Introduction: Spatial transcriptomics has become a powerful tool for interrogating a disease-specific transcriptome within the context of tissue architecture. In this study, we used spatial transcriptomics to investigate the molecular mechanisms underlying COVID-19 in the lungs and heart. Methods: We performed sequencing-based spatial transcriptomics (ST), using 10X Genomics’ Visium, on 6 paired fatal COVID-19 lung and heart tissue samples and 2 control samples per organ. With the gene by spot matrix, we performed dimensional reduction, clustering (Louvain), differential expression (Wilcoxon Rank sum test), and pathway analysis (GSEA). spacexr was used to estimate the cell type composition of ST spots. Results: By histology and ST, COVID-19 tissue was defined by a loss in parenchymal cells and increased inflammation ( fig. 1E ). In both the heart and lung, we found a cluster of ST spots specific to fatal COVID-19 ( fig. 1A, B ; P<0.001). Between the two organs, these clusters shared genes and pathways relating to tissue remodeling, B cell action, and complement pathway activation ( fig. 1C, D ). Response to wounding and blood vessel endothelial cell migration pathways were distinct to the heart cluster, also explaining the increased capillary cell weight in the COVID-19 heart compared to the control ( fig. 1D, E ; P=0.0167). Conclusion: Our results suggest that there is a shared spatial niche between the heart and lungs in COVID-19 infection and highlight the importance of studying multiple organs in understanding the disease. These findings provide new insights into the molecular basis of COVID-19 and have the potential to inform the development of novel therapies for this disease. Figure 1 (A) UMAP colored by (left) clusters and (right) disease. (B) Cluster proportions between COVID-19 and control tissue (C) Scatter plot of shared differentially expressed genes. (D) GSEA pathway score. (E) Spatial mapping of clusters and cell-type deconvolution.

Introduction and Aims: While primarily a pulmonary disease, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can affect multiple organ systems, including the vasculature. Thus far studies have successfully characterised the whole organ compartment, however, our understanding of the mechanisms associated with COVID-19 infection and its impact on pulmonary microvasculature (pMV) remains incomplete. In human pulmonary microvascular endothelial cells (HPMVEC) treated with plasma from patients hospitalised with COVID-19, we aimed to evaluate transcriptional, morphological and functional changes, and to provide a mechanism for the excess mortality in patients with COVID-19 and how it may inform therapeutic advancement. Methods and Results: Plasma acquired from hospitalised patients with COVID-19 was compared to plasma from patients hospitalised without COVID-19 but with other acute illnesses, such as COPD. Exposure to plasma from patients with COVID-19 caused a significant functional decline in HPMVEC, with a decrease in both cell viability on the WST-1 cell-proliferation assay (n=75, p= 0.013) and cell-to-cell barrier function measured by electric cell-substrate impedance sensing (n=3, p = 0.033) . High content imaging using the cell painting image-based assay showed a phenotypic change in nuclear (n=4, p=0.019) and nucleic acid (n=4, p=0.022) morphology, indicative of a deterioration in core biological processes including protein transcription and metabolic output. Comparative deep RNA-sequencing of HPMVECs treated with COVID-19 plasma (n=5) confirmed upregulation of genes, notably IFIT5 and SFMBT1 . These genes are known to negatively regulate transcription and respond to viral infection, respectively, further demonstrating the phenotypic and transcriptional decline in the pulmonary microvasculature in patients with COVID-19. Conclusion and Impact: Our findings suggest that core biological processes are disrupted in the lung microvascular endothelium during severe COVID-19 disease contributing to aggravated disease. From a translational perspective, this knowledge could provide insight into possible treatment opportunities and prognostic tools that may directly target the dysregulated endothelium.

