natriuretic peptides (BNPs), galectin-3 (Gal-3), high-sensitivity cardiac troponin I (hsTn1), high-sensitivity C-reactive protein (hs-CRP), and soluble urokinase plasminogen activator receptor (sUPAR)]. METHODS: Among a cross-sectional population-based cohort of older adults, longitudinal LA strain including reservoir strain (εs), conduit strain (εe), and booster strain (εa) as well as peak strain rates (SRs, SRe, SRa) were determined using CMR and studied in association with blood biomarkers. RESULTS: We studied 243 community adults (42.8% female, mean age 70.3 ± 9.5 years). In bivariate analysis, εe and SRe were reduced in gradation with increasing risk factors (all p values <0.0001). Corresponding levels of sUPAR (ng/mL) were quantitatively higher in older adults with <2 risk factors (2.5 ± 1.6 vs. 1.7 ± 1.3, p = 0.0005), in those with ≥2 risk factors (3.3 ± 2.4 vs. 1.7 ± 1.3, p < 0.0001), compared to young adults; including between older adults with ≥2 risk factors and older adults with <2 risk factors (3.3 ± 2.4 vs. 2.5 ± 1.6, p = 0.017). Based on multivariate analysis, sUPAR was significantly associated with both εe (OR 1.52, p = 0.006) and SRe decline (OR 1.5, p = 0.019). The associations between Gal-3 and εe reduction (OR 1.2, p = 0.022) and between BNP and SRe decline were generally weaker (OR 1.03, p = 0.027). The addition of sUPAR to a model consisting of age, risk factors, Gal-3, and BNPs increased the area under the curve of εe from 0.72 to 0.77 (p = 0.015). CONCLUSION: By advanced CMR imaging, a panel of circulating biomarkers comprising galectin, MMP-9 and sUPAR were associated with left atrial dysfunction in older adults. Higher levels of Gal-3 and MMP-9 may be suggestive of fibrotic mechanisms in left atrial aging while impairments in left atrial strain seen in association with circulating sUPAR may be related to immune activation in the left atrium in response to left atrial remodeling and fibrotic processes.

Hyaluronidase-1-mediated glycocalyx impairment underlies endothelial abnormalities in polypoidal choroidal vasculopathy.

BACKGROUND: Polypoidal choroidal vasculopathy (PCV), a subtype of age-related macular degeneration (AMD), is a global leading cause of vision loss in older populations. Distinct from typical AMD, PCV is characterized by polyp-like dilatation of blood vessels and turbulent blood flow in the choroid of the eye. Gold standard anti-vascular endothelial growth factor (anti-VEGF) therapy often fails to regress polypoidal lesions in patients. Current animal models have also been hampered by their inability to recapitulate such vascular lesions. These underscore the need to identify VEGF-independent pathways in PCV pathogenesis. RESULTS: We cultivated blood outgrowth endothelial cells (BOECs) from PCV patients and normal controls to serve as our experimental disease models. When BOECs were exposed to heterogeneous flow, single-cell transcriptomic analysis revealed that PCV BOECs preferentially adopted migratory-angiogenic cell state, while normal BOECs undertook proinflammatory cell state. PCV BOECs also had a repressed protective response to flow stress by demonstrating lower mitochondrial functions. We uncovered that elevated hyaluronidase-1 in PCV BOECs led to increased degradation of hyaluronan, a major component of glycocalyx that interfaces between flow stress and vascular endothelium. Notably, knockdown of hyaluronidase-1 in PCV BOEC improved mechanosensitivity, as demonstrated by a significant 1.5-fold upregulation of Krüppel-like factor 2 (KLF2) expression, a flow-responsive transcription factor. Activation of KLF2 might in turn modulate PCV BOEC migration. Barrier permeability due to glycocalyx impairment in PCV BOECs was also reversed by hyaluronidase-1 knockdown. Correspondingly, hyaluronidase-1 was detected in PCV patient vitreous humor and plasma samples. CONCLUSIONS: Hyaluronidase-1 inhibition could be a potential therapeutic modality in preserving glycocalyx integrity and endothelial stability in ocular diseases with vascular origin.

