Assessing Molecular Regulation of Vascular Permeability Using a VEGF-Inducible Zebrafish Model.

Vascular endothelial growth factor (VEGF) stimulates vascular permeability in a variety of human pathologies, such as cancer, ischemic stroke, cardiovascular disease, retinal conditions, and COVID-19-associated pulmonary edema, sepsis, acute lung injury, and acute respiratory distress syndrome. Comprehensive investigation of the molecular mechanisms of VEGF-induced vascular permeability has been hindered by the lack of in vivo models that easily facilitate genetic manipulation studies in real time. To address this need, we generated a heat-inducible VEGF transgenic zebrafish model of vascular permeability. Here, we describe how this zebrafish model can be used to monitor VEGF-induced vascular permeability through live in vivo imaging to identify genetic regulators that play key roles in vascular barrier integrity in physiological conditions and human disease processes.

Renin-Angiotensin System: An Important Player in the Pathogenesis of Acute Respiratory Distress Syndrome.

Acute respiratory distress syndrome (ARDS) is characterized by massive inflammation, increased vascular permeability and pulmonary edema. Mortality due to ARDS remains very high and even in the case of survival, acute lung injury can lead to pulmonary fibrosis. The renin-angiotensin system (RAS) plays a significant role in these processes. The activities of RAS molecules are subject to dynamic changes in response to an injury. Initially, increased levels of angiotensin (Ang) II and des-Arg9-bradykinin (DABK), are necessary for an effective defense. Later, augmented angiotensin converting enzyme (ACE) 2 activity supposedly helps to attenuate inflammation. Appropriate ACE2 activity might be decisive in preventing immune-induced damage and ensuring tissue repair. ACE2 has been identified as a common target for different pathogens. Some Coronaviruses, including SARS-CoV-2, also use ACE2 to infiltrate the cells. A number of questions remain unresolved. The importance of ACE2 shedding, associated with the release of soluble ACE2 and ADAM17-mediated activation of tumor necrosis factor-α (TNF-α)-signaling is unclear. The roles of other non-classical RAS-associated molecules, e.g., alamandine, Ang A or Ang 1-9, also deserve attention. In addition, the impact of established RAS-inhibiting drugs on the pulmonary RAS is to be elucidated. The unfavorable prognosis of ARDS and the lack of effective treatment urge the search for novel therapeutic strategies. In the context of the ongoing SARS-CoV-2 pandemic and considering the involvement of humoral disbalance in the pathogenesis of ARDS, targeting the renin-angiotensin system and reducing the pathogen's cell entry could be a promising therapeutic strategy in the struggle against COVID-19.

The SARS-CoV-2 spike protein alters barrier function in 2D static and 3D microfluidic in-vitro models of the human blood-brain barrier.

As researchers across the globe have focused their attention on understanding SARS-CoV-2, the picture that is emerging is that of a virus that has serious effects on the vasculature in multiple organ systems including the cerebral vasculature. Observed effects on the central nervous system include neurological symptoms (headache, nausea, dizziness), fatal microclot formation and in rare cases encephalitis. However, our understanding of how the virus causes these mild to severe neurological symptoms and how the cerebral vasculature is impacted remains unclear. Thus, the results presented in this report explored whether deleterious outcomes from the SARS-CoV-2 viral spike protein on primary human brain microvascular endothelial cells (hBMVECs) could be observed. The spike protein, which plays a key role in receptor recognition, is formed by the S1 subunit containing a receptor binding domain (RBD) and the S2 subunit. First, using postmortem brain tissue, we show that the angiotensin converting enzyme 2 or ACE2 (a known binding target for the SARS-CoV-2 spike protein), is ubiquitously expressed throughout various vessel calibers in the frontal cortex. Moreover, ACE2 expression was upregulated in cases of hypertension and dementia. ACE2 was also detectable in primary hBMVECs maintained under cell culture conditions. Analysis of cell viability revealed that neither the S1, S2 or a truncated form of the S1 containing only the RBD had minimal effects on hBMVEC viability within a 48 h exposure window. Introduction of spike proteins to invitro models of the blood-brain barrier (BBB) showed significant changes to barrier properties. Key to our findings is the demonstration that S1 promotes loss of barrier integrity in an advanced 3D microfluidic model of the human BBB, a platform that more closely resembles the physiological conditions at this CNS interface. Evidence provided suggests that the SARS-CoV-2 spike proteins trigger a pro-inflammatory response on brain endothelial cells that may contribute to an altered state of BBB function. Together, these results are the first to show the direct impact that the SARS-CoV-2 spike protein could have on brain endothelial cells; thereby offering a plausible explanation for the neurological consequences seen in COVID-19 patients.

