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The number of coronavirus disease 2019 (COVID‐19) cases has been increasing significantly, and the disease has evolved into a global pandemic, posing an unprecedented challenge to the healthcare community. Angiotensin‐converting enzyme 2, the binding and entry receptor of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) in hosts, is also expressed on pulmonary vascular endothelium; thus, pulmonary vasculature is a potential target in COVID‐19. Indeed, pulmonary vascular thickening is observed by early clinical imaging, implying a tropism of SARS‐CoV‐2 for pulmonary vasculature. Recent studies reported that COVID‐19 is associated with vascular endothelial damage and dysfunction along with inflammation, coagulopathy, and microthrombosis; all of these pathologic changes are the hallmarks of pulmonary vascular diseases. Notwithstanding the not fully elucidated effects of COVID‐19 on pulmonary vasculature, the vascular endotheliopathy that occurs after infection is attributed to direct infection and indirect damage mainly caused by renin‐angiotensin‐aldosterone system imbalance, coagulation cascade, oxidative stress, immune dysregulation, and intussusceptive angiogenesis. Degradation of endothelial glycocalyx exposes endothelial cell (EC) surface receptors to the vascular lumen, which renders pulmonary ECs more susceptible to SARS‐CoV‐2 infection. The present article reviews the potential pulmonary vascular pathophysiology and clinical presentations in COVID‐19 to provide a basis for clinicians and scientists, providing insights into the development of therapeutic strategies targeting pulmonary vasculature., SARS‐CoV‐2 infection‐induced pulmonary vascular injury and potential treatments target the pulmonary vascular system.

Angiogenesis supplies oxygen and nutrients to growing tumors. Inhibiting angiogenesis may stop tumor growth, but vascular endothelial growth factor inhibitors have limited effect in most tumors. This limited effect may be explained by an additional, less vascular endothelial growth factor-driven form of angiogenesis known as intussusceptive angiogenesis. The importance of intussusceptive angiogenesis in human tumors is not known. Epifluorescence and confocal microscopy was used to visualize intravascular pillars, the hallmark structure of intussusceptive angiogenesis, in tumors. Human malignant melanoma metastases, patient-derived melanoma xenografts in mice (PDX), and genetically engineered v-raf murine sarcoma viral oncogene homolog B1 (BRAF)-induced, phosphatase and TENsin homolog deleted on chromosome 10 (PTEN)-deficient (BPT) mice (BrafCA/+Ptenf/fTyr-Cre+/0-mice) were analyzed for pillars. Gene expression in human melanoma metastases and PDXs was analyzed by RNA sequencing. Matrix metalloproteinase 9 (MMP9) protein expression and T-cell and macrophage infiltration in tumor sections were determined with multiplex immunostaining. Intravascular pillars were detected in human metastases but rarely in PDXs and not in BPT mice. The expression of MMP9 mRNA was higher in human metastases compared with PDXs. High expression of MMP9 protein as well as infiltration of macrophages and T-cells were detected in proximity to intravascular pillars. MMP inhibition blocked formation of pillars, but not tubes or tip cells, invitro. In conclusion, intussusceptive angiogenesis may contribute to the growth of human melanoma metastases. MMP inhibition blocked pillar formation invitro and should be further investigated as a potential anti-angiogenic drug target in metastatic melanoma.

Introduction: Tumor agiogenesis is generally classified in to two types, namely, sprouting and intussusceptive angiogenesis. The formation of new blood vessels from the already existing is termed as sprouting angiogenesis. Many growth factors including endothelial growth factor (VEGF) contribute the formation of new vessels at the tumor sites. On other hand, the tumor able to split the existing blood vessels, this type is termed as intussuscepted angiogenesis and discovered in human colon adenocarcinoma xenograft. In intussuscepted angiogenesis, an existing blood vessel splits into two new blood vessels by formation of trans vascular pillars. Objective: The present study aimed to analyse the nicrovascular density on breast cancer patients using CD 34 immunostaining method. Results: This mis-regulation may lead to the development of cancers. Histological grading with Grade II were more with 19 (63.3%) cases followed by Grade I with 4 (13.3%) and Grade III with 3 (10%) cases. There was increase in mean vascular density in Grade II when compared with Grade I and Grade III. However no significant correlation was observed statistically with a P value of 0.176. Using different antibodies such as CD34, CD31, Factor VIII and CD105 to microvessels differentiation was highlighted. Conclusion: The results showed that the anti-CD34 monoclonal antibody is more sensitive than the anti- CD31 antibody in calculation of MVD in breast cancer as mentioned in previous studies.

Cirrhosis describes the development of excess fibrous tissue around regenerative nodules in response to chronic liver injury and usually leads to irreversible organ damage and end-stage liver disease. During the development of cirrhosis, the formation of collagenous scar tissue is paralleled by a reorganization and remodeling of the hepatic vascular system. To date, macrovascular remodeling in various cirrhosis models has been examined using three-dimensional (3D) imaging modalities, while microvascular changes have been studied mainly by two-dimensional (2D) light microscopic and electron microscopic imaging. Here, we report on the application of high-resolution 3D synchrotron radiation-based microtomography (SRμCT) for the study of the sinusoidal and capillary blood vessel system in three murine models of advanced parenchymal and biliary hepatic fibrosis. SRμCT facilitates the characterization of microvascular architecture and identifies features of intussusceptive angiogenesis in progressive liver fibrosis in a non-destructive 3D manner.