Introduction: Spatial transcriptomics has become a powerful tool for interrogating a disease-specific transcriptome within the context of tissue architecture. In this study, we used spatial transcriptomics to investigate the molecular mechanisms underlying COVID-19 in the lungs and heart. Methods: We performed sequencing-based spatial transcriptomics (ST), using 10X Genomics’ Visium, on 6 paired fatal COVID-19 lung and heart tissue samples and 2 control samples per organ. With the gene by spot matrix, we performed dimensional reduction, clustering (Louvain), differential expression (Wilcoxon Rank sum test), and pathway analysis (GSEA). spacexr was used to estimate the cell type composition of ST spots. Results: By histology and ST, COVID-19 tissue was defined by a loss in parenchymal cells and increased inflammation ( fig. 1E ). In both the heart and lung, we found a cluster of ST spots specific to fatal COVID-19 ( fig. 1A, B ; P<0.001). Between the two organs, these clusters shared genes and pathways relating to tissue remodeling, B cell action, and complement pathway activation ( fig. 1C, D ). Response to wounding and blood vessel endothelial cell migration pathways were distinct to the heart cluster, also explaining the increased capillary cell weight in the COVID-19 heart compared to the control ( fig. 1D, E ; P=0.0167). Conclusion: Our results suggest that there is a shared spatial niche between the heart and lungs in COVID-19 infection and highlight the importance of studying multiple organs in understanding the disease. These findings provide new insights into the molecular basis of COVID-19 and have the potential to inform the development of novel therapies for this disease. Figure 1 (A) UMAP colored by (left) clusters and (right) disease. (B) Cluster proportions between COVID-19 and control tissue (C) Scatter plot of shared differentially expressed genes. (D) GSEA pathway score. (E) Spatial mapping of clusters and cell-type deconvolution.

Aim Myocardial infarction remains the leading cause of heart failure. The adult human heart lacks the capacity to undergo endogenous regeneration. New blood vessel growth is integral to regenerative medicine necessitating a comprehensive understanding of the pathways that regulate vascular regeneration. We sought to define the transcriptomic dynamics of coronary endothelial cells following ischaemic injuries in the developing and adult mouse and human heart and to identify new mechanistic insights and targets for cardiovascular regeneration. Methods and results We carried out a comprehensive meta-analysis of integrated single-cell RNA-sequencing data of coronary vascular endothelial cells from the developing and adult mouse and human heart spanning healthy and acute and chronic ischaemic cardiac disease. We identified species-conserved gene regulatory pathways aligned to endogenous neovascularization. We annotated injury-associated temporal shifts of the endothelial transcriptome and validated four genes: VEGF-C, KLF4, EGR1, and ZFP36. Moreover, we showed that ZFP36 regulates human coronary endothelial cell proliferation and defined that VEGF-C administration in vivo enhances clonal expansion of the cardiac vasculature post-myocardial infarction. Finally, we constructed a coronary endothelial cell meta-atlas, CrescENDO, to empower future in-depth research to target pathways associated with coronary neovascularization. Conclusion We present a high-resolution single-cell meta-atlas of healthy and injured coronary endothelial cells in the mouse and human heart, revealing a suite of novel targets with great potential to promote vascular regeneration, and providing a rich resource for therapeutic development.

Background Myocardial infarction (MI) is the leading cause of heart failure. The adult human heart, unlike mouse or early neonatal hearts, lacks the capability to undergo extensive regeneration. Rapid re-establishment of blood flow post MI is vital for limiting tissue damage and preserving cardiac function. A better understanding of the mechanisms underpinning cardiovascular regeneration in adult hearts is needed. Recent technologies including single cell RNA sequencing (scRNA-seq) and spatial transcriptomics (ST) have empowered studies of healthy and diseased tissue at unprecedented resolution. Methods and Results First, we established an EC-specific multispectral lineage-tracing mouse model (Pdgfb-iCreERT2-R26R-Brainbow2.1) and assessed EC clonal proliferation in the adult heart post MI. We discovered a significant increase in clone size in the MI hearts compared to the healthy controls (cells per clone = 4.0 ± 2.1 vs. 10.3 ± 10.6, P < 0.0001), demonstrating that the structural integrity of adult endothelium following MI was maintained through clonal proliferation by resident ECs in the infarct border region. We then isolated the Pdgfb-lineage ECs from the healthy (12,780) and injured (15,818) hearts through FACS, performed scRNA-seq and downstream analysis, and defined ten transcriptionally discrete heterogeneous EC states and associated pathways that might impact upon cardiovascular regeneration. Next, high-quality scRNA-seq data from 10 curated studies of the mouse and human hearts were integrated for a cross-species systematic meta-analysis. Coronary ECs were enriched in silico based on the expression of 45 endothelial markers and analysed using Seurat. Unsupervised clustering of integrated neonatal and adult mouse coronary ECs revealed 15 transcriptionally distinct clusters. The subsequent DEG analysis identified the Vegfc pathway as a program that can potentially augment adult cardiovascular regeneration in the neonatal heart. The integration of the mouse and human coronary EC data and the DEG analysis identified 41 commonly upregulated genes after ischaemic injuries, including KLF4, EGR1 and ZFP36. Further, spatial transcriptomics analysis of MI patient-derived heart tissues revealed the elevation of these conserved targets in the damaged tissues in the acute phase. We validated the upregulation of these targets in the injured human coronary ECs (% KLF4+ CD31+ EC = 29.7 ± 7.5% versus 7.3 ± 6.4%, P = 0.0009;% EGR1+ CD31+ EC = versus 10.1 ± 3.5% versus 3.4 ± 2.5%, P = 0.004; ZFP36 expression was high the diseased tissue but minimal in control hearts). In vitro siRNA knockdown of ZFP36 in cultured human cardiac microvascular endothelial cells (HCMECs) showed that cell proliferation was significantly inhibited compared to the control siRNA treatment (Fold change of%EdU+ HCMECs = 0.84 ± 0.19 vs 0.25 ± 0.12, P = 0.0007). In vivo, we used the multi-spectral MI mouse model and showed that the administration of rhVEGF-C significantly increased neovascularisation in the infarct border in the adult mouse heart compared to the PBS treated controls (vascular clone volume (μm3) = 3072 ± 491.2 versus 426 ± 105, P = 0.02). Conclusion We have successfully developed and implemented a robust framework, using meta-analysis of scRNA-seq, spatial transcriptomics, tissue section immunofluorescence, primary human cell culture, and multispectral MI mouse model, to collectively identify, assess, and validate novel mechanisms and targets potential to promote vascular regeneration.

