Beyond Viral Control: Tenofovir's Hidden Impact on Blood–Brain Barrier Endothelial Cells

ABSTRACT

The blood–brain barrier (BBB) is a dynamic endothelial interface that protects the brain from harmful agents while regulating molecular exchange. Human immunodeficiency virus (HIV) compromises BBB integrity, promoting neurological damage. Antiretroviral (ARV) therapies suppress HIV replication, preventing immune system deterioration and progression to AIDS. Although Tenofovir‐based ARV regimens are vital for HIV treatment and prevention, their impact on cerebrovascular function remains unclear.

Aim

This study examined Tenofovir's effects on murine brain endothelial cells using an in vitro BBB model.

Methods

Brain endothelial cells (bEnd.5) were treated with Tenofovir Disoproxil Fumarate (TDF; 9.8–98 ng/mL) or Tenofovir Alafenamide (TAF; 1–10 ng/mL) for 24–96 h. Cell proliferation, cell cycle progression (via flow cytometry) and monolayer permeability (via Transendothelial Electrical Resistance) were evaluated.

Results

Both TDF and TAF treatments suppressed cell division by S‐phase disruption of the cell cycle and increased monolayer permeability.

Conclusion

These findings suggest that prolonged TDF or TAF exposure compromises BBB integrity by inhibiting endothelial cell division and altering barrier function, which could have implications for HIV‐ARV‐induced neurodegeneration in individuals receiving long‐term Tenofovir‐based therapy.

1. Introduction

The blood–brain barrier (BBB) is a complex system composed of a continuous endothelial cell layer that maintains the homeostatic environment in the central nervous system (CNS) by physiologically protecting the brain from toxins and harmful chemicals and separating the blood from the brain's extracellular fluid [1, 2]. Endothelial cells are the main component of the BBB; they create the tubular structure of brain capillaries and maintain barrier integrity, with the abluminal surface covered by basement membranes made of extracellular matrix [3]. These endothelial tubes are encircled by pericytes, astrocyte end‐feet, and neurons, forming the neurovascular unit [4]. Tight junctions seal off the paracellular spaces between cells [5]. This restricts the passive transport of molecules into the brain and prevents them from easily accessing the fluid environment of cerebral tissues, resulting in extremely high transendothelial electrical resistance (TEER) across brain capillaries [6]. This is the basis of the regulation of the homeostatic neural parenchyma of the CNS, and if compromised, can lead to both cognitive and neural degenerative pathologies. HIV infection can disrupt BBB function through inflammatory cytokine release, dysregulation of tight junction proteins, and activation of brain microvascular endothelial cells, thereby increasing permeability and facilitating viral trafficking to the brain [7, 8]. Antiretrovirals (ARVs) are medications prescribed to treat HIV infection and prevent disease progression by suppressing viral replication, thereby limiting immune system deterioration and the progression from HIV to AIDS [9]. In 2024, an estimated 630 000 people worldwide died from AIDS related causes, a substantial decline from 1.4 million in 2010, coinciding with an increase in access to ARV therapy from approximately 7.7 million people in 2010 to 31.6 million in 2024 [10]. These statistics do not include persons at risk of contracting HIV who are treated with various formulations of HIV‐specific ARVs [11].

Among key ARV agents, Tenofovir Disoproxil Fumarate (TDF) and Tenofovir Alafenamide (TAF) are both prodrugs of Tenofovir but differ significantly in pharmacokinetic and safety profiles [12]. Both are also utilized in pre‐exposure prophylaxis (PrEP), a preventative strategy aimed at reducing the risk of HIV infection in high‐risk individuals [11]. TDF, an earlier prodrug, is associated with higher systemic exposure and risk of renal and bone toxicity, whereas TAF offers enhanced lymphatic and cellular uptake with lower plasma levels, resulting in improved renal and bone safety profiles while maintaining antiviral efficacy [13]. However, little to no studies have reported on the long‐term chronic effects of HIV‐ARV treatment on the primary regulatory component of the BBB, the capillary endothelium.

This study employed an in vitro BBB model to investigate the effects of TDF and TAF on brain capillary endothelial cells (bEnd.5 cells). The experimental parameters assessed included rates of cell proliferation, cell cycle dynamics, and endothelial monolayer permeability.