Epigenomes of Human Hearts Reveal New Genetic Variants Relevant for Cardiac Disease and Phenotype.

RATIONALE: Identifying genetic markers for heterogeneous complex diseases such as heart failure is challenging and requires prohibitively large cohort sizes in genome-wide association studies to meet the stringent threshold of genome-wide statistical significance. On the other hand, chromatin quantitative trait loci, elucidated by direct epigenetic profiling of specific human tissues, may contribute toward prioritizing subthreshold variants for disease association. OBJECTIVE: Here, we captured noncoding genetic variants by performing epigenetic profiling for enhancer H3K27ac chromatin immunoprecipitation followed by sequencing in 70 human control and end-stage failing hearts. METHODS AND RESULTS: We have mapped a comprehensive catalog of 47 321 putative human heart enhancers and promoters. Three thousand eight hundred ninety-seven differential acetylation peaks (FDR [false discovery rate], 5%) pointed to pathways altered in heart failure. To identify cardiac histone acetylation quantitative trait loci (haQTLs), we regressed out confounding factors including heart failure disease status and used the G-SCI (Genotype-independent Signal Correlation and Imbalance) test1 to call out 1680 haQTLs (FDR, 10%). RNA sequencing performed on the same heart samples proved a subset of haQTLs to have significant association also to gene expression (expression quantitative trait loci), either in cis (180) or through long-range interactions (81), identified by Hi-C (high-throughput chromatin conformation assay) and HiChIP (high-throughput protein centric chromatin) performed on a subset of hearts. Furthermore, a concordant relationship between the gain or disruption of TF (transcription factor)-binding motifs, inferred from alternative alleles at the haQTLs, implied a surprising direct association between these specific TF and local histone acetylation in human hearts. Finally, 62 unique loci were identified by colocalization of haQTLs with the subthreshold loci of heart-related genome-wide association studies datasets. CONCLUSIONS: Disease and phenotype association for 62 unique loci are now implicated. These loci may indeed mediate their effect through modification of enhancer H3K27 acetylation enrichment and their corresponding gene expression differences (bioRxiv: https://doi.org/10.1101/536763). Graphical Abstract: A graphical abstract is available for this article.

Hypercoagulability, endotheliopathy, and inflammation approximating 1 year after recovery: Assessing the long-term outcomes in COVID-19 patients.

Sustained hypercoagulability and endotheliopathy are present in convalescent COVID-19 patients for up to 4 months from recovery. The hemostatic, endothelial, and inflammatory profiles of 39 recovered COVID-19 patients were evaluated up to 16 months after recovery from COVID-19. These values were compared with a control group of healthy volunteers (n = 124). 39 patients (71.8% males, median age 43 years) were reviewed at a mean of 12.7 ± 3.6 months following recovery. One patient without cardiovascular risk factors had post COVID-19 acute ischaemic limb. Elevated D-dimer and Factor VIII levels above normal ranges were noted in 17.9% (7/39) and 48.7% (19/39) of patients respectively, with a higher median D-dimer 0.34 FEU µg/mL (IQR 0.28, 0.46) (p < .001) and Factor VIII 150% (IQR 171, 203) (p = .004), versus controls. Thrombin generation (Thromboscreen) showed a higher median endogenous thrombin potential (ETP) of 1352 nM*min (IQR 1152, 1490) (p = .002) and a higher median peak height of 221.4 nM (IQR 170.2, 280.4) (p = 0.01) and delayed lag time 2.4 min (1.42-2.97) (p = 0.0002) versus controls. Raised vWF:Ag and ICAM-1 levels were observed in 17.9% (7/39) and 7.7% (3/39) of patients respectively, with a higher median VWF:Ag 117% (IQR 86, 154) (p = 0.02) and ICAM-1 54.1 ng/mL (IQR 43.8, 64.1) (p = .004) than controls. IL-6 was noted to be raised in 35.9% (14/39) of patients, with a higher median IL-6 of 1.5 pg/mL (IQR 0.6, 3.0) (p = 0.004) versus controls. Subgroup analysis stratifying patients by COVID-19 severity and COVID-19 vaccination preceding SARS-CoV-2 infection did not show statistically significant differences. Hypercoagulability, endothelial dysfunction, and inflammation are still detectable in some patients approximately 1 year after recovery from COVID-19.