Cardiac injury leads to the loss of cardiomyocytes, which are rapidly replaced by the proliferation of the surviving cells in zebrafish, but not in mammals. In both the regenerative zebrafish and non-regenerative mammals, cardiac injury induces a sustained macrophage response. Macrophages are required for cardiomyocyte proliferation during zebrafish cardiac regeneration, but the mechanisms whereby macrophages facilitate this crucial process are fundamentally unknown. Using heartbeat-synchronized live imaging, RNA sequencing, and macrophage-null genotypes in the larval zebrafish cardiac injury model, we characterize macrophage function and reveal that these cells activate the epicardium, inducing cardiomyocyte proliferation. Mechanistically, macrophages are specifically recruited to the epicardial-myocardial niche, triggering the expansion of the epicardium, which upregulates vegfaa expression to induce cardiomyocyte proliferation. Our data suggest that epicardial Vegfaa augments a developmental cardiac growth pathway via increased endocardial notch signaling. The identification of this macrophage-dependent mechanism of cardiac regeneration highlights immunomodulation as a potential strategy for enhancing mammalian cardiac repair.

Ischaemic heart disease remains a leading global cause of death. Restoring blood perfusion in the peri-infarct border region may limit further infarct expansion and promote cardiac regeneration. However, the pathways driving endogenous vascular regeneration following myocardial infarction (MI) remain poorly understood. We aimed to investigate the origin, clonal dynamics and transcriptional profiles of resident coronary endothelial cells (EC) in the post-ischaemic adult mouse heart and to apply single cell RNA-sequencing (scRNAseq) to identify and validate novel targets with a potential role in neovasculogenesis following MI. MI was induced in an EC-specific multispectral lineage-tracing mouse, ‘Pdgfb-iCreERT2-R26R-Brainbow2.1’ by permanent ligation of the left anterior descending coronary artery. Blood vessel formation via clonal proliferation by resident coronary vascular EC was significantly upregulated in the ischaemic border at 7 days post-MI, compared to the healthy heart (Pdgfb-Confetti+ EC per clone = 4.0 ± 2.1 versus 10.3 ± 10.6, P<0.0001). ScRNAseq analyses revealed 10 transcriptionally discrete heterogeneous EC states in the ischaemic heart and revealed molecular pathways through which each cluster was likely to mediate neovasculogenesis following MI. Plasmalemma vesicle–associated protein (Plvap) gene expression was upregulated in MI in a cluster specific manner, indicating its potential relevance to neovasculogenic pathways. We further showed that PLVAP protein expression was EC-specific and significantly greater in the infarct border of the post-ischaemic mouse heart compared to the healthy heart (% PLVAP+ EC = 70.5 ± 19.9% versus 38.7 ± 28.2%, P=0.002). PLVAP expression was also significantly increased in EC adjacent to regions of fibrosis and scarring in the ischaemic human heart, compared to healthy human hearts (% PLVAP+ EC = 36.9.8 ± 10.1% versus 12.7 ± 12.1%, P=0.02). Moreover, in vitro silencing using RNAi in HUVECS showed that PLVAP plays a role in regulating EC proliferation (% EdU+ HUVEC = 60.7 ± 3.9% versus. 21.1 ± 11.0%, P = 0.0038). In summary, we present a single cell gene expression atlas of cardiac resident EC, which can be interrogated to define the transcriptional profile of post-MI ECs and subsequently identify novel targets, such as PLVAP, with a potential role in augmenting endogenous myocardial perfusion following ischaemia.

