Left ventricular hypertrophy and metabolic resetting in the Notch3-deficient adult mouse heart.

The heart depends on a functional vasculature for oxygenation and transport of nutrients, and it is of interest to learn how primary impairment of the vasculature can indirectly affect cardiac function and heart morphology. Notch3-deficiency causes vascular smooth muscle cell (VSMC) loss in the vasculature but the consequences for the heart remain largely elusive. Here, we demonstrate that Notch3-/- mice have enlarged hearts with left ventricular hypertrophy and mild fibrosis. Cardiomyocytes were hypertrophic but not hyperproliferative, and the expression of several cardiomyocyte markers, including Tnt2, Myh6, Myh7 and Actn2, was altered. Furthermore, expression of genes regulating the metabolic status of the heart was affected: both Pdk4 and Cd36 were downregulated, indicating a metabolic switch from fatty acid oxidation to glucose consumption. Notch3-/- mice furthermore showed lower liver lipid content. Notch3 was expressed in heart VSMC and pericytes but not in cardiomyocytes, suggesting that a perturbation of Notch signalling in VSMC and pericytes indirectly impairs the cardiomyocytes. In keeping with this, Pdgfbret/ret mice, characterized by reduced numbers of VSMC and pericytes, showed left ventricular and cardiomyocyte hypertrophy. In conclusion, we demonstrate that reduced Notch3 or PDGFB signalling in vascular mural cells leads to cardiomyocyte dysfunction.

Effectiveness and Safety of Remdesivir in Treating Hospitalised Patients with COVID-19: A Propensity Score Analysis of Real-Life Data from a Monocentric Observational Study in Times of Health Emergency.

BACKGROUND AND OBJECTIVES: Remdesivir is an antiviral agent, which was shown to be safe and effective in treating early COVID-19, but its favourable impact in hospitalised patients with non-critical disease is still under investigation. The present study aimed to assess the effectiveness and safety of remdesivir as a treatment for hospitalised patients with COVID-19 by a propensity score analysis of observational data. METHODS: In this monocentric retrospective cohort study, the effectiveness and safety of a 5-day course of remdesivir (200 mg intravenously at Day 1, then 100 mg from Days 2-5) in association with the standard of care were assessed in comparison with the standard of care only. The primary endpoint was the proportion of recovery on Day 14. RESULTS: Of 3662 eligible inpatients who tested positive for the severe acute respiratory syndrome coronavirus 2 genome by nasopharyngeal swab at admission, 861 (24%) non-critical patients were included in a propensity score analysis and 281 (33%) were exposed to remdesivir. In total, 242/281 (86.1%) and 435/580 (75.0%) patients recovered in exposed and non-exposed, respectively, with a relative improvement of 11.1% (95% CI + 5.8 to 16.5%; unadjusted odds ratio: 2.07, 95% CI 1.40-3.05, p = 0.0001; after adjustment by propensity score weighting, odds ratio: 1.92, 95% CI 1.30-2.83, p = 0.001). In treated patients, 1 (0.03%) anaphylactic reaction and 1 (0.03%) acute reaction during drug injection were reported, and 24 (8.5%) patients stopped the treatment due to adverse reactions. No significant differences were found with respect to the secondary efficacy endpoints (in-hospital all-cause death, need for intensive care treatments, clinical improvement score at Day 28) and safety endpoints (any and serious adverse reactions). CONCLUSION: A 5-day course of remdesivir in association with the standard of care effectively promoted recovery from COVID-19 among non-critical in-hospital patients and had an acceptable safety profile.

Molecular anatomy of adult mouse leptomeninges.

