Ceramide Mediates COVID-19 Vascular Injury and ARDS

Background: A series of cases of pneumonia of unknown etiology reported in late 2019 in Wuhan, China were found to be due to infection with a novel betacoronavirus, SARS-CoV-2. The clinical syndrome, COVID-19, rapidly became a pandemic affecting more than 38 million people with >600,000 deaths in the U.S. Severe viral pneumonia leading to Acute Respiratory Distress Syndrome (CoV2-ARDS) is a major driver of morbidity and mortality in COVID-19, and is present in >90% of COVID-19 fatalities. Endothelial damage in the lung leads to CoV2-ARDS with injured endothelial cells releasing pro-inflammatory cytokines, intensifying lung injury, hypoxic vasoconstriction, and hypoxemia. Although SARS-CoV-2 can directly infect endothelial cells, the virus does not replicate there; associated endothelial injury is most likely indirect or downstream of direct cellular infection; the detailed mechanisms of this injury are not yet well understood. This gap explains in part why no current direct-acting antiviral nor immune modulator appears to target the vascular damage unique to CoV2-ARDS. Rationale: Infection of host cells with SARS-CoV-2 leads to unopposed angiotensin 2 (Ang-2) signaling that culminates in vascular endothelial cell damage by activation of acid sphingomyelinase (ASMase), leading to ceramide generation at the endothelial plasma membrane. Plasma membrane ceramide elevations above a certain threshold lead to formation therein of macrodomains (>1 µm in length) we term ceramide-rich platforms, structures that in turn trigger endothelial injury, ultimately culminating in apoptosis. C16:0 ceramide, a ceramide species formed at high levels in patients with CoV2-ARDS, is uniquely capable of ceramide-rich platform generation compared with other naturally occurring ceramide species and is the focus of this application. The ASMase/ceramide pathway can also be activated by oxidative stress, and inflammatory/stress mediators that characterize severe viral pneumonia, amplifying the Ang2 response. Ongoing investigations into the levels of circulating C16:0 ceramide in COVID-19 patient serum and at autopsy in lung specimens from patients that have succumbed to CoV2-ARDS demonstrate a specific, marked C16:0 ceramide elevation. These studies provide strong support for the hypothesis that microvascular injury from human coronavirus infection is ceramide-mediated, as endothelial cells are a primary source of circulating C16:0 ceramides. Further, these studies suggest that COVID-19 complications including CoV2-ARDS might be amenable to treatment by an anti-ceramide antibody under development in our program. We propose that inhibition of the ASMase/ceramide pathway will effectively prevent and treat lung injury in COVID-19 ARDS. This insight could lead to novel therapeutics targeting this signaling pathway. Hypothesis: We hypothesize that Ang2-triggered ASMase activation in lung endothelial cells is critical for SARS-CoV-2 induced pulmonary vascular dysfunction and acute lung injury (ALI). Specific Aims: We will address our hypothesis using mechanistic complementary approaches and clinically relevant models of disease that will provide a specific and therapeutically targetable molecular ordering of events that cause lung microvascular damage and ALI in COVID-19 designed to determine whether: SA1: SARS-CoV2-generated Ang2 requires ASMase activation to cause human lung microvascular endothelial cell dysfunction. We will investigate the mechanism and role of ASMase activation in Ang2-induced human lung microvascular endothelial cell injury and barrier dysfunction. SA2: Ceramide-rich platform formation is critical for Ang2-induced activation of human lung microvascular endothelium. Using monoclonal anti-ceramide Abs, we will study impact of ceramide-rich platform disruption on Ang2-induced auto-activation of plasma membrane NADPH oxidase, and other signaling proteins that cause endothelial injury and dysfunction. SA3: Inhibition of endothelial ASMase and ceramide signaling alleviates SARS-CoV-2-induced ALI. We will use complementary preclinical animal models pertinent to SARS-CoV-2-induced lung injury to test the therapeutic benefit of ASMase inhibition and anti-ceramide antibodies on ALI outcomes examining: (a) Ang2-induced pulmonary edema in mice; (b) SARS-CoV-2-Spike/S1 protein augmentation of acid aspiration-induced ALI in mice; and (c) SARS-CoV-2 (mouse adapted or wild-type) lung infection in mice or hamsters, respectively. We will complement data obtained with these models by measuring sphingolipidomic changes indicative of ASMase-ceramide activation of pulmonary vascular injury and dysfunction (apoptosis, oxidative stress, increased permeability). Study Design and Variables: Using complementary methods and models that are relevant to human ARDS, our research design aims to demonstrate the hypothesis that ASMase activation-mediated ceramide-rich platform formation is a critical event in the vascular injury that drives the ALI triggered by SARS-CoV-2. We will use both a reductionistic approach focusing on Ang2-triggered events and ceramide-rich platform-mediated signaling in primary human lung microvascular endothelial cells (HLMVECs), a molecular event in the vasculature that occurs downstream and as a consequence of SARS-CoV-2 infection of the lung epithelium. These cell-based studies will be complemented by a comprehensive in vivo approach that targets ASMase and ceramide-rich platforms in complex animal models of COVID-19, in laboratory mice and hamsters. The key variables will be ASMase activation and ceramide-rich platform formation, events that will be targeted with pharmacological, molecular, and biological approaches. Long-Term and Short-Term Impacts to the Field and Patient Care: COVID-19 Impact: Successful outcomes of experiments proposed here could allow for rapid re-deployment of existing clinical candidates for the treatment of CoV2-ARDS. An effective direct therapy for CoV2-ARDS has remained elusive. Many areas of the U.S. have low vaccination rates. American Defense personnel must also operate abroad, where vaccination rates can also be very low. Even where vaccination rates are high, increasing numbers of breakthrough cases are appearing among the vaccinated. These factors support a clear need for new, directing acting-treatments for COVID-19 and CoV2-ARDS. Commercial Impact: Memorial Sloan Kettering Cancer Center (MSKCC) licensee Ceramedix Holding, LLC is developing two therapeutic anti-ceramide candidates: a small-chain variable fragment (scFv) and a full-length antibody. These molecules are currently in preclinical development for a host of indications mediated by microvascular injury, including microvascular complications of hematopoietic stem cell transplantation (HSCT) in partnership with Sergio Giralt, MD, Chair of the Transplant Service at MSKCC. It is anticipated that clinical trials will be initiated in 2022. Long-Term Impacts: In the long term, a ceramide-targeting therapeutic could have profound impact in numerous defense and civilian indications. For Department of Defense (DOD) and public health medical countermeasures, ceramide-targeting therapeutics are also being investigated for radiation-induced injury focusing on the Gastrointestinal-Acute Radiation Syndrome (GI-ARS). Ceramide-targeting therapeutics are also being developed for the treatment of diabetic retinopathy, acute graft versus host disease (GvHD), interstitial lung disease (ILD), idiopathic pulmonary fibrosis (IPD), and cystic fibrosis (CF). Defense Impacts: Therapies based on scFvs have profound advantages over full-length antibodies. scFv therapies can be delivered by simple subcutaneous (SC) or intramuscular (IM) injection, rather than by infusion. These administration routes offer significant advantages in austere settings or during mass casualty events. Relevance to PRMRP Topic Area: This proposal is responsive to the Respiratory Health Topic Area and Area of Encouragement. Specifically, with "development and/or testing of novel and/or innovative treatments to prevent acute lung injury (ALI)/acute respiratory distress syndrome (ARDS)." This proposal also aligns with the Area of Encouragement related to "acute and chronic lung injury/disorders due to viral infections, such as SARS-CoV-2." Less