Ischaemic heart disease (IHD) remains one of the leading causes of death worldwide. The global burden of IHD remains extremely high with over 126 million people living with the disease and 8.9 million deaths in 2017. Efficient and rapid restoration of a functional blood vessel network is critical to prevent further cardiomyocyte death in patients following myocardial infarction (MI). The primary clinical revascularization strategies are percutaneous coronary intervention or coronary artery bypass grafting. Despite timely reperfusion of the myocardium in the majority of cases, the number of patients that will go on to develop heart failure as a consequence of left ventricular impairment following acute MI continues to rise worldwide. Hence, new therapeutic approaches to enhance myocardial perfusion and promote cardiac repair and regeneration are urgently required. Formation of new vascular networks is a critical early step during cardiac regeneration in early neonatal mice, generating a functional framework for subsequent regeneration of the myocardium. However, in most adult mammals, regenerative ability in the heart rapidly declines in the days following birth. Despite this, even in adulthood, the heart postMI has been shown to be an active site of angiogenesis, albeit at a level that is insufficient to support adequate tissue repair or regeneration. Therefore, significant research has aimed to understand and thereby intervene in order to facilitate endogenous neovascularization in the injured adult mammalian heart. Vector-assisted gene delivery of pro-angiogenic factors holds therapeutic promise for enhancement of myocardial neovascularization. Pre-clinical studies have shown improved cardiac perfusion and function after administration of the pro-angiogenic vascular endothelial growth factor (VEGF) after MI. However, human gene therapy clinical trials aiming to improve patient outcomes in IHD through VEGF delivery have reported mixed results so far. Therefore, a deeper understanding of the mechanisms that regulate myocardial neovascularization may help to explain previous disappointing outcomes in humans, and to identify strategies to improve future pro-angiogenic clinical strategies. Kocijan et al. tackle the potential reasons underpinning insufficient endogenous angiogenesis in the adult heart. Previously, the authors have successfully promoted new blood vessel formation using delivery of VEGF in skeletal muscle after hindlimb ischaemia (HLI). However, when the same strategy was assessed in the ischaemic myocardium, no substantive angiogenic response was detected. In the current study, they aimed to understand whether restricted angiogenic responses in the adult heart could be enlightened through comparative analysis between responses in skeletal and cardiac muscle, together with mechanistic insight into tumour angiogenesis. They used a lineage tracing mouse model to label sprouting endothelial cells (EC), driven by apelin. Apelin expression is normally low in the adult vasculature but is increased in response to hypoxia and is associated with sprouting angiogenesis. As an angiogenic trigger, a gene therapy approach was used by directly injecting human AAV-VEGF165 into either the tibialis anterior or the left ventricular wall of uninjured adult mice. As a result, VEGF induced apelin expression in both heart and skeletal muscle ECs although in the heart, apelinpositive ECs largely remained quiescent and failed to proliferate sufficiently to form new vessels. Conversely, in skeletal muscle, apelinexpressing ECs proliferated and underwent effective angiogenesis. This was driven by Notch signalling, indicated by an increased expression of Notch target genes Hes1, Hey1, and Notch1, an effect that was not evident in the heart. This highlights important fundamental differences in the functional responses of the vascular endothelium to pro-angiogenic stimuli in diverse tissue settings. This is likely due to differences in environmental cues as well as inherent molecular and cellular heterogeneity in the endothelium between these tissues. Importantly, single-cell transcriptional profiling has been used to successfully delineate endothelial cell heterogeneity in multiple organs and tissues, and also in response to ischaemic injury in the adult mouse heart. To further understand the differential responses observed in vivo, the authors isolated cardiac and skeletal muscle ECs and, following exposure to VEGF, quantified proliferation and angiogenesis in an ex vivo spheroid model. Interestingly, despite VEGF inducing apelin expression in ECs from both tissues, only those from skeletal muscle were capable of forming angiogenic sprouts. Following interrogation of the well-characterized tip/stalk cell signalling pathway they showed differences in the expression of key genes such as DLL4 and Notch with cardiac ECs having reduced expression.