Leptomeninges, consisting of the pia mater and arachnoid, form a connective tissue investment and barrier enclosure of the brain. The exact nature of leptomeningeal cells has long been debated. In this study, we identify five molecularly distinct fibroblast-like transcriptomes in cerebral leptomeninges; link them to anatomically distinct cell types of the pia, inner arachnoid, outer arachnoid barrier, and dural border layer; and contrast them to a sixth fibroblast-like transcriptome present in the choroid plexus and median eminence. Newly identified transcriptional markers enabled molecular characterization of cell types responsible for adherence of arachnoid layers to one another and for the arachnoid barrier. These markers also proved useful in identifying the molecular features of leptomeningeal development, injury, and repair that were preserved or changed after traumatic brain injury. Together, the findings highlight the value of identifying fibroblast transcriptional subsets and their cellular locations toward advancing the understanding of leptomeningeal physiology and pathology.

A robust and efficient microvascular isolation method for multimodal characterization of the mouse brain vasculature.

Studying disease-related changes in the brain vasculature is warranted due to its crucial role in supplying oxygen and nutrients and removing waste and due to the anticipated vascular dysfunction in brain diseases. To this end, we have developed a protocol for fast and simple isolation of brain vascular fragments without the use of transgenic reporters. We used it to isolate and analyze 22,515 cells by single-cell RNA sequencing. The cells distributed into 23 distinct clusters corresponding to all known vascular and perivascular cell types in the brain. Western blot analysis also suggested that the protocol is suitable for proteomic analysis. We further adapted it for the establishment of primary cell cultures. The protocol generated highly reproducible results. In conclusion, we have developed a simple and robust brain vascular isolation protocol suitable for different experimental modalities, such as single-cell analyses, western blotting, and primary cell culture.

Active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common monogenic form of familial small vessel disease; no preventive or curative therapy is available. CADASIL is caused by mutations in the NOTCH3 gene, resulting in a mutated NOTCH3 receptor, with aggregation of the NOTCH3 extracellular domain (ECD) around vascular smooth muscle cells. In this study, we have developed a novel active immunization therapy specifically targeting CADASIL-like aggregated NOTCH3 ECD. Immunizing CADASIL TgN3R182C150 mice with aggregates composed of CADASIL-R133C mutated and wild-type EGF1-5 repeats for a total of 4 months resulted in a marked reduction (38-48%) in NOTCH3 deposition around brain capillaries, increased microglia activation and lowered serum levels of NOTCH3 ECD. Active immunization did not impact body weight, general behavior, the number and integrity of vascular smooth muscle cells in the retina, neuronal survival, or inflammation or the renal system, suggesting that the therapy is tolerable. This is the first therapeutic study reporting a successful reduction of NOTCH3 accumulation in a CADASIL mouse model supporting further development towards clinical application for the benefit of CADASIL patients.

Notch signalling in healthy and diseased vasculature.

Notch signalling is an evolutionarily highly conserved signalling mechanism governing differentiation and regulating homeostasis in many tissues. In this review, we discuss recent advances in our understanding of the roles that Notch signalling plays in the vasculature. We describe how Notch signalling regulates different steps during the genesis and remodelling of blood vessels (vasculogenesis and angiogenesis), including critical roles in assigning arterial and venous identities to the emerging blood vessels and regulation of their branching. We then proceed to discuss how experimental perturbation of Notch signalling in the vasculature later in development affects vascular homeostasis. In this review, we also describe how dysregulated Notch signalling, as a consequence of direct mutations of genes in the Notch pathway or aberrant Notch signalling output, contributes to various types of vascular disease, including CADASIL, Snedden syndrome and pulmonary arterial hypertension. Finally, we point out some of the current knowledge gaps and identify remaining challenges in understanding the role of Notch in the vasculature, which need to be addressed to pave the way for Notch-based therapies to cure or ameliorate vascular disease.

Specification of CNS macrophage subsets occurs postnatally in defined niches.