The interest on the role of angiogenesis in the pathogenesis and progression of human interstitial lung diseases is growing, with conventional sprouting (SA) and non-sprouting intussusceptive angiogenesis (IA) being differently represented in specific pulmonary injury patterns. The role of viruses as key regulators of angiogenesis is known for several years. A significantly enhanced amount of new vessel growth, through a mechanism of IA, has been reported in lungs of patients who died from Covid-19; among the angiogenesis-related genes, fibroblast growth factor 2 (FGF2) was found to be upregulated. These findings are intriguing. FGF2 plays a role in some viral infections: the upregulation is involved in the MERS-CoV-induced strong apoptotic response crucial for its highly lytic replication cycle in lung cells, whereas FGF2 is protective against the acute lung injury induced by H1N1 influenza virus, improving the lung wet-to-dry weight ratio. FGF2 plays a role also in regulating IA, acting on pericytes (crucial for the formation of intraluminal pillars), and endothelium, and FGF2-induced angiogenesis may be promoted by inflammation and hypoxia. IA is a faster and probably more efficient process than SA, able to modulate vascular remodeling through pruning of redundant or inefficient blood vessels. We can speculate that IA might have the function of restoring a functional vascular plexus consequently to extensive endothelialitis and alveolar capillary micro-thrombosis observed in Covid-19. Anti-Vascular endothelial growth factor (anti-VEGF) strategies are currently investigated for treatment of severe and critically ill Covid-19 patients, but also FGF2, and its expression and/or signaling, might represent a promising target.

Pericytes are essential components of capillaries in many tissues and organs, contributing to vessel stability and integrity, with additional contributions to microvascular function still being discovered. We review current and foundational studies identifying pericyte differentiation mechanics and their roles in the earliest stages of vessel formation. Recent advances in pericyte-focused tools and models have illuminated critical aspects of pericyte biology including their roles in vascular development. Pericytes likely collaborate with endothelial cells undergoing vasculogenesis, initiating direct interactions during sprouting and intussusceptive angiogenesis. Pericytes also provide important regulation of vascular growth including mechanisms underlying vessel pruning, rarefaction, and subsequent regrowth. A phenotypic transition likely occurs as pericytes shift from roles in vascular development to supporting vessel maturation, homeostasis, and physiology. We provide a forward-looking perspective on pericyte-focused studies of vascular development and applications aimed at basic and clinically relevant insights into pericyte biology.

This third study of the author is based on findings of the previous studies. The first study showed how Becker et. al. constructed the SARS CoV-2 Spike Protein in 2008, with approaches of reverse genetics, and inserted into the backbone of the SARS CoV from 2006 (1). The second study showed, how the three US research groups, involved in the synthetic production of the SARS CoV-2 spike protein, developed and matured the SARS CoV-2 virus by decades of laboratory experiments on recombinant human-animal coronavirus RNA through targeted RNA manipulation (2). This overview aims to investigate the publications on whether the constructed SARS CoV-2 virus can be transmitted between individuals. Requirements for infection and transmission of a virus are translation and replication of viral genome in the host. The findings show that the public has been deliberately deceived with false statements in publications to create the narrative of a bat-evolved virus that can be transmitted between humans. But in fact, a spike protein antigen RNA, called SARS CoV-2, was constructed that has neither the efficient function of translation or replication. The only function of the antigen is to enter the human cells to manipulate the human genome. The antigen is then activated by recurrent external injections misleadingly called vaccines, in order to initiate the spike protein antigen synthesis with a sensitive RNA Probe. Consequently, the entry of synthetic foreign genes into the human genome activates an alert system through a cascade of exacerbated immune reactions, which initiates a self destruction mechanism that leads to exodus of the individual. This prevents, at the price of human life, alien genetic material from being incorporated into the human genome and passed to next generations., This is the english version of the following study "Kann das SARS CoV-2 ��bertragen werden?" published December 1, 2021. The author declares no conflict of interests., {"references":["1. M.A. Emergence of synthetic recombinant bat-human spike protein in the Wuhan SARS CoV-2. Zenodo 2020.https://doi.org/10.5281/zenodo.4532195.","2. M.A. The Story about the constructed SARS COV-2 Virus - A Review of three Research Groups.2021;https://doi.org/10.5281/zenodo.4570941.","3. Lan T. Phan, Thuong V. Nguyen, Quang C. Luong, Thinh V. Nguyen, Hieu T. Nguyen, Hung Q. Le, Thuc T. Nguyen, Thang M. Cao, Quang D. Pham,Importation and Human-to-Human Transmission of a Novel Coronavirus in Vietnam. N Engl J med 2020; 382 (9).","4. 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Diese dritte Studie baut auf Erkenntnissen der vorherigen Studien auf. In der ersten Studie wurde nachgewiesen, wie Becker et. al. im Jahre 2008 das SARS CoV-2 Spike Protein durch Verfahren des Reverse Genetics hergestellt und in das Rückgrat des SARS CoV aus dem Jahre 2006 eingefügt haben(1). Die zweite Studie zeigte, dass die drei US-Forschungsgruppen, die an der künstlichen Herstellung des SARS CoV-2 Spike Proteins beteiligt waren, durch Jahrzehnte langen Laborexperimenten an rekombinanten Mensch-Tier Coronavirus RNA, das SARS CoV-2 Virus durch gezielte RNA Manipulation entwickelt und ausgereift haben(2). Diese Arbeit hat sich zur Aufgabe gemacht, die wissenschaftlichen Veröffentlichungen auf die Fragestellung zu untersuchen, ob das künstlich erstellte SARS CoV-2 Virus überhaupt zwischen Menschen übertragen werden kann. Die notwendige Voraussetzung zur Infektion und Übertragung einer Viruserkrankung ist die Virusvermehrung durch Transkription und Replikation des viralen Genoms im Wirtskörper. Die Studienlage zeigt, dass die Öffentlichkeit gezielt durch falsche Behauptungen in wissenschaftlichen Publikationen getäuscht wurde, um das Narrativ eines von Fledermäusen abstammenden Virus zu erschaffen, welches zwischen Menschen übertragen werden könnte. Tatsächlich wurde eine künstliche RNA mit einem Spike Protein Antigen, genannt SARS CoV-2, erstellt, welches weder eine effiziente Transkriptions- noch Replikationsfähigkeit hat. Dabei hat das Antigen zur Aufgabe in die menschlichen Zellen einzudringen, um dort Manipulationen am Genom vorzunehmen. Das Antigen wird dann durch externe Injektionen, hierbei irreführend Impfungen genannt, aktiviert, um die Spike Proteinsynthese, die durch rezidivierende Injektionen mit einer Spike Protein sensitiven RNA Probe auszulösen. Die Folgen eines Eindringens künstlich fremden Genmaterials in das menschliche Genom ist die Auslösung von Warnmechanismen, die durch eine Kaskade von exazerbierten Immunreaktionen, eine Art Selbstzerstörungsmechanismus einleitet und zum Exodus führen. Somit wird der Einbau fremden Genmaterials ins menschliche Genom, und die Vererbung an nächste Generationen, auf Kosten menschlichen Lebens, verhindert.

