Single-cell RNA sequencing of cystic fibrosis liver disease explants reveals endothelial complement activation.

BACKGROUND & AIMS: Cystic fibrosis (CF) is considered a multisystemic disorder in which CF-associated liver disease (CFLD) is the third most common cause of mortality. Currently, no effective treatment is available for CFLD because its pathophysiology is still unclear. Interestingly, CFLD exhibits identical vascular characteristics as non-cirrhotic portal hypertension, recently classified as porto-sinusoidal vascular disorders (PSVD). METHODS: Since endothelial cells (ECs) are an important component in PSVD, we performed single-cell RNA sequencing (scRNA-seq) on four explant livers from CFLD patients to identify differential endothelial characteristics which could contribute to the disease. We comprehensively characterized the endothelial compartment and compared it with publicly available scRNA-seq datasets from cirrhotic and healthy livers. Key gene signatures were validated ex vivo on patient tissues. RESULTS: We found that ECs from CF liver explants are more closely related to healthy than cirrhotic patients. In CF patients we also discovered a distinct population of liver sinusoidal ECs-coined CF LSECs-upregulating genes involved in the complement cascade and coagulation. Finally, our immunostainings further validated the predominant periportal location of CF LSECs. CONCLUSIONS: Our work showed novel aspects of human liver ECs at the single-cell level thereby supporting endothelial involvement in CFLD, and reinforcing the hypothesis that ECs could be a driver of PSVD. Therefore, considering the vascular compartment in CF and CFLD may help developing new therapeutic approaches for these diseases.

PARP inhibition promotes endothelial-like traits in melanoma cells and modulates pericyte coverage dynamics during vasculogenic mimicry.

Vasculogenic mimicry (VM) describes the ability of highly aggressive tumor cells to develop pseudovascular structures without the participation of endothelial cells. PARP1 is implicated in the activation of hypoxia-inducible factors, which are crucial in tumor neovascularization. We have explored the role of hypoxia and PARP inhibition in VM. In uveal melanoma xenografts, the PARP inhibitor olaparib improved in vivo pericyte coverage specifically of VM channels. This was concomitant with reduced metastasis in olaparib-treated VM+ tumors. PARP inhibition and hypoxia modulated melanoma tube formation in vitro, inducing a more sparse and regular tubular architecture. Whole-transcriptome profiling revealed that olaparib treatment under hypoxic conditions modulated the expression of genes implicated in vasculogenesis during tube formation, enhancing the endothelial-like phenotype of VM+ uveal melanoma cells. PARP inhibition, especially during hypoxia, upregulated PDGFß, which is essential for pericyte recruitment. Our study indicates that PARP inhibitors may enhance the endothelial characteristics of VM+ cells, modulate pericyte coverage, and reduce metastatic spread in VM+ melanoma. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

The pulmonary vasculature in lethal COVID-19 and idiopathic pulmonary fibrosis at single-cell resolution.

AIMS: Severe acute respiratory syndrome coronavirus-2 infection causes COVID-19, which in severe cases evokes life-threatening acute respiratory distress syndrome (ARDS). Transcriptome signatures and the functional relevance of non-vascular cell types (e.g. immune and epithelial cells) in COVID-19 are becoming increasingly evident. However, despite its known contribution to vascular inflammation, recruitment/invasion of immune cells, vascular leakage, and perturbed haemostasis in the lungs of severe COVID-19 patients, an in-depth interrogation of the endothelial cell (EC) compartment in lethal COVID-19 is lacking. Moreover, progressive fibrotic lung disease represents one of the complications of COVID-19 pneumonia and ARDS. Analogous features between idiopathic pulmonary fibrosis (IPF) and COVID-19 suggest partial similarities in their pathophysiology, yet, a head-to-head comparison of pulmonary cell transcriptomes between both conditions has not been implemented to date. METHODS AND RESULTS: We performed single-nucleus RNA-sequencing on frozen lungs from 7 deceased COVID-19 patients, 6 IPF explant lungs, and 12 controls. The vascular fraction, comprising 38 794 nuclei, could be subclustered into 14 distinct EC subtypes. Non-vascular cell types, comprising 137 746 nuclei, were subclustered and used for EC-interactome analyses. Pulmonary ECs of deceased COVID-19 patients showed an enrichment of genes involved in cellular stress, as well as signatures suggestive of dampened immunomodulation and impaired vessel wall integrity. In addition, increased abundance of a population of systemic capillary and venous ECs was identified in COVID-19 and IPF. COVID-19 systemic ECs closely resembled their IPF counterparts, and a set of 30 genes was found congruently enriched in systemic ECs across studies. Receptor-ligand interaction analysis of ECs with non-vascular cell types in the pulmonary micro-environment revealed numerous previously unknown interactions specifically enriched/depleted in COVID-19 and/or IPF. CONCLUSIONS: This study uncovered novel insights into the abundance, expression patterns, and interactomes of EC subtypes in COVID-19 and IPF, relevant for future investigations into the progression and treatment of both lethal conditions.