All tissue-resident macrophages of the central nervous system (CNS)-including parenchymal microglia, as well as CNS-associated macrophages (CAMs1) such as meningeal and perivascular macrophages2-7-are part of the CNS endogenous innate immune system that acts as the first line of defence during infections or trauma2,8-10. It has been suggested that microglia and all subsets of CAMs are derived from prenatal cellular sources in the yolk sac that were defined as early erythromyeloid progenitors11-15. However, the precise ontogenetic relationships, the underlying transcriptional programs and the molecular signals that drive the development of distinct CAM subsets in situ are poorly understood. Here we show, using fate-mapping systems, single-cell profiling and cell-specific mutants, that only meningeal macrophages and microglia share a common prenatal progenitor. By contrast, perivascular macrophages originate from perinatal meningeal macrophages only after birth in an integrin-dependent manner. The establishment of perivascular macrophages critically requires the presence of arterial vascular smooth muscle cells. Together, our data reveal a precisely timed process in distinct anatomical niches for the establishment of macrophage subsets in the CNS.

Trends in all-cause mortality of hospitalized patients due to SARS-CoV-2 infection from a monocentric cohort in Milan (Lombardy, Italy).

Background: Robust data on case fatality rate (CFR) among inpatients with COVID-19 are still lacking, and the role of patient characteristics in in-hospital deaths remains under-investigated. This study quantified the overall CFR and described its trend in a cohort of hospitalized patients with SARS-CoV-2 in Italy. Admission to ICU, death, or discharge were the secondary outcomes. Methods: This retrospective study is based on administrative health data and electronic case records of inpatients consecutively admitted to Niguarda Hospital between 21 February and 8 November 2020. Results: An overall CFR of 18% was observed. CFR was significantly reduced during the second wave of contagion (1 June to 30 September, 16%) compared with the first wave (21 February to 31 May, 21% p = 0.015). Such reduction was mainly observed among male inpatients between 40 and 80 years with limited comorbidities. Admission to ICU was associated with a high risk of mortality in both waves. The incidence of severe disease and the need for ICU admission were lower in the second wave. Conclusion: CFR in SARS-CoV-2 inpatients was demonstrated to decrease over time. This reduction may partly reflect the changes in hospital strategy and clinical practice. The reasons for this improvement should be further investigated to plan an exit strategy in case of future outbreaks. Key messages: What is already known on this topic Before the advent of anti-COVID-19 vaccines, a multi-wave pattern of contagion was observed, and this trend conditioned the inpatient case fatality rate (CFR), which varied over time accordingly to the waves of contagion.Only preliminary results on the in-hospital mortality trend are available, along with a partial analysis of its determinants. Consequently, robust data on CFR among inpatients with SARS-CoV-2 infection are still lacking, and the role of patient characteristics in in-hospital deaths remains under-investigated. What this study adds This study shows that the in-hospital mortality in patients with SARS-CoV-2 infection decreases over time.Such reduction was mainly observed among male inpatients between 40 and 80 years with limited comorbidities. Admission to ICU was invariably associated with a high risk of mortality during the whole study period (21 February to 8 November 2020), but the incidence of severe disease and the need for ICU admission were lower in the second wave of contagions (1 October to 8 November 2020). This reduction may partly reflect the impact of changes in hospital strategy and clinical practice. The reasons for this improvement should be further investigated to inform the response to future outbreaks and to plan exit strategy by prioritizing high-risk populations. Supplementary Information: The online version contains supplementary material available at 10.1007/s10389-021-01675-y.

Heart Rate in Patients with SARS-CoV-2 Infection: Prevalence of High Values at Discharge and Relationship with Disease Severity.