Diese dritte Studie baut auf Erkenntnissen der vorherigen Studien auf. In der ersten Studie wurde nachgewiesen, wie Becker et. al. im Jahre 2008 das SARS CoV-2 Spike Protein durch Verfahren des Reverse Genetics hergestellt und in das R��ckgrat des SARS CoV aus dem Jahre 2006 eingef��gt haben(1). Die zweite Studie zeigte, dass die drei US-Forschungsgruppen, die an der k��nstlichen Herstellung des SARS CoV-2 Spike Proteins beteiligt waren, durch Jahrzehnte langen Laborexperimenten an rekombinanten Mensch-Tier Coronavirus RNA, das SARS CoV-2 Virus durch gezielte RNA Manipulation entwickelt und ausgereift haben(2). Diese Arbeit hat sich zur Aufgabe gemacht, die wissenschaftlichen Ver��ffentlichungen auf die Fragestellung zu untersuchen, ob das k��nstlich erstellte SARS CoV-2 Virus ��berhaupt zwischen Menschen ��bertragen werden kann. Die notwendige Voraussetzung zur Infektion und ��bertragung einer Viruserkrankung ist die Virusvermehrung durch Transkription und Replikation des viralen Genoms im Wirtsk��rper. Die Studienlage zeigt, dass die ��ffentlichkeit gezielt durch falsche Behauptungen in wissenschaftlichen Publikationen get��uscht wurde, um das Narrativ eines von Flederm��usen abstammenden Virus zu erschaffen, welches zwischen Menschen ��bertragen werden k��nnte. Tats��chlich wurde eine k��nstliche RNA mit einem Spike Protein Antigen, genannt SARS CoV-2, erstellt, welches weder eine effiziente Transkriptions- noch Replikationsf��higkeit hat. Dabei hat das Antigen zur Aufgabe in die menschlichen Zellen einzudringen, um dort Manipulationen am Genom vorzunehmen. Das Antigen wird dann durch externe Injektionen, hierbei irref��hrend Impfungen genannt, aktiviert, um die Spike Proteinsynthese, die durch rezidivierende Injektionen mit einer Spike Protein sensitiven RNA Probe auszul��sen. Die Folgen eines Eindringens k��nstlich fremden Genmaterials in das menschliche Genom ist die Ausl��sung von Warnmechanismen, die durch eine Kaskade von exazerbierten Immunreaktionen, eine Art Selbstzerst��rungsmechanismus einleitet und zum Exodus f��hren. Somit wird der Einbau fremden Genmaterials ins menschliche Genom, und die Vererbung an n��chste Generationen, auf Kosten menschlichen Lebens, verhindert., The author declares no conflict of interests., {"references":["1. M.A. Emergence of synthetic recombinant bat-human spike protein in the Wuhan SARS CoV-2. Zenodo 2020.https://doi.org/10.5281/zenodo.4532195.","2. M.A. The Story about the constructed SARS COV-2 Virus - A Review of three Research Groups.2021;https://doi.org/10.5281/zenodo.4570941.","3. Lan T. Phan, Thuong V. Nguyen, Quang C. Luong, Thinh V. Nguyen, Hieu T. Nguyen, Hung Q. Le, Thuc T. Nguyen, Thang M. Cao, Quang D. Pham,Importation and Human-to-Human Transmission of a Novel Coronavirus in Vietnam. N Engl J med 2020; 382 (9).","4. Chaolin Huang, Yeming Wang, Xingwang Li, Lili Ren, Jianping Zhao, Yi Hu, Li Zhang, Guohui Fan, Jiuyang Xu, Xiaoying Gu,Zhenshun Cheng, Ting Yu, Jiaan Xia, Yuan Wei, Wenjuan Wu, Xuelei Xie, Wen Yin, Hui Li, Min Liu, Yan Xiao, Hong Gao, Li Guo, Jungang Xie,Guangfa Wang, Rongmeng Jiang, Zhancheng Gao, Qi Jin, Jianwei Wang, Bin Cao, Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. The Lancet 2020; 395: 497-506.","5. World Health Organization. Classifications https://www.who.int/standards/classifications/classification-of-diseases, 2021 [accessed 28.11.2021]","6. Sufang Tian, Yong Xiong, Huan Liu, Li Niu, Jianchun Guo, Meiyan Liao, Shu-Yuan Xiao, Pathological study of the 2019 novel coronavirus disease (COVID-19) through postmortem core biopsies.Modern Pathology 2020; 33:1007–1014.","7. https://www.gatesfoundation.org/How-We-Work/Quick-Links/Grants-Database#q/k=chinese%20academy&page=2, 2021 [accessed 2.3.2021]","8. 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American journal of respiratory and critical care medicine : AJRCCM 205(1), 121-125 (2022). doi:10.1164/rccm.202103-0594IM, Published by American Thoracic Society, New York, NY