Single cell atlas identifies lipid-processing and immunomodulatory endothelial cells in healthy and malignant breast.

Since a detailed inventory of endothelial cell (EC) heterogeneity in breast cancer (BC) is lacking, here we perform single cell RNA-sequencing of 26,515 cells (including 8433 ECs) from 9 BC patients and compare them to published EC taxonomies from lung tumors. Angiogenic ECs are phenotypically similar, while other EC subtypes are different. Predictive interactome analysis reveals known but also previously unreported receptor-ligand interactions between ECs and immune cells, suggesting an involvement of breast EC subtypes in immune responses. We also identify a capillary EC subtype (LIPEC (Lipid Processing EC)), which expresses genes involved in lipid processing that are regulated by PPAR-γ and is more abundant in peri-tumoral breast tissue. Retrospective analysis of 4648 BC patients reveals that treatment with metformin (an indirect PPAR-γ signaling activator) provides long-lasting clinical benefit and is positively associated with LIPEC abundance. Our findings warrant further exploration of this LIPEC/PPAR-γ link for BC treatment.

Generation of vessel co-option lung metastases mouse models for single-cell isolation of metastases-derived cells and endothelial cells.

Tumor vessel co-option, a process in which cancer cells "hijack" pre-existing blood vessels to grow and invade healthy tissue, is poorly understood but is a proposed resistance mechanism against anti-angiogenic therapy (AAT). Here, we describe protocols for establishing murine renal (RENCA) and breast (4T1) cancer lung vessel co-option metastases models. Moreover, we outline a reproducible protocol for single-cell isolation from murine lung metastases using magnetic-activated cell sorting as well as immunohistochemical stainings to distinguish vessel co-option from angiogenesis. For complete details on the use and execution of this protocol, please refer to Teuwen et al. (2021).

Tumor vessel co-option probed by single-cell analysis.

Tumor vessel co-option is poorly understood, yet it is a resistance mechanism against anti-angiogenic therapy (AAT). The heterogeneity of co-opted endothelial cells (ECs) and pericytes, co-opting cancer and myeloid cells in tumors growing via vessel co-option, has not been investigated at the single-cell level. Here, we use a murine AAT-resistant lung tumor model, in which VEGF-targeting induces vessel co-option for continued growth. Single-cell RNA sequencing (scRNA-seq) of 31,964 cells reveals, unexpectedly, a largely similar transcriptome of co-opted tumor ECs (TECs) and pericytes as their healthy counterparts. Notably, we identify cell types that might contribute to vessel co-option, i.e., an invasive cancer-cell subtype, possibly assisted by a matrix-remodeling macrophage population, and another M1-like macrophage subtype, possibly involved in keeping or rendering vascular cells quiescent.

Transcriptomic analysis of CFTR-impaired endothelial cells reveals a pro-inflammatory phenotype.