The most common arrhythmia associated with COronaVIrus-related Disease (COVID) infection is sinus tachycardia. It is not known if high Heart Rate (HR) in COVID is simply a marker of higher systemic response to sepsis or if its persistence could be related to a long-term autonomic dysfunction. The aim of our work is to assess the prevalence of elevated HR at discharge in patients hospitalized for COVID-19 and to evaluate the variables associated with it. We enrolled 697 cases of SARS-CoV2 infection admitted in our hospital after February 21 and discharged within 23 July 2020. We collected data on clinical history, vital signs, laboratory tests and pharmacological treatment. Severe disease was defined as the need for Intensive Care Unit (ICU) admission and/or mechanical ventilation. Median age was 59 years (first-third quartile 49, 74), and male was the prevalent gender (60.1%). 84.6% of the subjects showed a SARS-CoV-2 related pneumonia, and 13.2% resulted in a severe disease. Mean HR at admission was 90 ± 18 bpm with a mean decrease of 10 bpm to discharge. Only 5.5% of subjects presented HR > 100 bpm at discharge. Significant predictors of discharge HR at multiple linear model were admission HR (mean increase = ß = 0.17 per bpm, 95% CI 0.11; 0.22, p < 0.001), haemoglobin (ß = -0.64 per g/dL, 95% CI -1.19; -0.09, p = 0.023) and severe disease (ß = 8.42, 95% CI 5.39; 11.45, p < 0.001). High HR at discharge in COVID-19 patients is not such a frequent consequence, but when it occurs it seems strongly related to a severe course of the disease.

Novel Cysteine-Sparing Hypomorphic NOTCH3 A1604T Mutation Observed in a Family With Migraine and White Matter Lesions.

OBJECTIVE: To conduct a clinical study of a family with neurologic symptoms and findings carrying a novel NOTCH3 mutation and to analyze the molecular consequences of the mutation. METHODS: We analyzed a family with complex neurologic symptoms by MRI and neurologic examinations. Exome sequencing of the NOTCH3 locus was conducted, and whole-genome sequencing was performed to identify COL4A1, COL4A2, and HTRA1 mutations. Cell lines expressing the normal or NOTCH3A1604T receptors were analyzed to assess proteolytic processing, cell morphology, receptor routing, and receptor signaling. RESULTS: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common hereditary form of cerebral small vessel disease (SVD) and caused by mutations in the NOTCH3 gene. Most CADASIL mutations alter the number of cysteine residues in the extracellular domain of the NOTCH3 receptor, but in this article, we describe a family in which some members carry a novel cysteine-sparing NOTCH3 mutation (c.4810 G>A, p.Ala1604Thr). Two of 3 siblings heterozygous for the NOTCH3A1604T mutation presented with migraine and white matter lesions (WMLs), the latter of a type related to but distinct from what is normally observed in CADASIL. Two other members instead carried a novel COL4A1 missense mutation (c.4795 G>A; p.(Ala1599Thr)). The NOTCH3A1604T receptor was aberrantly processed, showed reduced presence at the cell surface, and less efficiently activated Notch downstream target genes. CONCLUSIONS: We identify a family with migraine and WML in which some members carry a cysteine-sparing hypomorphic NOTCH3 mutation. Although a causal relationship is not established, we believe that the observations contribute to the discussion on dysregulated Notch signaling in cerebral SVDs.

The infantile myofibromatosis NOTCH3 L1519P mutation leads to hyperactivated ligand-independent Notch signaling and increased PDGFRB expression.

Infantile myofibromatosis (IMF) is a benign tumor form characterized by the development of nonmetastatic tumors in skin, bone, muscle and sometimes viscera. Autosomal dominant forms of IMF are caused by mutations in the PDGFRB gene, but a family carrying a L1519P mutation in the NOTCH3 gene has also recently been identified. In this report, we address the molecular consequences of the NOTCH3L1519P mutation and the relationship between the NOTCH and PDGFRB signaling in IMF. The NOTCH3L1519P receptor generates enhanced downstream signaling in a ligand-independent manner. Despite the enhanced signaling, the NOTCH3L1519P receptor is absent from the cell surface and instead accumulates in the endoplasmic reticulum. Furthermore, the localization of the NOTCH3L1519P receptor in the bipartite, heterodimeric state is altered, combined with avid secretion of the mutated extracellular domain from the cell. Chloroquine treatment strongly reduces the amount of secreted NOTCH3L1519P extracellular domain and decreases signaling. Finally, NOTCH3L1519P upregulates PDGFRB expression in fibroblasts, supporting a functional link between Notch and PDGF dysregulation in IMF. Collectively, our data define a NOTCH3-PDGFRB axis in IMF, where an IMF-mutated NOTCH3 receptor elevates PDGFRB expression. The functional characterization of a ligand-independent gain-of-function NOTCH3 mutation is important for Notch therapy considerations for IMF, including strategies aimed at altering lysosome function.