Single-Cell Transcriptome of Wet AMD Patient-Derived Endothelial Cells in Angiogenic Sprouting.

Age-related macular degeneration (AMD) is a global leading cause of visual impairment in older populations. 'Wet' AMD, the most common subtype of this disease, occurs when pathological angiogenesis infiltrates the subretinal space (choroidal neovascularization), causing hemorrhage and retinal damage. Gold standard anti-vascular endothelial growth factor (VEGF) treatment is an effective therapy, but the long-term prevention of visual decline has not been as successful. This warrants the need to elucidate potential VEGF-independent pathways. We generated blood out-growth endothelial cells (BOECs) from wet AMD and normal control subjects, then induced angiogenic sprouting of BOECs using a fibrin gel bead assay. To deconvolute endothelial heterogeneity, we performed single-cell transcriptomic analysis on the sprouting BOECs, revealing a spectrum of cell states. Our wet AMD BOECs share common pathways with choroidal neovascularization such as extracellular matrix remodeling that promoted proangiogenic phenotype, and our 'activated' BOEC subpopulation demonstrated proinflammatory hallmarks, resembling the tip-like cells in vivo. We uncovered new molecular insights that pathological angiogenesis in wet AMD BOECs could also be driven by interleukin signaling and amino acid metabolism. A web-based visualization of the sprouting BOEC single-cell transcriptome has been created to facilitate further discovery research.

Regulatory Light Chains in Cardiac Development and Disease.

The role of regulatory light chains (RLCs) in cardiac muscle function has been elucidated progressively over the past decade. The RLCs are among the earliest expressed markers during cardiogenesis and persist through adulthood. Failing hearts have shown reduced RLC phosphorylation levels and that restoring baseline levels of RLC phosphorylation is necessary for generating optimal force of muscle contraction. The signalling mechanisms triggering changes in RLC phosphorylation levels during disease progression remain elusive. Uncovering this information may provide insights for better management of heart failure patients. Given the cardiac chamber-specific expression of RLC isoforms, ventricular RLCs have facilitated the identification of mature ventricular cardiomyocytes, opening up possibilities of regenerative medicine. This review consolidates the standing of RLCs in cardiac development and disease and highlights knowledge gaps and potential therapeutic advancements in targeting RLCs.

Use of the Ciliation Index to Distinguish Invasive Melanoma From Associated Conventional Melanocytic Nevi.

Our understanding of melanoma precursors and progression to melanoma has developed as a result of advances in the field of molecular diagnostics. We now better understand the potential for genetic heterogeneity within a single lesion. Combined tumors can pose a diagnostic challenge when deciding the line between benign and malignant, which in turn has direct implications for patient management. Primary cilia (PC) are ubiquitous sensory organelles that have essential functions in cellular proliferation, differentiation, and development. The ciliation index (percentage of ciliated melanocytes) has been shown to reliably differentiate melanoma, which fail to ciliate, from melanocytic nevi, which retain PC. We therefore analyzed the potential for using the ciliation index to differentiate benign and malignant components in combined melanocytic lesions. We collected patient samples (n = 10) of unequivocal combined lesions with both melanoma and associated nevus components. Melanocytes were highlighted with SOX10 and costained with gamma-Tubulin and acetylated alpha-Tubulin to highlight the basal body and cilium, respectively. The number of melanocytes retaining cilia under high-power microscopy was examined. The melanoma component had average of 4% ciliation (SD: 7%), whereas the associated nevus component was significantly higher with 59% ciliation (SD: 17%). These data show that PC may be a reliable means of distinguishing benign from malignant components within a single tumor. The ciliation index may be a helpful tool in distinguishing challenging cases of combined lesions of melanoma in situ with a dermal nevus component from invasive melanoma, thus promoting improved staging and clinical management.