Cystic fibrosis (CF) is a life-threatening disorder characterised by decreased pulmonary mucociliary and pathogen clearance, and an exaggerated inflammatory response leading to progressive lung damage. CF is caused by bi-allelic pathogenic variants of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a chloride channel. CFTR is expressed in endothelial cells (ECs) and EC dysfunction has been reported in CF patients, but a role for this ion channel in ECs regarding CF disease progression is poorly described.We used an unbiased RNA sequencing approach in complementary models of CFTR silencing and blockade (by the CFTR inhibitor CFTRinh-172) in human ECs to characterise the changes upon CFTR impairment. Key findings were further validated in vitro and in vivo in CFTR-knockout mice and ex vivo in CF patient-derived ECs.Both models of CFTR impairment revealed that EC proliferation, migration and autophagy were downregulated. Remarkably though, defective CFTR function led to EC activation and a persisting pro-inflammatory state of the endothelium with increased leukocyte adhesion. Further validation in CFTR-knockout mice revealed enhanced leukocyte extravasation in lung and liver parenchyma associated with increased levels of EC activation markers. In addition, CF patient-derived ECs displayed increased EC activation markers and leukocyte adhesion, which was partially rescued by the CFTR modulators VX-770 and VX-809.Our integrated analysis thus suggests that ECs are no innocent bystanders in CF pathology, but rather may contribute to the exaggerated inflammatory phenotype, raising the question of whether normalisation of vascular inflammation might be a novel therapeutic strategy to ameliorate the disease severity of CF.

Single-Cell RNA Sequencing Maps Endothelial Metabolic Plasticity in Pathological Angiogenesis.

Endothelial cell (EC) metabolism is an emerging target for anti-angiogenic therapy in tumor angiogenesis and choroidal neovascularization (CNV), but little is known about individual EC metabolic transcriptomes. By single-cell RNA sequencing 28,337 murine choroidal ECs (CECs) and sprouting CNV-ECs, we constructed a taxonomy to characterize their heterogeneity. Comparison with murine lung tumor ECs (TECs) revealed congruent marker gene expression by distinct EC phenotypes across tissues and diseases, suggesting similar angiogenic mechanisms. Trajectory inference predicted that differentiation of venous to angiogenic ECs was accompanied by metabolic transcriptome plasticity. ECs displayed metabolic transcriptome heterogeneity during cell-cycle progression and in quiescence. Hypothesizing that conserved genes are important, we used an integrated analysis, based on congruent transcriptome analysis, CEC-tailored genome-scale metabolic modeling, and gene expression meta-analysis in cross-species datasets, followed by in vitro and in vivo validation, to identify SQLE and ALDH18A1 as previously unknown metabolic angiogenic targets.

Unraveling the transcriptional determinants of liver sinusoidal endothelial cell specialization.

Liver sinusoidal endothelial cells (LSECs) are the first liver cells to encounter waste macromolecules, pathogens, and toxins in blood. LSECs are highly specialized to mediate the clearance of these substances via endocytic scavenger receptors and are equipped with fenestrae that mediate the passage of macromolecules toward hepatocytes. Although some transcription factors (TFs) are known to play a role in LSEC specialization, information about the specialized LSEC signature and its transcriptional determinants remains incomplete.Based on a comparison of liver, heart, and brain endothelial cells (ECs), we established a 30-gene LSEC signature comprising both established and newly identified markers, including 7 genes encoding TFs. To evaluate the LSEC TF regulatory network, we artificially increased the expression of the 7 LSEC-specific TFs in human umbilical vein ECs. Although Zinc finger E-box-binding protein 2, homeobox B5, Cut-like homolog 2, and transcription factor EC (TCFEC) had limited contributions, musculoaponeurotic fibrosarcoma (C-MAF), GATA binding protein 4 (GATA4), and MEIS homeobox 2 (MEIS2) emerged as stronger inducers of LSEC marker expression. Furthermore, a combination of C-MAF, GATA4, and MEIS2 showed a synergistic effect on the increase of LSEC signature genes, including liver/lymph node-specific ICAM-3 grabbing non-integrin (L-SIGN) (or C-type lectin domain family member M (CLEC4M)), mannose receptor C-Type 1 (MRC1), legumain (LGMN), G protein-coupled receptor 182 (GPR182), Plexin C1 (PLXNC1), and solute carrier organic anion transporter family member 2A1 (SLCO2A1). Accordingly, L-SIGN, MRC1, pro-LGMN, GPR182, PLXNC1, and SLCO2A1 protein levels were elevated by this combined overexpression. Although receptor-mediated endocytosis was not significantly induced by the triple TF combination, it enhanced binding to E2, the hepatitis C virus host-binding protein. We conclude that C-MAF, GATA4, and MEIS2 are important transcriptional regulators of the unique LSEC fingerprint and LSEC interaction with viruses. Additional factors are however required to fully recapitulate the molecular, morphological, and functional LSEC fingerprint.NEW & NOTEWORTHY Liver sinusoidal endothelial cells (LSECs) are the first liver cells to encounter waste macromolecules, pathogens, and toxins in the blood and are highly specialized. Although some transcription factors are known to play a role in LSEC specialization, information about the specialized LSEC signature and its transcriptional determinants remains incomplete. Here, we show that Musculoaponeurotic Fibrosarcoma (C-MAF), GATA binding protein 4 (GATA4), and Meis homeobox 2 (MEIS2) are important transcriptional regulators of the unique LSEC signature and that they affect the interaction of LSECs with viruses.