Single-cell RNA sequencing of mouse brain and lung vascular and vessel-associated cell types.

Vascular diseases are major causes of death, yet our understanding of the cellular constituents of blood vessels, including how differences in their gene expression profiles create diversity in vascular structure and function, is limited. In this paper, we describe a single-cell RNA sequencing (scRNA-seq) dataset that defines vascular and vessel-associated cell types and subtypes in mouse brain and lung. The dataset contains 3,436 single cell transcriptomes from mouse brain, which formed 15 distinct clusters corresponding to cell (sub)types, and another 1,504 single cell transcriptomes from mouse lung, which formed 17 cell clusters. In order to allow user-friendly access to our data, we constructed a searchable database (http://betsholtzlab.org/VascularSingleCells/database.html). Our dataset constitutes a comprehensive molecular atlas of vascular and vessel-associated cell types in the mouse brain and lung, and as such provides a strong foundation for future studies of vascular development and diseases.

Author Correction: A molecular atlas of cell types and zonation in the brain vasculature.

In Fig. 1b of this Article, 'Csf1r' was misspelt 'Csfr1'. In addition, in Extended Data Fig. 11b, owing to an error during figure formatting, the genes listed in the first column shifted down three rows below the first gene on the list, causing a mismatch between the gene names and their characteristics. These errors have been corrected online, and the original Extended Data Fig. 11b is provided as Supplementary Information to the accompanying Amendment.

NOTCH3 inactivation increases triple negative breast cancer sensitivity to gefitinib by promoting EGFR tyrosine dephosphorylation and its intracellular arrest.

Notch dysregulation has been implicated in numerous tumors, including triple-negative breast cancer (TNBC), which is the breast cancer subtype with the worst clinical outcome. However, the importance of individual receptors in TNBC and their specific mechanism of action remain to be elucidated, even if recent findings suggested a specific role of activated-Notch3 in a subset of TNBCs. Epidermal growth factor receptor (EGFR) is overexpressed in TNBCs but the use of anti-EGFR agents (including tyrosine kinase inhibitors, TKIs) has not been approved for the treatment of these patients, as clinical trials have shown disappointing results. Resistance to EGFR blockers is commonly reported. Here we show that Notch3-specific inhibition increases TNBC sensitivity to the TKI-gefitinib in TNBC-resistant cells. Mechanistically, we demonstrate that Notch3 is able to regulate the activated EGFR membrane localization into lipid rafts microdomains, as Notch3 inhibition, such as rafts depletion, induces the EGFR internalization and its intracellular arrest, without involving receptor degradation. Interestingly, these events are associated with the EGFR tyrosine dephosphorylation at Y1173 residue (but not at Y1068) by the protein tyrosine phosphatase H1 (PTPH1), thus suggesting its possible involvement in the observed Notch3-dependent TNBC sensitivity response to gefitinib. Consistent with this notion, a nuclear localization defect of phospho-EGFR is observed after combined blockade of EGFR and Notch3, which results in a decreased TNBC cell survival. Notably, we observed a significant correlation between EGFR and NOTCH3 expression levels by in silico gene expression and immunohistochemical analysis of human TNBC primary samples. Our findings strongly suggest that combined therapies of TKI-gefitinib with Notch3-specific suppression may be exploited as a drug combination advantage in TNBC treatment.

A molecular atlas of cell types and zonation in the brain vasculature.

