D28-18 Transcriptomic Profile of Human Immunodeficiency Virus-Mediated Disruptions of Pulmonary Artery Endothelial and Smooth Muscle Cell Crosstalk in 2D and 3D Vascular Models

Rationale

Vascular homeostasis relies on coordinated crosstalk between endothelial cells (EC) and smooth muscle cells (SMC) to maintain vascular integrity and function. Disruption of these interactions by inflammatory or infectious stimuli, including Human Immunodeficiency Virus (HIV), promotes endothelial dysfunction and vascular remodeling, contributing to the increased risk of pulmonary vascular disease in people living with HIV. Further definition of how vascular-immune cell crosstalk shapes pulmonary vascular responses during HIV exposure will address a critical knowledge gap in this field.

Methods

Herein, we co-cultured primary Human Pulmonary Artery Endothelial Cells (HPAEC) and Human Pulmonary Artery Smooth Muscle Cells (PASMC). These cells were cultured either separately with HIV-infected or uninfected T cells (EC-T cell and SMC-T cell cocultures), or together with T cells in a triculture model (EC-SMC-T cell) implemented in both 2D and 3D formats. T cells were infected with an HIV-R5 construct expressing DsRed. Phenotypic and molecular changes were assessed through confocal fluorescent microscopy, qPCR, and bulk RNA sequencing.

Results

We observed a significant increase in the expression of apoptosis-related markers (BAX, BCL2, and P21) in infected cultures across both models. This was accompanied by a marked reduction in proliferation and cell-cycle progression markers (Ki67, PCNA, CDK2, and Cyclin A2), indicating a shift toward a stress-induced, less proliferative phenotype driven by interactions with infected T cells. In parallel, infected cultures showed robust upregulation of pro-inflammatory cytokines (IL-6, IL-8) and endothelial adhesion molecules (ICAM-1, VCAM-1, and EDN1), consistent with infection-induced inflammatory activation and early features of vascular dysfunction. Transcriptomic profiling by bulk RNA-seq revealed broad differential gene expression and pathway-level changes consistent with enhanced inflammatory signaling, stress responses, and impaired cell-cycle regulation, corroborating the targeted qPCR findings. Bulk RNA-seq analysis revealed distinct transcriptomic profiles between 2D and 3D cultures, with 3D tricultures exhibiting attenuated inflammatory and stress-related pathways and partial preservation of proliferative and homeostatic gene programs compared with 2D systems, consistent with a more physiologically relevant response to HIV exposure.

Conclusions

These findings show that 3D cultures better recapitulate the in vivo pulmonary microenvironment and modulate cellular stress and inflammatory responses to HIV. Bulk RNA-seq provided an unbiased transcriptomic framework that extended targeted analyses and clarified how HIV reshapes vascular-immune crosstalk, underscoring the value of physiologically relevant in vitro models.

This abstract is funded by: NIH