The formation of new blood vessels and the establishment of vascular networks are crucial during brain development, in the adult healthy brain, as well as in various diseases of the central nervous system. Here, we describe a step-by-step protocol for our recently developed method that enables hierarchical imaging and computational analysis of vascular networks in postnatal and adult mouse brains. The different stages of the procedure include resin-based vascular corrosion casting, scanning electron microscopy, synchrotron radiation and desktop microcomputed tomography imaging, and computational network analysis. Combining these methods enables detailed visualization and quantification of the 3D brain vasculature. Network features such as vascular volume fraction, branch point density, vessel diameter, length, tortuosity and directionality as well as extravascular distance can be obtained at any developmental stage from the early postnatal to the adult brain. This approach can be used to provide a detailed morphological atlas of the entire mouse brain vasculature at both the postnatal and the adult stage of development. Our protocol allows the characterization of brain vascular networks separately for capillaries and noncapillaries. The entire protocol, from mouse perfusion to vessel network analysis, takes ~10 d. This protocol uses vascular corrosion casting, hierarchical synchrotron radiation microcomputed tomography imaging and computational image analysis to assess the 3D vascular network architecture in the entire postnatal and adult mouse brain.

Coronavirus disease-2019 (COVID-19), the disease caused by severe acute respiratory syndrome-coronavirus-2, has claimed more than 4.4 million lives worldwide (as of 20 August 2021). Severe cases of the disease often result in respiratory distress due to cytokine storm, and mechanical ventilation is required. Although, the lungs are the primary organs affected by the disease, more evidence on damage to the heart, kidney, and liver is emerging. A common link in these connections is the cardiovascular network. Inner lining of the blood vessels, called endothelium, is formed by a single layer of endothelial cells. Several clinical manifestations involving the endothelium have been reported, such as its activation via immunomodulation, endotheliitis, thrombosis, vasoconstriction, and distinct intussusceptive angiogenesis (IA), a unique and rapid process of blood-vessel formation by splitting a vessel into two lumens. In fact, the virus directly infects the endothelium via TMPRSS2 spike glycoprotein priming to facilitate ACE-2-mediated viral entry. Recent studies have indicated a significant increase in remodeling of the pulmonary vascular bed via intussusception in patients with COVID-19. However, the lack of circulatory biomarkers for IA limits its detection in COVID-19 pathogenesis. In this review, we describe the implications of angiogenesis in COVID-19, unique features of the pulmonary vascular bed and its remodeling, and a rapid and non-invasive assessment of IA to overcome the technical limitations in patients with COVID-19.

SARS-CoV-2-associated acute respiratory distress syndrome (ARDS) and acute lung injury are life-threatening manifestations of severe viral infection. The pathogenic mechanisms that lead to respiratory complications, such as endothelialitis, intussusceptive angiogenesis, and vascular leakage remain unclear. In this study, by using an immunofluorescence assay and in situ RNA-hybridization, we demonstrate the capability of SARS-CoV-2 to infect human primary lung microvascular endothelial cells (HL-mECs) in the absence of cytopathic effects and release of infectious particles. Preliminary data point to the role of integrins in SARS-CoV-2 entry into HL-mECs in the absence of detectable ACE2 expression. Following infection, HL-mECs were found to release a plethora of pro-inflammatory and pro-angiogenic molecules, as assessed by microarray analyses. This conditioned microenvironment stimulated HL-mECs to acquire an angiogenic phenotype. Proteome analysis confirmed a remodeling of SARS-CoV-2-infected HL-mECs to inflammatory and angiogenic responses and highlighted the expression of antiviral molecules as annexin A6 and MX1. These results support the hypothesis of a direct role of SARS-CoV-2-infected HL-mECs in sustaining vascular dysfunction during the early phases of infection. The construction of virus-host interactomes will be instrumental to identify potential therapeutic targets for COVID-19 aimed to inhibit HL-mEC-sustained inflammation and angiogenesis upon SARS-CoV-2 infection.