An Integrated Gene Expression Landscape Profiling Approach to Identify Lung Tumor Endothelial Cell Heterogeneity and Angiogenic Candidates.

Heterogeneity of lung tumor endothelial cell (TEC) phenotypes across patients, species (human/mouse), and models (in vivo/in vitro) remains poorly inventoried at the single-cell level. We single-cell RNA (scRNA)-sequenced 56,771 endothelial cells from human/mouse (peri)-tumoral lung and cultured human lung TECs, and detected 17 known and 16 previously unrecognized phenotypes, including TECs putatively regulating immune surveillance. We resolved the canonical tip TECs into a known migratory tip and a putative basement-membrane remodeling breach phenotype. Tip TEC signatures correlated with patient survival, and tip/breach TECs were most sensitive to vascular endothelial growth factor blockade. Only tip TECs were congruent across species/models and shared conserved markers. Integrated analysis of the scRNA-sequenced data with orthogonal multi-omics and meta-analysis data across different human tumors, validated by functional analysis, identified collagen modification as a candidate angiogenic pathway.

HIF-1α metabolically controls collagen synthesis and modification in chondrocytes.

Endochondral ossification, an important process in vertebrate bone formation, is highly dependent on correct functioning of growth plate chondrocytes1. Proliferation of these cells determines longitudinal bone growth and the matrix deposited provides a scaffold for future bone formation. However, these two energy-dependent anabolic processes occur in an avascular environment1,2. In addition, the centre of the expanding growth plate becomes hypoxic, and local activation of the hypoxia-inducible transcription factor HIF-1α is necessary for chondrocyte survival by unidentified cell-intrinsic mechanisms3-6. It is unknown whether there is a requirement for restriction of HIF-1α signalling in the other regions of the growth plate and whether chondrocyte metabolism controls cell function. Here we show that prolonged HIF-1α signalling in chondrocytes leads to skeletal dysplasia by interfering with cellular bioenergetics and biosynthesis. Decreased glucose oxidation results in an energy deficit, which limits proliferation, activates the unfolded protein response and reduces collagen synthesis. However, enhanced glutamine flux increases α-ketoglutarate levels, which in turn increases proline and lysine hydroxylation on collagen. This metabolically regulated collagen modification renders the cartilaginous matrix more resistant to protease-mediated degradation and thereby increases bone mass. Thus, inappropriate HIF-1α signalling results in skeletal dysplasia caused by collagen overmodification, an effect that may also contribute to other diseases involving the extracellular matrix such as cancer and fibrosis.

Serine Synthesis via PHGDH Is Essential for Heme Production in Endothelial Cells.

The role of phosphoglycerate dehydrogenase (PHGDH), a key enzyme of the serine synthesis pathway (SSP), in endothelial cells (ECs) remains poorly characterized. We report that mouse neonates with EC-specific PHGDH deficiency suffer lethal vascular defects within days of gene inactivation, due to reduced EC proliferation and survival. In addition to nucleotide synthesis impairment, PHGDH knockdown (PHGDHKD) caused oxidative stress, due not only to decreased glutathione and NADPH synthesis but also to mitochondrial dysfunction. Electron transport chain (ETC) enzyme activities were compromised upon PHGDHKD because of insufficient heme production due to cellular serine depletion, not observed in other cell types. As a result of heme depletion, elevated reactive oxygen species levels caused EC demise. Supplementation of hemin in PHGDHKD ECs restored ETC function and rescued the apoptosis and angiogenesis defects. These data argue that ECs die upon PHGDH inhibition, even without external serine deprivation, illustrating an unusual importance of serine synthesis for ECs.