Cerebrovascular disease is the third most common cause of death in developed countries, but our understanding of the cells that compose the cerebral vasculature is limited. Here, using vascular single-cell transcriptomics, we provide molecular definitions for the principal types of blood vascular and vessel-associated cells in the adult mouse brain. We uncover the transcriptional basis of the gradual phenotypic change (zonation) along the arteriovenous axis and reveal unexpected cell type differences: a seamless continuum for endothelial cells versus a punctuated continuum for mural cells. We also provide insight into pericyte organotypicity and define a population of perivascular fibroblast-like cells that are present on all vessel types except capillaries. Our work illustrates the power of single-cell transcriptomics to decode the higher organizational principles of a tissue and may provide the initial chapter in a molecular encyclopaedia of the mammalian vasculature.

Randomized Controlled Trial of Family Therapy in Advanced Cancer Continued Into Bereavement.

PURPOSE: Systematic family-centered cancer care is needed. We conducted a randomized controlled trial of family therapy, delivered to families identified by screening to be at risk from dysfunctional relationships when one of their relatives has advanced cancer. PATIENTS AND METHODS: Eligible patients with advanced cancer and their family members screened above the cut-off on the Family Relationships Index. After screening 1,488 patients or relatives at Memorial Sloan Kettering Cancer Center or three related community hospice programs, 620 patients (42%) were recruited, which represented 170 families. Families were stratified by three levels of family dysfunction (low communicating, low involvement, and high conflict) and randomly assigned to one of three arms: standard care or 6 or 10 sessions of a manualized family intervention. Primary outcomes were the Complicated Grief Inventory-Abbreviated (CGI) and Beck Depression Inventory-II (BDI-II). Generalized estimating equations allowed for clustered data in an intention-to-treat analysis. RESULTS: On the CGI, a significant treatment effect (Wald χ(2) = 6.88; df = 2; P = .032) and treatment by family-type interaction was found (Wald χ(2) = 20.64; df = 4; P < .001), and better outcomes resulted from 10 sessions compared with standard care for low-communicating and high-conflict groups compared with low-involvement families. Low-communicating families improved by 6 months of bereavement. In the standard care arm, 15.5% of the bereaved developed a prolonged grief disorder at 13 months of bereavement compared with 3.3% of those who received 10 sessions of intervention (Wald χ(2) = 8.31; df = 2; P =.048). No significant treatment effects were found on the BDI-II. CONCLUSION: Family-focused therapy delivered to high-risk families during palliative care and continued into bereavement reduced the severity of complicated grief and the development of prolonged grief disorder.

Challenges in providing family-centered support to families in palliative care.

BACKGROUND: Supporting the family-as-a-whole presents challenges in palliative care, although family meetings are increasingly used in routine practice. The Family Focused Grief Therapy (FFGT) Model guides clinicians in using a range of intervention strategies. AIM: To examine the therapists' techniques used in assessing 'at risk' families in palliative care to better illuminate what helps and what remains challenging. METHOD: Recorded sessions 1 and 2 were coded using the FFGT fidelity coding measure, with its glossary of definitions. Inter-rater reliability between three coders was satisfactory at 88%. Frequencies of strategy utilization were computed, with extraction of examples of both successful and problematic approaches. SETTING/PARTICIPANTS: From within a larger study of family therapy during palliative care at a comprehensive cancer center, the first two sessions (n = 144) delivered to 74 families (299 individuals) by 32 therapists were coded and analyzed. RESULTS: Therapists readily explored the story of illness and families' ways of coping (97%) and assessed communication and cohesiveness in the majority. Exploration of relational patterns occurred in 89% of sessions, use of a genogram in 80%, understanding members' roles in 65% and family values and beliefs in 62%. Less use was made of summaries (39%), family mottos (34%), exploration of family conflict (35%) and the formalization of a comprehensive family treatment plan (20%). CONCLUSIONS: Challenges exist in therapy with difficult families. Therapy in the home brings special issues. Therapists can apply most of the interventions prescribed by the FFGT model.