Vascular underpinning of COVID-19.

COVID-19 management guidelines have largely attributed critically ill patients who develop acute respiratory distress syndrome, to a systemic overproduction of pro-inflammatory cytokines. Cardiovascular dysfunction may also represent a primary phenomenon, with increasing data suggesting that severe COVID-19 reflects a confluence of vascular dysfunction, thrombosis and dysregulated inflammation. Here, we first consolidate the information on localized microvascular inflammation and disordered cytokine release, triggering vessel permeability and prothrombotic conditions that play a central role in perpetuating the pathogenic COVID-19 cascade. Secondly, we seek to clarify the gateways which SARS-CoV-2, the causative COVID-19 virus, uses to enter host vascular cells. Post-mortem examinations of patients' tissues have confirmed direct viral endothelial infection within several organs. While there have been advances in single-cell RNA sequencing, endothelial cells across various vascular beds express low or undetectable levels of those touted SARS-CoV-2 entry factors. Emerging studies postulate alternative pathways and the apicobasal distribution of host cell surface factors could influence endothelial SARS-CoV-2 entry and replication. Finally, we provide experimental considerations such as endothelial polarity, cellular heterogeneity in organoids and shear stress dynamics in designing cellular models to facilitate research on viral-induced endothelial dysfunctions. Understanding the vascular underpinning of COVID-19 pathogenesis is crucial to managing outcomes and mortality.

Pulmonary Edema in COVID19-A Neural Hypothesis.

In COVID-19, lung manifestations present as a slowly evolving pneumonia with insidious early onset interstitial pulmonary edema that undergoes acute exacerbation in the late stages and microvascular thrombosis. Currently, these manifestations are considered to be only consequences of pulmonary SARS-CoV-2 virus infection. We are proposing a new hypothesis that neurogenic insult may also play a major role in the pathogenesis of these manifestations. SARS-CoV-2 mediated inflammation of the nucleus tractus solitarius (NTS) may play a role in the acute exacerbation of pulmonary edema and microvascular clotting in COVID-19 patients.

The vascular endothelium: the cornerstone of organ dysfunction in severe SARS-CoV-2 infection.

In severe SARS-CoV-2 infections, emerging data including recent histopathological studies have emphasized the crucial role of endothelial cells (ECs) in vascular dysfunction, immunothrombosis, and inflammation.Histopathological studies have evidenced direct viral infection of ECs, endotheliitis with diffuse endothelial inflammation, and micro- and macrovascular thrombosis both in the venous and arterial circulations. Venous thrombotic events, particularly pulmonary embolism, with elevated D-dimer and coagulation activation are highly prevalent in COVID-19 patients. The pro-inflammatory cytokine storm, with elevated levels of interleukin-6 (IL-6), IL-2 receptor, and tumor necrosis factor-α, could also participate in endothelial dysfunction and leukocyte recruitment in the microvasculature. COVID-19-induced endotheliitis may explain the systemic impaired microcirculatory function in different organs in COVID-19 patients. Ongoing trials directly and indirectly target COVID-19-related endothelial dysfunctions: i.e., a virus-cell entry using recombinant angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS-2) blockade, coagulation activation, and immunomodulatory therapies, such as anti-IL-6 strategies. Studies focusing on endothelial dysfunction in COVID-19 patients are warranted as to decipher their precise role in severe SARS-CoV-2 infection and organ dysfunction and to identify targets for further interventions.