Mosaic inactivation of CCM2 in humans causes cerebral cavernous malformations (CCMs) containing adjacent dilated blood-filled multi-cavernous lesions. We used CRISPR-Cas9 mutagenesis to induce mosaic inactivation of zebrafish ccm2 resulting in a novel lethal multi-cavernous lesion in the embryonic caudal venous plexus (CVP) caused by obstruction of blood flow by intraluminal pillars. These pillars mimic those that mediate intussusceptive angiogenesis; however, in contrast to the normal process, the pillars failed to fuse to split the pre-existing vessel in two. Abortive intussusceptive angiogenesis stemmed from mosaic inactivation of ccm2 leading to patchy klf2a overexpression and resultant aberrant flow signaling. Surviving adult fish manifested histologically typical hemorrhagic CCM. Formation of mammalian CCM requires the flow-regulated transcription factor KLF2; fish CCM and the embryonic CVP lesion failed to form in klf2a null fish indicating a common pathogenesis with the mammalian lesion. These studies describe a zebrafish CCM model and establish a mechanism that can explain the formation of characteristic multi-cavernous lesions.

Novel Coronavirus Disease 2019 (Covid-19) is associated with multi-systemic derangement, including circulatory dysfunction with features of endothelial dysfunction, microangiopathic thrombosis and angiocentric inflammation. Recently, intussusceptive angiogenesis has been implicated in the pathogenesis of the disease. Herein, we conducted a narrative review according to the SANRA guidelines to review and discuss data regarding splitting angiogenesis including mechanisms, drivers, regulators and putative roles. Relevant angiogenic features in Covid-19, including their potential role in inflammation, endothelial dysfunction and permeability, as well as their use as prognostic markers and therapeutic roles are reviewed. Splitting angiogenesis in Covid-19 involve hypoxia, hypoxia-inducible factors, classic angiogenic mediators, such as the Vascular Endothelial Growth Factor (VEGF), Angiopoietins, hyperinflammation and cytokine storm, and dysregulation of the Renin-Angiotensin-Aldosterone System, which combined, interact to promote intussusception. Data regarding the use of angiogenic mediators as prognostic tools is summarized and suggest that angiopoietins and VEGF are elevated in Covid-19 patients and predictors of adverse outcomes. Finally, we reviewed the scarce data regarding angiogenic mediators as therapeutic targets. These preliminary findings suggest a potential benefit of bevacizumab as an add-on therapy.

The vascular endothelium is characterized by a remarkable level of plasticity, which is the driving force not only of physiological repair/remodeling of adult tissues but also of pathological angiogenesis. The resulting heterogeneity of endothelial cells (ECs) makes targeting the endothelium challenging, no less because many EC phenotypes are yet to be identified and functionally inventorized. Efforts to map the vasculature at the single-cell level have been instrumental to capture the diversity of EC types and states at a remarkable depth in both normal and pathological states. Here, we discuss new EC subtypes and functions emerging from recent single-cell studies in health and disease. Interestingly, such studies revealed distinct metabolic gene signatures in different EC phenotypes, which deserve further consideration for therapy. We highlight how this metabolic targeting strategy could potentially be used to promote (for tissue repair) or block (in tumor) angiogenesis in a tissue or even vascular bed-specific manner. ispartof: ANGIOGENESIS vol:24 issue:2 pages:311-326 ispartof: location:Germany status: published

Introduction: As the management of COVID-19 has continued to evolve, the question as to whether delaying intubation is beneficial or harmful for patients with COVID-19 induced hypoxic respiratory failure has yet to be answered. Early reports suggested that patients may benefit from early intubation during a period of severe hypoxia;later management shifted towards delaying intubation as much as possible using non-invasive ventilation. Additionally, the pathophysiologic implications of timing of intubation are poorly understood, including the potential for pulmonary vascular thrombosis and intussusceptive angiogenesis. This study examines the differences in outcomes and respiratory mechanics between subjects who are intubated earlier versus later in their COVID-19 disease course. Study Design and Methods: Retrospective single-center cohort study of subjects intubated for COVID-19 ARDS between March and June 2020. Patients were stratified by time to intubation: 30 subjects were intubated 4-24 hours after presentation and 24 subjects were intubated 5-10 days after presentation. Data regarding baseline characteristics, hospitalization, ventilator mechanics and outcomes were extracted and analyzed. 10 clinically available CT scans for these patients were manually reviewed to identify evidence of pulmonary vascular thrombosis and intussusceptive angiogenesis. Results: Median time from symptom onset to intubation was significantly different between the Early and Late Intubation Cohorts, with the latter being intubated later in the course of their illness (7.9 days vs 11.8 days;p=0.04). The Early Intubation Cohort had a lower mortality rate than the Late Intubation Cohort (6% vs 30%, p < 0.001) without significantly different ventilator mechanics at the time of intubation. However, the Late Intubation Cohort was noted to have higher dead space ratio (0.40 vs 0.52;p = 0.03). On review of CT scans, the Late Intubation Cohort also had more segments with dilated and tortuous peripheral vessels on imaging (2 segments vs 5 segments). Interpretation: As our approaches to treatment of COVID-19 continue to evolve, the decision of timing of intubation remains paramount. While non-invasive ventilation may allow for delaying intubation, it is possible that there are downstream effects of delayed intubation that should be considered.