Determination of critical shear stress for maturation of human pluripotent stem cell-derived endothelial cells towards an arterial subtype.

Human pluripotent stem cell-derived endothelial cells (hPSC-ECs) present an attractive alternative to primary EC sources for vascular grafting. However, there is a need to mature them towards either an arterial or venous subtype. A vital environmental factor involved in the arteriovenous specification of ECs during early embryonic development is fluid shear stress; therefore, there have been attempts to employ adult arterial shear stress conditions to mature hPSC-ECs. However, hPSC-ECs are naïve to fluid shear stress, and their shear responses are still not well understood. Here, we used a multiplex microfluidic platform to systematically investigate the dose-time shear responses on hPSC-EC morphology and arterial-venous phenotypes over a range of magnitudes coincidental with physiological levels of embryonic and adult vasculatures. The device comprised of six parallel cell culture chambers that were individually linked to flow-setting resistance channels, allowing us to simultaneously apply shear stress ranging from 0.4 to 15 dyne/cm 2 . We found that hPSC-ECs required up to 40 hr of shear exposure to elicit a stable phenotypic change. Cell alignment was visible at shear stress <1 dyne/cm 2 , which was independent of shear stress magnitude and duration of exposure. We discovered that the arterial markers NOTCH1 and EphrinB2 exhibited a dose-dependent increase in a similar manner beyond a threshold level of 3.8 dyne/cm 2 , whereas the venous markers COUP-TFII and EphB4 expression remained relatively constant across different magnitudes. These findings indicated that hPSC-ECs were sensitive to relatively low magnitudes of shear stress, and a critical level of ~4 dyne/cm 2 was sufficient to preferentially enhance their maturation into an arterial phenotype for future vascular tissue engineering applications.

Depletion of primary cilium in acral melanoma.

BACKGROUND: A eukaryotic cell's primary cilium (PC) is critical for cell signaling, migration and homeostasis. Primary cilium dysfunction has been demonstrated in several malignancies, but whether primary cilia loss occurs in acral melanoma has remained unknown. To address this, we examined the ciliation index (% melanocytes containing a PC) of patient-derived, biopsy-proven acral melanoma and compared these to benign acral nevi. METHODS: We generated a pilot initiative study that included six acral melanomas and seven acral nevi derived from the foot. Using fluorescent immunohistochemistry, we calculated ciliation indexes of Sox10+ melanocytes. RESULTS: Average ciliation index for acral nevi was 74.0% (SE of the mean [SEM] 3.3%) vs 9.3% for acral melanoma (SEM 5.7%), finding a statistically significant difference between the groups (P-value <.001, two tailed t test). CONCLUSION: The data show a significant loss of primary cilia in malignant acral melanoma vs benign acral nevi, suggesting that cilia may play an important role during acral melanoma formation. Our data, which should be validated by a larger study with longer follow-up period, suggest that examining ciliation index may be a useful diagnostic test when distinguishing benign acral nevi from melanoma.

Value of soluble Urokinase plasminogen activator receptor over age as a biomarker of impaired myocardial relaxation.