Blockade of Myeloid-Derived Suppressor Cell Expansion with All-Trans Retinoic Acid Increases the Efficacy of Antiangiogenic Therapy.

Intrinsic and adaptive resistance hampers the success of antiangiogenic therapies (AAT), especially in breast cancer where this treatment modality has proven largely ineffective. Therefore, novel strategies to improve the efficacy of AAT are warranted. Solid tumors such as breast cancer are characterized by a high infiltration of myeloid-derived suppressor cells (MDSC), which are key drivers of resistance to AAT. Therefore, we hypothesized that all-trans retinoic acid (ATRA), which induces differentiation of MDSC into mature cells, could improve the therapeutic effect of AAT. ATRA increased the efficacy of anti-VEGFR2 antibodies alone and in combination with chemotherapy in preclinical breast cancer models. ATRA reverted the anti-VEGFR2-induced accumulation of intratumoral MDSC, alleviated hypoxia, and counteracted the disorganization of tumor microvessels. Mechanistic studies indicate that ATRA treatment blocked the AAT-induced expansion of MDSC secreting high levels of vessel-destabilizing S100A8. Thus, concomitant treatment with ATRA holds the potential to improve AAT in breast cancer and possibly other tumor types.Significance: Increasing the therapeutic efficiency of antiangiogenic drugs by reducing resistance-conferring myeloid-derived suppressor cells might improve breast cancer treatment.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/12/3220/F1.large.jpg Cancer Res; 78(12); 3220-32. ©2018 AACR.

Tumor vessel disintegration by maximum tolerable PFKFB3 blockade.

Blockade of the glycolytic activator PFKFB3 in cancer cells (using a maximum tolerable dose of 70 mg/kg of the PFKFB3 blocker 3PO) inhibits tumor growth in preclinical models and is currently being tested as a novel anticancer treatment in phase I clinical trials. However, a detailed preclinical analysis of the effects of such maximum tolerable dose of a PFKFB3 blocker on the tumor vasculature is lacking, even though tumor endothelial cells are hyper-glycolytic. We report here that a high dose of 3PO (70 mg/kg), which inhibits cancer cell proliferation and reduces primary tumor growth, causes tumor vessel disintegration, suppresses endothelial cell growth for protracted periods, (model-dependently) aggravates tumor hypoxia, and compromises vascular barrier integrity, thereby rendering tumor vessels more leaky and facilitating cancer cell intravasation and dissemination. These findings contrast to the effects of a low dose of 3PO (25 mg/kg), which induces tumor vessel normalization, characterized by vascular barrier tightening and maturation, but reduces cancer cell intravasation and metastasis. Our findings highlight the importance of adequately dosing a glycolytic inhibitor for anticancer treatment.

Inhibition of MicroRNA-146a and Overexpression of Its Target Dihydrolipoyl Succinyltransferase Protect Against Pressure Overload-Induced Cardiac Hypertrophy and Dysfunction.

BACKGROUND: Cardiovascular diseases remain the predominant cause of death worldwide, with the prevalence of heart failure continuing to increase. Despite increased knowledge of the metabolic alterations that occur in heart failure, novel therapies to treat the observed metabolic disturbances are still lacking. METHODS: Mice were subjected to pressure overload by means of angiotensin-II infusion or transversal aortic constriction. MicroRNA-146a was either genetically or pharmacologically knocked out or genetically overexpressed in cardiomyocytes. Furthermore, overexpression of dihydrolipoyl succinyltransferase (DLST) in the murine heart was performed by means of an adeno-associated virus. RESULTS: MicroRNA-146a was upregulated in whole heart tissue in multiple murine pressure overload models. Also, microRNA-146a levels were moderately increased in left ventricular biopsies of patients with aortic stenosis. Overexpression of microRNA-146a in cardiomyocytes provoked cardiac hypertrophy and left ventricular dysfunction in vivo, whereas genetic knockdown or pharmacological blockade of microRNA-146a blunted the hypertrophic response and attenuated cardiac dysfunction in vivo. Mechanistically, microRNA-146a reduced its target DLST-the E2 subcomponent of the α-ketoglutarate dehydrogenase complex, a rate-controlling tricarboxylic acid cycle enzyme. DLST protein levels significantly decreased on pressure overload in wild-type mice, paralleling a decreased oxidative metabolism, whereas DLST protein levels and hence oxidative metabolism were partially maintained in microRNA-146a knockout mice. Moreover, overexpression of DLST in wild-type mice protected against cardiac hypertrophy and dysfunction in vivo. CONCLUSIONS: Altogether we show that the microRNA-146a and its target DLST are important metabolic players in left ventricular dysfunction.