Objectives:. Determine the variation in outcomes and respiratory mechanics between the subjects who are intubated earlier versus later in their coronavirus disease 2019 course. Design:. Retrospective cohort study. Setting:. Northwestern Memorial Hospital ICUs. Patients:. All patients intubated for coronavirus disease 2019 between March 2020 and June 2020. Interventions:. Patients were stratified by time to intubation: 30 subjects were intubated 4–24 hours after presentation and 24 subjects were intubated 5–10 days after presentation. Baseline characteristics, hospitalization, ventilator mechanics, and outcomes were extracted and analyzed. Ten clinically available CT scans were manually reviewed to identify evidence of pulmonary vascular thrombosis and intussusceptive angiogenesis. Measurements and Main Results:. Median time from symptom onset to intubation was significantly different between the early and late intubation cohorts, with the latter being intubated later in the course of their illness (7.9 vs 11.8 d; p = 0.04). The early intubation cohort had a lower mortality rate than the late intubation cohort (6% vs 30%, p < 0.001) without significantly different respiratory mechanics at the time of intubation. The late intubation cohort was noted to have higher dead space ratio (0.40 vs 0.52; p = 0.03). On review of CT scans, the late intubation cohort also had more dilated peripheral segments on imaging (two segments vs five segments). Conclusions:. The question as to whether delaying intubation is beneficial or harmful for patients with coronavirus disease 2019-induced hypoxemic respiratory failure has yet to be answered. As our approaches to coronavirus disease 2019 continue to evolve, the decision of timing of intubation remains paramount. Although noninvasive ventilation may allow for delaying intubation, it is possible that there are downstream effects of delayed intubation that should be considered, including the potential for pulmonary vascular thrombosis and intussusceptive angiogenesis with delayed intubation.

The precise mechanisms of SDF‐1 (CXCL12) in angiogenesis are not fully elucidated. Recently, we showed that Notch inhibition induces extensive intussusceptive angiogenesis by recruitment of mononuclear cells and it was associated with increased levels of SDF‐1 and CXCR4. In the current study, we demonstrated SDF‐1 expression in liver sinusoidal vessels of Notch1 knockout mice with regenerative hyperplasia by means of intussusception, but we did not detect any SDF‐1 expression in wild‐type mice with normal liver vessel structure. In addition, pharmacological inhibition of SDF‐1/CXCR4 signalling by AMD3100 perturbs intussusceptive vascular growth and abolishes mononuclear cell recruitment in the chicken area vasculosa. In contrast, treatment with recombinant SDF‐1 protein increased microvascular density by 34% through augmentation of pillar number compared to controls. The number of extravasating mononuclear cells was four times higher after SDF‐1 application and two times less after blocking this pathway. Bone marrow‐derived mononuclear cells (BMDC) were recruited to vessels in response to elevated expression of SDF‐1 in endothelial cells. They participated in formation and stabilization of pillars. The current study is the first report to implicate SDF‐1/CXCR4 signalling in intussusceptive angiogenesis and further highlights the stabilizing role of BMDC in the formation of pillars during vascular remodelling.

Aims Hepatic capillaries are lined with specialized liver sinusoidal endothelial cells (LSECs) which support macromolecule passage to hepatocytes and prevent fibrosis by keeping hepatic stellate cells (HSCs) quiescent. LSEC specialization is co-determined by transcription factors. The zinc-finger E-box-binding homeobox (Zeb)2 transcription factor is enriched in LSECs. Here, we aimed to elucidate the endothelium-specific role of Zeb2 during maintenance of the liver and in liver fibrosis. Methods and results To study the role of Zeb2 in liver endothelium we generated EC-specific Zeb2 knock-out (ECKO) mice. Sequencing of liver EC RNA revealed that deficiency of Zeb2 results in prominent expression changes in angiogenesis-related genes. Accordingly, the vascular area was expanded and the presence of pillars inside ECKO liver vessels indicated that this was likely due to increased intussusceptive angiogenesis. LSEC marker expression was not profoundly affected and fenestrations were preserved upon Zeb2 deficiency. However, an increase in continuous EC markers suggested that Zeb2-deficient LSECs are more prone to dedifferentiation, a process called ‘capillarization’. Changes in the endothelial expression of ligands that may be involved in HSC quiescence together with significant changes in the expression profile of HSCs showed that Zeb2 regulates LSEC–HSC communication and HSC activation. Accordingly, upon exposure to the hepatotoxin carbon tetrachloride (CCl4), livers of ECKO mice showed increased capillarization, HSC activation, and fibrosis compared to livers from wild-type littermates. The vascular maintenance and anti-fibrotic role of endothelial Zeb2 was confirmed in mice with EC-specific overexpression of Zeb2, as the latter resulted in reduced vascularity and attenuated CCl4-induced liver fibrosis. Conclusion Endothelial Zeb2 preserves liver angioarchitecture and protects against liver fibrosis. Zeb2 and Zeb2-dependent genes in liver ECs may be exploited to design novel therapeutic strategies to attenuate hepatic fibrosis.