2. Methods and Materials

2.1. Experimental Design

Figure 1 depicts the study design for ARV's long‐term effects on the bEnd.5 cell line. Once the in vitro blood–brain barrier (BBB) model was established, Tenofovir's (please note this is the collective term used throughout the study for both the tenofovir disoproxil fumarate [TDF] and tenofovir alafenamide [TAF]) effects on brain endothelial cells were examined. The effects of ARV's were observed by treating bEnd.5 cells with selected concentrations of TDF and TAF for 24–96 h. All media were replaced daily to maintain consistent treatment concentrations and to provide adequate metabolic substrates necessary for normal cell function. The concentrations used were chosen to reflect the plasma levels observed in HIV patients [14]. Cell proliferation (live and dead cells) and cell suppression were recorded after exposure to TDF and TAF. Other experiments were also employed, such as flow cytometry to determine at which phase cell cycle progression was being halted and trans‐endothelial electrical resistance (TEER) across the bEnd.5 monolayers to examine how these treatments affected the permeability.

FIGURE 1.

1. Establishment of the in vitro BBB model: Various seeding densities of bEnd.5 cells (1000, 1500, 2000, 2500 cells/well) were systematically employed to obtain the optimal cell concentration conducive to consistent monolayer functionality. Following experimentation, it was found that 1500 cells/well represented the optimal cell density for bEnd.5 cells. 2. Exposure to TDF or TAF: Selected concentrations of TDF (9.8–98 ng/mL) or TAF (1–10 ng/mL) were administered to bEnd.5 cells. 3. Physiological Assessment: Experiments were conducted to evaluate bEnd.5 cell proliferation and suppression utilizing Trypan Blue staining. Flow cytometry was used to see where TDF or TAF exposure halted cell cycle progression, and TEER measurements were taken to see how TDF or TAF exposure affected the permeability of the BBB model.

2.2. Cell Line and Bio‐Reagents

Mouse (BALB/c) brain microvascular endothelial cells (bEnd.5) were obtained from Sigma‐Aldrich (96091930, St. Louis, MI, USA) in 2021. After initial seeding and routine testing for mycoplasma (Lonza's MycoAlert Mycoplasma Detection Assays: cat. no. LT07‐218), the presence of the bEnd.5 cell line was authenticated by morphological analysis using phase‐contrast microscopy, and showed typical bEnd.5 cobblestone‐like endothelial appearance. Further authentication using immuno‐fluorescent staining and Western blot analysis confirmed that cells retain brain endothelial characteristics for both occludin and claudin‐5 (brain endothelial cell‐specific marker).

These bEnd.5 cells were cultured and maintained in accordance with the manufacturer's specifications, maintained in Gibco Dulbecco's Modified Eagle Media: Nutrient mixture F‐12 (Gibco, Thermofisher, cat. no: 31330038) supplemented with 10% FBS (Gibco, Thermofisher, cat. no: 01190046M), 1% NEAAs (Gibco, Thermofisher, cat. no: 11140050), 1% sodium pyruvate (Gibco, Thermofisher, cat. no: 11360039), and 1% antibiotic (Gibco, Thermofisher, cat. no: 15140122). Cells were harvested using 0.25% EDTA–trypsin (Gibco, Thermofisher, cat. no: 25200072). Pharmaceutical‐grade bio‐reagents included Tenofovir Disoproxil Fumarate (TDF) powder (Adcock Ingram Limited, Midrand, South Africa), Tenofovir Alafenamide (TAF) powder (LGC Standards Ltd., TRC‐T018555‐5MG, South Africa), phosphate buffer (Whitehead Scientific BE17‐51Q, South Africa) and Trypan Blue solution (Gibco, Thermofisher, cat. no: 15250061).

The bEnd.5 cells were cultured and maintained in complete DMEM/F12 in humidified 5% CO₂ air at 37°C until they reached 75%–80% confluence.

2.3. Trypan Blue Exclusion Assay

This method is based on the principle that viable cells, with intact membranes, exclude certain dyes, while non‐viable cells with compromised membranes readily take them up [15].

In this assay, bEnd.5 cells were seeded in triplicate in 12‐well plates at a seeding density of 1500 cells/well and incubated for 24 h, allowing the cells to attach. Monitoring was done using an inverted microscope (Eclipse‐Ts2‐Ls, Nikon, Amsterdam, The Netherlands). After confirming attachment, the media was replaced with treatment media as indicated in the experimental design, and the control group's media was replenished with fresh media. Cells were terminated by washing with PBS and trypsinization with 0.25% EDTA–trypsin every 24 h for 96 h. The cells were centrifuged at 1845g for 5 min, after which the supernatant was discarded, and the pellet was resuspended in 400 μL fresh, unsupplemented media. Cells were then stained by mixing 20 μL of cell suspension and 20 μL trypan blue dye (0.4%) ensuring a 1:1 ratio. Ten microliters of this cell suspension was loaded onto a hemocytometer and counted using a Countess III specialized counter (Cat. No. AMQAX2000, ThermoFisher Scientific, South Africa). Total cell number, live cell number, and dead cell number were recorded simultaneously by the Countess. The suppression of each sample was then calculated (total number of cells in the control − total number of cells in the treatment group) and recorded.

