Three-dimensional in vitro model to reproduce COVID-19-induced endothelial dysfunction

In a recent study posted to the bioRxiv* preprint server, researchers developed a three-dimensional (3D) in vitro tissue model to assess the effects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on the vascular endothelial cells.

In vitro models are required to improve understanding of coronavirus disease 2019 (COVID-19) pathogenicity. Previously, in vitro, two-dimensional (2D) tissue models to study SARS-CoV-2 infection impacts on the endothelium have been developed with reduced cell adhesion molecule expression, endothelial barrier disruption, and enhanced inflammatory cytokine secretion in endothelial cells. However, due to the inability to simulate vascular networks, 2D models cannot model in vivo conditions as precisely as 3D models.

The authors of the present study previously used human-induced pluripotent stem cells (hiPSCs) that were differentiated into endothelial progenitors (EPs) and encapsulated within collagen hydrogel structures to assess angiogenesis.

About the study

In the present study, researchers extended their previous analysis by creating a 3D model to study vascular network disruption in COVID-19.

For the analysis, hiPSCs derived from human dermal foreskin fibroblasts (DF19-19-9-11T) were cultured and differentiated into hiPSC-EPs. Subsequently, the cluster of differentiation (CD)34⁺ hiPSC^–EPs were isolated by FACS (fluorescence-activated cell sorting) analysis and the CD34⁺ hiPSC^–EP cells were encapsulated into a collagen hydrogel structure.

The encapsulated hydrogels were cultured for a week to form capillary networks. After five days of encapsulation, the CD34⁺-hiPSC-EP-laden collagen hydrogels were SARS-CoV-2 spike (S) protein (CSP)-treated to simulate SARS-CoV-2 infections. Cytokine release assays were performed to measure inflammatory cytokine expression after CSP treatment. The vessel-like networks generated within CD34⁺ iPSC^–EP-laden collagen hydrogels were visualized by immunocytochemistry.

The cells were also treated with dexamethasone and visualized by confocal microscopy. The vessel length, vascular connectivity, and vessel lumen diameters were analyzed based on a previously developed computational pipeline. SARS-CoV-2 messenger ribonucleic acid (mRNA) from the hydrogels was subjected to qRT-PCR (quantitative reverse transcription–polymerase chain reaction) analysis to quantify mRNA expression based on the cycle threshold (Ct) values.

Further, the team investigated whether CD34⁺-hiPSCs forming vascular networks within the collagen hydrogels expressed angiotensin-converting enzyme 2 (ACE2). In addition, the expression of several endothelial genes such as KDR (kinase insert domain receptor), CD31, CDH5 (cadherin 5), and CD34 were evaluated to track endothelial maturation over the one-week culture period and correlate the levels to ACE2 expression.

Results

Treatment with CSP significantly reduced the proportion of vessel-forming cells and vessel network connectivity in the hiPSCs-derived EPs encapsulated in collagen hydrogels. Elevated inflammatory cytokine expression was observed in accordance with clinical findings, and 17 ng/mL dexamethasone treatment reduced vascular dysfunction in CSP-treated cells. Even in the absence of immunological cells, the team could use the 3D in vitro angiogenesis model to simulate the SARS-CoV-2 infection-induced dysfunction of endothelial cells representative of clinical COVID-19 presentations.

Following CSP treatment, the vascular network showed reduced density and more cells with an altered rounded morphology. CSP treatment (10 µg/mL) led to 22%, 33% and 29% reductions in vessel endpoints, vessel branch points, and vessel links, respectively. The corresponding reductions observed on using CSP in 100 µg/mL concentration were 17%, 24%, and 22%, respectively.

Vascular network connectivity loss was observed among CSP-treated collagen hydrogels with a 77% reduction in the proportion of cells that were a part of the largest vessel network. When CSP was added on the first day, 19% and 31% reductions were observed in vessel branch points and volume fractions of collagen hydrogels comprising cells that form vessels, respectively. The corresponding reductions observed when CSP was added on the fifth day were 59% and 66%, respectively, indicating higher CSP toxicity when added on the fifth day after vascular networks started to form.

ACE2 expression was highest on day 0 and reduced by 62% within a week post-CSP treatment. The team observed 2.7-fold and 6.7-fold reductions in CD34 expression on the first and fifth days, respectively. Contrastingly, CD31 expression increased by two-fold, four-fold, and seven-fold on days 1, 5, and 7, respectively, and CDH5 expression increased by two-fold and three-fold on the fifth and seventh day, respectively.

CSP treatment significantly increased interleukin-8 (IL-8) and chemokine C-X-C motif ligand 1 (CXCL1) release by two-fold and three-fold, respectively. Both cytokines are produced by endothelial cells and are upregulated in COVID-19. The findings indicated that CSP treatment created an inflammatory state comparable to the cytokine storm documented in severe COVID-19 cases.

Overall, the study findings showed successful reproduction of COVID-19-induced endothelial dysfunction observed in clinical settings using CSP-treated hiPSC-EP-laden collagen hydrogels. CSP treatment caused significant vascular disruption, concerning vascular network cells and vascular network connectivity, which was inhibited by dexamethasone.

*Important notice

medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Written by

Pooja Toshniwal Paharia

Dr. based clinical-radiological diagnosis and management of oral lesions and conditions and associated maxillofacial disorders.