Viral respiratory diseases constitute the most common reasons for hospitalization with more than half of all acute illnesses worldwide. Progressive respiratory failure with pronounced diffuse alveolar damage has been identified as the primary cause of death in COVID-19. COVID-19 pneumonia shares common histopathological hallmarks with influenza (H1N1)-related ARDS, like diffuse alveolar damage (DAD) with edema, hemorrhage, and intra-alveolar fibrin deposition. The lungs with COVID-19 pneumonia revealed perivascular inflammation, an endothelial injury, microangiopathy, and an aberrant blood vessel neoformation by intussusceptive angiogenesis. While this pronounced angiocentric inflammation is likely be found - to varying degrees - in numerous other organs, e.g., the heart, COVID-19 is hypothesized to be not just a pulmonary, but rather a systemic "vascular disease."Neben der jährlich saisonal auftretenden Influenzaendemie stellt das sich pandemisch verbreitende SARS-CoV-2-Virus die weltweiten Gesundheitssysteme als vaskuläre Multiorganerkrankung COVID-19 vor ganz neue medizinische und sozioökonomische Herausforderungen. Bei beiden Viruserkrankungen kommt es insbesondere bei schweren Krankheitsverläufen zu einem fulminanten Lungenversagen, dem „acute respiratory distress syndrome“ (ARDS), welchem morphologisch meist ein unspezifischer diffuser Alveolarschaden mit hyalinen Membranen, Fibrinexsudaten und einem intraalveolären Ödem zugrunde liegt. Neben einer Infektion des Alveolarepithels und von Makrophagen durch das SARS-CoV-2-Virus scheint die COVID-19-Erkrankung zusätzlich durch eine vaskulozentrische Entzündungsreaktion mit Mikroangiopathie und aberranter Blutgefäßneubildung durch die sog. intussuszeptive Angiogenese charakterisiert zu sein. Zudem finden sich wahrscheinlich auf dem Boden dieser primär systemischen angiozentrischen Entzündung in vielen weiteren Organen, z. B. dem Herzen, im unterschiedlichen Ausmaß Gewebeschädigungen.

Intussusceptive angiogenesis (IA) is an important physiological form of angiogenesis in which an existing vessel splits in two by the formation of an intraluminal tissue pillar. The presence of these intraluminal pillars form the hallmark of ongoing IA in growing vascular beds. However, their visualization is technically challenging. The goal of this systematic review was to investigate which techniques are being used to identify intraluminal pillars and to formulate important points to keep in mind when studying IA. A systematic literature search resulted in 154 evaluated articles of which the majority (65%) provided sufficient data to unambiguously demonstrate the presence of intraluminal pillars. Scanning electron microscopy imaging of vascular corrosion casts and serial sectioning of ultrathin sections are the most used techniques. New methods such as serial block face scanning electron microscopy and micro computed tomography (mu CT) are gaining importance. Moreover, our results indicate that IA was studied in a variety of animals and tissues. IA is a biologically very relevant form of angiogenesis. Techniques to visualize intraluminal pillars need to have a minimal resolution of 1 mu m and should provide information on the 3D-nature of the pillars. Optimally, several techniques are combined to demonstrate ongoing IA.

eLife 10, e71359 (2021). doi:10.7554/eLife.71359, For the first time, we have used phase-contrast X-ray tomography to characterize the three-dimensional (3d) structure of cardiac tissue from patients who succumbed to Covid-19. By extending conventional histopathological examination by a third dimension, the delicate pathological changes of the vascular system of severe Covid-19 progressions can be analyzed, fully quantified and compared to other types of viral myocarditis and controls. To this end, cardiac samples with a cross-section of 3.5mm were scanned at a laboratory setup as well as at a parallel beam setup at a synchrotron radiation facility the synchrotron in a parallel beam configuration. The vascular network was segmented by a deep learning architecture suitable for 3d datasets (V-net), trained by sparse manual annotations. Pathological alterations of vessels, concerning the variation of diameters and the amount of small holes, were observed, indicative of elevated occurrence of intussusceptive angiogenesis, also confirmed by high-resolution cone beam X-ray tomography and scanning electron microscopy. Furthermore, we implemented a fully automated analysis of the tissue structure in the form of shape measures based on the structure tensor. The corresponding distributions show that the histopathology of Covid-19 differs from both influenza and typical coxsackie virus myocarditis., Published by eLife Sciences Publications, Cambridge

Cerebral small-vessel diseases (SVDs) often follow a progressive course. Little is known about the function of angiogenesis, which potentially induces regression of SVDs. Here, we investigated angiogenesis in a mouse model of incontinentia pigmenti (IP), a genetic disease comprising features of SVD. IP is caused by inactivating mutations of Nemo, the essential component of NF-κB signaling. When deleting Nemo in the majority of brain endothelial cells ( NemobeKOmice), the transcriptional profile of vessels indicated cell proliferation. Brain endothelial cells expressed Ki67 and showed signs of DNA synthesis. In addition to cell proliferation, we observed sprouting and intussusceptive angiogenesis in NemobeKOmice. Angiogenesis occurred in all segments of the vasculature and in proximity to vessel rarefaction and tissue hypoxia. Apparently, NEMO was required for productive angiogenesis because endothelial cells that had escaped Nemo inactivation showed a higher proliferation rate than Nemo-deficient cells. Therefore, newborn endothelial cells were particularly vulnerable to ongoing recombination. When we interfered with productive angiogenesis by inducing ongoing ablation of Nemo, mice did not recover from IP manifestations but rather showed severe functional deficits. In summary, the data demonstrate that angiogenesis is present in this model of SVD and suggest that it may counterbalance the loss of vessels.