The role of fatty acid ß-oxidation in lymphangiogenesis.

Lymphatic vessels are lined by lymphatic endothelial cells (LECs), and are critical for health. However, the role of metabolism in lymphatic development has not yet been elucidated. Here we report that in transgenic mouse models, LEC-specific loss of CPT1A, a rate-controlling enzyme in fatty acid ß-oxidation, impairs lymphatic development. LECs use fatty acid ß-oxidation to proliferate and for epigenetic regulation of lymphatic marker expression during LEC differentiation. Mechanistically, the transcription factor PROX1 upregulates CPT1A expression, which increases acetyl coenzyme A production dependent on fatty acid ß-oxidation. Acetyl coenzyme A is used by the histone acetyltransferase p300 to acetylate histones at lymphangiogenic genes. PROX1-p300 interaction facilitates preferential histone acetylation at PROX1-target genes. Through this metabolism-dependent mechanism, PROX1 mediates epigenetic changes that promote lymphangiogenesis. Notably, blockade of CPT1 enzymes inhibits injury-induced lymphangiogenesis, and replenishing acetyl coenzyme A by supplementing acetate rescues this process in vivo.

Glycolytic regulation of cell rearrangement in angiogenesis.

During vessel sprouting, endothelial cells (ECs) dynamically rearrange positions in the sprout to compete for the tip position. We recently identified a key role for the glycolytic activator PFKFB3 in vessel sprouting by regulating cytoskeleton remodelling, migration and tip cell competitiveness. It is, however, unknown how glycolysis regulates EC rearrangement during vessel sprouting. Here we report that computational simulations, validated by experimentation, predict that glycolytic production of ATP drives EC rearrangement by promoting filopodia formation and reducing intercellular adhesion. Notably, the simulations correctly predicted that blocking PFKFB3 normalizes the disturbed EC rearrangement in high VEGF conditions, as occurs during pathological angiogenesis. This interdisciplinary study integrates EC metabolism in vessel sprouting, yielding mechanistic insight in the control of vessel sprouting by glycolysis, and suggesting anti-glycolytic therapy for vessel normalization in cancer and non-malignant diseases.

Atherosclerosis Alters Loading-Induced Arterial Damage: Implications for Robotic Surgery.

BACKGROUND: Lack of intra-operative haptic information during robotic surgery increases the risk for unintended tissue overload and damage. Knowledge about the acute and chronic fundamental relationship between force load and induced damage in healthy and diseased arteries is crucial to enable intra-operative haptic feedback or shared autonomy and improve patient safety. METHODS: Arteries of wildtype and atherosclerotic mice were clamped in vivo for 2 minutes (0.0N, 0.6N or 1.27N). Histological analysis (Verhoeff's-Van Gieson, Osteopontin, CD45, CD105) was performed immediately, or after 6 hours, 2 weeks or 1 month. Endothelium-dependent and-independent vasodilatation was assessed immediately or 1 month after clamping. RESULTS: Endothelium dependent vasodilatation is worse after clamping of wildtype arteries, but is restored after one month. Clamping also results in flattening of the innermost elastic membrane of both genotypes, which is reversed over time for wildtype arteries but not for vessels from atherosclerotic mice. Higher osteopontin content in wildtype and LDLR-/- mice after 2 weeks suggests a phenotypic switch of the medial smooth muscle cells (SMCs), an effect that is reversed after 1 month. While inflammation in the intima diminishes, medial CD45 content rises through time in both genotypes. CD105 staining shows that even manipulation without clamping results in endothelial cell loss in both LDLR+/+ and LDLR-/- mice. CONCLUSIONS: Arterial clamping induces different acute and long-term injury to the vessel wall of atherosclerotic and healthy arteries.