2024 AIChE Annual Meeting
Investigating the Effects of HIV Nef Sequence Variants on Pulmonary Endothelial Cell Function
While significant progress has been made to improve the quality, length of life, and health of people
living with human immunodeficiency virus (HIV), this patient group remains at an increased risk of
developing chronic vascular complications and comorbidities. The rising incidence rate of HIV-associated
pulmonary vascular disease (HIV-PVD) is a major cause of concern in HIV research, yet the molecular
mechanisms for HIV-PVD remain largely unknown. Here, we explored a potential mechanism of HIV-
PVD by examining the effects of HIV Nef on human pulmonary endothelial cell function in vitro.
Endothelial cells line the interior of all human vasculature and play a critical role in blood vessel
constriction/dilation, platelet direction, immune cell recruitment, and angiogenesis. Nef is an accessory
protein of HIV and a primary determinant of viral pathogenesis. This project investigated the effects of
HIV Nef on pulmonary endothelial cell function to determine its potential role in causing pulmonary
hypertension and PVD.
Here, we utilized banked Nef molecular constructs previously generated from patients with known cases
of HIV-associated pulmonary hypertension. Briefly, the plasmid DNA encoding these nefs were
expanded using Top10 competent cells spread on agar plates treated with ampicillin, kanamycin, and
G418. Colonies were picked, inoculated in Luria-Bertani broth, and pelleted. Plasmids were purified
using QIAprep Miniprep Kit (Qiagen), and plasmid DNA was confirmed on previously sequenced
plasmids with restriction endonucleases and gel electrophoresis. The unique nef sequence variants were
then purified as endotoxin-free plasmids using the Endofree Prep Maxi Kit (Qiagen) and transfected into
human pulmonary arterial endothelial cell (HPAEC) cultures. Transfected cultures were incubated at
37ºC, 5% CO2 overnight, and work is in progress to test three endothelial cell functional endpoints:
intracellular nitric oxide (NO) production (by live cell imaging), angiogenesis biomarkers, and cell
adhesion biomarkers (using cell supernatants). We expect to identify the mutational profiles of HIV Nef
that contributes to PVD in our in vitro models.
living with human immunodeficiency virus (HIV), this patient group remains at an increased risk of
developing chronic vascular complications and comorbidities. The rising incidence rate of HIV-associated
pulmonary vascular disease (HIV-PVD) is a major cause of concern in HIV research, yet the molecular
mechanisms for HIV-PVD remain largely unknown. Here, we explored a potential mechanism of HIV-
PVD by examining the effects of HIV Nef on human pulmonary endothelial cell function in vitro.
Endothelial cells line the interior of all human vasculature and play a critical role in blood vessel
constriction/dilation, platelet direction, immune cell recruitment, and angiogenesis. Nef is an accessory
protein of HIV and a primary determinant of viral pathogenesis. This project investigated the effects of
HIV Nef on pulmonary endothelial cell function to determine its potential role in causing pulmonary
hypertension and PVD.
Here, we utilized banked Nef molecular constructs previously generated from patients with known cases
of HIV-associated pulmonary hypertension. Briefly, the plasmid DNA encoding these nefs were
expanded using Top10 competent cells spread on agar plates treated with ampicillin, kanamycin, and
G418. Colonies were picked, inoculated in Luria-Bertani broth, and pelleted. Plasmids were purified
using QIAprep Miniprep Kit (Qiagen), and plasmid DNA was confirmed on previously sequenced
plasmids with restriction endonucleases and gel electrophoresis. The unique nef sequence variants were
then purified as endotoxin-free plasmids using the Endofree Prep Maxi Kit (Qiagen) and transfected into
human pulmonary arterial endothelial cell (HPAEC) cultures. Transfected cultures were incubated at
37ºC, 5% CO2 overnight, and work is in progress to test three endothelial cell functional endpoints:
intracellular nitric oxide (NO) production (by live cell imaging), angiogenesis biomarkers, and cell
adhesion biomarkers (using cell supernatants). We expect to identify the mutational profiles of HIV Nef
that contributes to PVD in our in vitro models.
This work was supported by an NIH/NHLBI Grant R01 HL172709 to SA and funding from Texas Tech
University Honors College to EH and MN.