BACKGROUND: SuPAR is a biomarker that reflects the level of immune activation. As inflammation plays an important role in the ageing process of the cardiovascular system, we hypothesized that suPAR might be a useful predictive biomarker of the ageing heart. METHODS: We performed conventional and tissue Doppler echocardiography and measured plasma suPAR levels. RESULTS: We studied community adults (n=120, 37.5% female) (mean age: 70.3±9.3 years) without known cardiovascular disease (CVD). Participants with impaired myocardial relaxation were older (84% vs 59% were aged ≥71 years, p=0.002), with more diabetes mellitus (27% vs 11%, p=0.034). SuPAR levels were higher among participants with impaired myocardial relaxation (3.9 ng/ml vs 3.0 ng/ml, p=0.015). At the univariate level, older age (OR 3.6; 95%CI 1.6, 8.5; p=0.003), diabetes mellitus (OR 3.04; 95%CI 1.1, 8.8; p=0.04), systolic blood pressure (OR 1.03; 95%CI 1.001, 1.1; p=0.041) and suPAR levels ≥3.00ng/ml (OR 3.4; 95%CI 1.16, 7.4; p=0.002) were associated with impaired myocardial relaxation. In multivariable regression analysis, only older age (OR 2.8; 95%CI 1.1, 6.7; p=0.026) and suPAR (OR 2.7; 95%CI 1.2, 6.1; p=0.018) remained independently associated with impaired myocardial relaxation. Receiver operating characteristics (ROC) curve analysis revealed an area under the curve (AUC) value of 0.63 (95% CI 0.54, 0.71) for model that included age alone. Addition of suPAR significantly increased AUC value to 0.70 (95%CI 0.60, 0.79), which was significantly larger than the model with age alone (p=0.016). CONCLUSION: We demonstrate additional ability of suPAR, over age, to predict impaired myocardial relaxation. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02791139 (Registered May 31, 2016).

Loss of primary cilia correlates with cytologic severity in dysplastic melanocytic nevi.

BACKGROUND: Primary cilia are ubiquitously present in cell surface organelles with essential functions in cellular proliferation, differentiation and development. We have previously shown that cilia in melanoma in situ, invasive melanoma and metastatic melanoma are nearly completely lost, whereas benign nevi are ciliated. Dysplastic nevi (DN) have a wide range of histopathologic features from mild (low-grade) to severe (high-grade) cytologic atypia and represent a key clinical and histopathologic marker for melanomagenesis. We sought to identify whether cilia are retained in these melanocytic lesions and whether increasing degree of dysplasia correlates with loss of ciliation. METHODS: We measured the percentage of ciliated melanocytes (ciliation index) in DN with mild (n = 9) and severe dysplasia (n = 10). We identified the primary cilium and basal bodies by immunofluorescence staining of sections with acetylated alpha-tubulin and gamma-tubulin, respectively. RESULTS: Our results showed a significant decrease in the ciliation index from mild dysplastic (55%) to severe DN (14%) (p = 0.005). CONCLUSIONS: These data support the hypothesis that primary cilium loss may play a role in the underlying biology of severe DN. The ciliation index is a novel quantitative tool that may increase the reproducibility in grading severity of dysplasia for diagnostic and clinical management of melanocytic neoplasms.

Immunohistochemical analysis of lichenoid reactions in patients treated with anti-PD-L1 and anti-PD-1 therapy.

BACKGROUND: Recent advances in the immunotherapeutic treatment of cancer have led to the development of multiple new directed therapies including monoclonal antibodies that block the immune checkpoint T-cell receptor programmed death 1 (PD-1) and the PD-1 ligand, programmed death ligand 1 (PD-L1). Various immune-related toxicities have been associated with these drugs including, most commonly, skin rashes. METHODS: Five cases of lichenoid dermatitis, including one case of lichenoid mucositis and one case of lichen sclerosus, associated with anti-PD-L1 and anti-PD1 therapy were compared with three biopsies of non-drug-related lichen planus (LP) and three lichen planus-like keratoses (LPLK) used as controls. RESULTS: Histopathologic and immunophenotypic analysis of these lichenoid lesions demonstrated significantly greater histiocytic infiltrates than observed in control lichenoid reactions (p = 0.0134). We also observed increased spongiosis and epidermal necrosis. No significant differences were seen in expression of CD3, CD4:CD8, CD20, PD-1, CD25, Foxp3, CXCL13 and PD-L1 expression. CONCLUSIONS: These findings expand the literature of immune-related toxicities of PD-L1 and PD-1 blockade to include lichenoid dermatitis and lichenoid mucositis. Of note, these cutaneous side effects were amenable to topical treatment, without the need for medication dose reduction or discontinuation.