Anti-angiogenic therapies (AATs) have been widely used for cancer treatment. But the beneficial effects of AATs are short, because AAT-induced tumor revascularization facilitates the tumor relapse. In this mini-review, we described different forms of tumor neovascularization and revascularization including sprouting angiogenesis, vessel co-option, intussusceptive angiogenesis, and vasculogenic mimicry, all of which are closely mediated by vascular endothelial growth factor (VEGF), angiopoietins, matrix metalloproteinases, and exosomes. We also summarized the current findings for the resistance mechanisms of AATs including enhancement in pro-angiogenic cytokines, heterogeneity in tumor-associated endothelial cells (ECs), crosstalk between tumor cells and ECs, masking of extracellular vesicles, matrix stiffness and contributions from fibroblasts, macrophages and adipocytes in the tumor microenvironment. We highlighted the revascularization following AATs, particularly the role of exosome stimulating factors such as hypoxia and miRNA, and that of exosomal cargos such as cytokines, miRNAs, lncRNAs, and circRNAs from the tumor ECs in angiogenesis and revascularization. Finally, we proposed that renormalization of tumor ECs would be a more efficient cancer therapy than the current AATs.

The ongoing coronavirus disease 2019 (COVID-19) pandemic has affected millions worldwide and has been found to cause severe disease in patients with underlying comorbidities. In patients with known malignancies, in addition to constraints in routine healthcare, the risk of being susceptible to developing severe forms of the disease is of grave concern. While follow-up studies on survivors of the severe acute respiratory syndrome (SARS) 2003 outbreak revealed increased susceptibility to infections, tumours and cardiovascular abnormalities, recent studies implicating angiopoietin 2 in induction of inflammatory intussusceptive angiogenesis and diffuse alveolar damage in COVID-19 patients raises the possibility of progression of carcinogenetic processes in patients with known malignancies. Angiotensin converting enzyme-2 (ACE-2) mediated cellular entry of SARS-Cov2 leads to receptor shedding of ACE-2 and disrupts the renin angiotensin aldosterone axis (RAAS). This augments the pro-inflammatory and proliferative effects of RAAS, while attenuating the anti-inflammatory and anti-proliferative angiotensin 1-7 /Mas pathway. Angiopoietin-2, a molecule responsible for angiogenesis and cancer progression which corelates with tumour load in certain cancers, is upregulated by angiotensin 2-AT1 Receptor axis. Tumour microenvironment—comprising of various cells, blood vessels and extra cellular matrix which express the RAAS peptides—plays a key role in cancer initiation, progression and metastasis. Angiotensin 2 induces the formation of a desmoplastic environment, favouring cancer cell growth. ACE-2 downregulation causes bradykinin accumulation which may exert its proliferative action via mitogen activated protein kinase pathways which has established roles in cancers of breast and kidney. In addition to cytokine storm causing organ damage, acute inflammation in COVID-19 may also cause epithelial mesenchymal transition and heat shock protein 27 phosphorylation, both of which are key mediators in cancer signalling pathways. We hypothesise that SARS-Cov2, by impacting the RAAS and immune system, has the potential to cause tumour cell proliferation, apoptosis evasion and metastasis, thereby increasing the possibility of cancer progression in patients with known malignancies.

BACKGROUND: Endothelial cells (ECs) line the blood vessel lumen and respond to fluid shear stress. ECs are also responsible for initiating the formation of new blood vessels. These new vessels can form either by sprouting angiogenesis or intussusceptive, or splitting, angiogenesis. The latter is poorly understood but recent evidence suggests that intussusception occurs with altered blood flow. However, the biomechanical and biochemical mechanisms of intussusceptive angiogenesis remain largely unknown and in vitro models do not exist. The purpose of this thesis was to develop a three-dimensional EC culture model capable of forming transluminal pillars, a key step in angiogenesis, and to utilize this model to identify regulators of pillar formation. METHODS: I developed a novel 3D microvessel model using microfluidic channels of circumferentially lined with a confluent monolayer of endothelial cells that are exposed to range of shear stress from physiological to ultra-low. Cells were immunostained for VE-Cadherin, primary cilia, and phosphorylated VEGFR2 (pVEGFR2). A similar microfluidic model was used to co-culture HUVECs differentially treated with control or VEGFR2 siRNA and subjected to ultra-low shear stress to directly test the role of VEGFR2 in pillar formation. RESULTS: Endothelial cells exposed to ultra-low shear stress showed marked changes in morphology compared to cells exposed to physiological shear stress, including changes in elongation and alignment in the direction of flow, and cytoskeletal reorganization. Furthermore, abundance of primary cilia was increased in HUVECs exposed to ultra-low shear stress, alongside a decrease in the expression of pVEGFR2. A co-culture of HUVECs differentially transfected with control siRNA or VEGFR2 siRNA demonstrated changes in cell morphology dependent on VEGFR2 content. CONCLUSIONS: I established a 3D microfluidic culture model of endothelial cells that successfully yields transluminal endothelial pillars. Pillar formation was dependent on both altered shear stresses and expression and/or activities of shear sensors.

The human body contains 60 000 miles of blood vessels, including at least 19 billion capillaries, so that under physiological conditions cells are located no further than 100–200 μm from the nearest capillary. In those, endothelial cells and pericytes seem to play a pivotal role in COVID-19 by binding SARS-CoV-2 to the angiotensin-converting-enzyme 2 (ACE-2) [1, 2]. In the lung, the transmembrane ACE-2 receptor is predominantly expressed in endothelial cells, perivascular pericytes, and type 2 cells [2, 3]., Inflammation and Intussusceptive Angiogenesis in COVID-19