Aims A better understanding of the pathways that regulate regeneration of the coronary vasculature is of fundamental importance for the advancement of strategies to treat patients with heart disease. Here, we aimed to investigate the origin and clonal dynamics of endothelial cells (ECs) associated with neovascularization in the adult mouse heart following myocardial infarction (MI). Furthermore, we sought to define murine cardiac endothelial heterogeneity and to characterize the transcriptional profiles of pro-angiogenic resident ECs in the adult mouse heart, at single-cell resolution. Methods and results An EC-specific multispectral lineage-tracing mouse (Pdgfb-iCreER(T2)-R26R-Brainbow2.1) was used to demonstrate that structural integrity of adult cardiac endothelium following MI was maintained through clonal proliferation by resident ECs in the infarct border region, without significant contributions from bone marrow cells or endothelial-to-mesenchymal transition. Ten transcriptionally discrete heterogeneous EC states, as well as the pathways through which each endothelial state is likely to enhance neovasculogenesis and tissue regeneration following ischaemic injury were defined. Plasmalemma vesicle-associated protein (Plvap) was selected for further study, which showed an endothelial-specific and increased expression in both the ischaemic mouse and human heart, and played a direct role in regulating human endothelial proliferation in vitro. Conclusion We present a single-cell gene expression atlas of cardiac specific resident ECs, and the transcriptional hierarchy underpinning endogenous vascular repair following MI. These data provide a rich resource that could assist in the development of new therapeutic interventions to augment endogenous myocardial perfusion and enhance regeneration in the injured heart.

Aims: A better understanding of the pathways that regulate regeneration of the coronary vasculature is important for future strategies to treat patients with heart disease. We investigated (i) the clonal dynamics of endothelial cells (EC) associated with neovascularization in the ischemic border region (ii) transcriptional signatures of regenerative EC in the ischemic heart using single cell RNA-sequencing (iii) the functional relevance of selected targets. Methods: MI was induced in ‘EC-Confetti’ mice by coronary artery ligation. EC clonal proliferation was quantified or hearts dissociated for scRNAseq. Immunofluorescence staining for targets identified by scRNAseq was performed on cardiac tissue from patients with ischemic heart disease. EC proliferation was assessed in vitro following siRNA gene silencing. Results: EC-Confetti mice express YFP, RFP, GFP, or CFP specifically in EC. Fluorophores are inherited by EC progeny following proliferation, allowing quantitative clonal analysis. Clonal proliferation was significantly increased in the infarct border at 7 days post-MI compared to healthy hearts ( P <0.0001). Ten transcriptionally discrete EC clusters were defined following scRNAseq with 3 clusters predominantly composed of cells from the MI group, indicating their gene expression profiles may be relevant to neovasculogenic pathways. We selected plasmalemma vesicle associated protein (Plvap) for further study and confirmed EC-specific increased Plvap expression in ischemic border regions of human ( P =0.002) and mouse ( P =0.002) hearts, compared to healthy myocardium. siRNA gene silencing of Plvap significantly inhibited EC proliferation ( P = 0.0038), strong evidence that Plvap can directly modulate EC function. Conclusions: Generation of new blood vessels following ischemic injury in the mouse heart is predominantly mediated by clonal proliferation of resident EC. We present a gene expression atlas of resident cardiac EC, and the transcriptional hierarchy underpinning endogenous vascular repair following MI. This resource identifies novel targets, including Plvap, that may augment myocardial perfusion post-MI, and inform future design of strategies aimed at promoting vascular perfusion in ischemic heart disease.

Key Points Aberrations in genome maintenance and DNA repair genes including POT1 occur at a high frequency in Sézary syndrome. Candidate driver genes and affected pathways in Sézary syndrome show extensive heterogeneity but overlap with other mature T-cell lymphomas.