2.4. Cell Cycle Analysis by Flow Cytometry

In this study, the findings obtained from the cell proliferation assay prompted a detailed examination of the cell cycle dynamics of the brain endothelial cells (BECs) after exposure to varying concentrations of Tenofovir treatment media, employing flow cytometry for a 96‐h exposure only.

The bEnd.5 cells were seeded at a cell density of 18 000 cells in T25 flasks in a final volume of 3 mL in preparation for an exposure time of 96 h. The cells were incubated at 37°C and 5% CO₂ for 24 h to allow for attachment. After the 24‐h attachment period, the media were discarded, and the treatment groups were exposed daily to equal volumes (3 mL) of treatment media, which consisted of supplemented DMEM/F12 containing TDF or TAF (Figure 1). The control group was exposed daily to fresh DMEM/F12 only. At 96 h of exposure to treatment, the experiment was terminated by collecting the spent media as well as the harvested cells.

Spent and treatment media were collected in a 15 mL conical tube to recover any floating cells. Adherent cells were harvested by trypsinization. The tube was centrifuged for 5 min at 2500 rpm (664 g). The supernatant was discarded, and the pellet was washed with cold PBS while vortexing to reduce clumping and re‐pelleted under the same conditions. The PBS was discarded, and cold 70% ethanol was slowly added dropwise to the pellet while vortexing to prevent clumping. The cells were fixed in ethanol for at least 2 h at −20°C.

Before analysis, cells were centrifuged for 5 min at 2500 rpm, removing the ethanol and washing the pellet with cold PBS. Five hundred microliters DAPI (Invitrogen, Cat no. D1306) staining solution (prepared according to the manufacturer's instructions) was added to each sample. Cells were incubated at room temperature for 10 min before analysis. Cell cycle analysis was conducted using the CytExpert for DxFLEX 2.0 software, utilizing a 450/50 nm bandpass filter to detect the blue‐emitting fluorophore DAPI, which is excited by a 355 nm blue laser. Samples were analyzed in triplicate, and the DNA content of 20 000 events was analyzed to determine the distribution of cells across the different phases of the cell cycle (G1, S, and G2/M).

2.5. Trans‐Endothelial Electrical Resistance (TEER)

The principle of TEER involves measuring the electrical resistance across a monolayer of endothelial cells, where higher resistance values indicate stronger cell‐to‐cell junctions and better barrier integrity [16].

For TEER measurements, bEnd.5 cells, from the same cell culture population and passage, were seeded at a seeding density of 50 000 cells/insert and cultured on membrane inserts (MF‐Millipore, Serial Number: PIHA01250, Germany) with a pore size of 0.45 μm, filtration diameter of 12 mm, and a surface area of 0.6 cm². Inserts were placed in 12‐well plates, where a maximum of 300 μL media/treatment media was added to the apical compartment, and 500 μL media was added to the basolateral compartment. The bEnd.5 cells were cultured and grown in fresh cultured media for a 6‐day period to establish a confluent monolayer on the membrane insert. Based on previous studies and extensive pilot studies, this corresponds to where TEER across control monolayers plateaus, and reflects maximal barrier integrity and the establishment of intercellular junctions [8, 17]. Fresh supplemented media were administered daily to the control samples, while TDF and TAF were introduced to the treatment medium from day 6, once the confluent monolayer was established, as previously described [18, 19]. TEER measurements were conducted over a nine‐day period using a Millicell epithelial Volt‐Ohm meter (Millipore, Serial Number: 57318, 11B, Germany). During the data analysis, the resistance measured from the blank inserts (containing cultured media only) (without cells) was subtracted from the resistance of the inserts containing bEnd.5 cell monolayers. This resultant value was then multiplied by the surface area of the insert.

2.6. Statistical Analysis

All outcomes are reported as the mean ± standard error of the mean (SEM), with corresponding error bars representing the extent of data variability. To ensure statistical reproducibility, all experiments were conducted in triplicate (n = 3) and repeated in duplicate. Statistical analyses were performed using GraphPad Prism 9.5.0 software (Dotmatics, California, USA). Prior to employing parametric or non‐parametric statistical tests, normality testing was conducted. For parametric data, one‐way analysis of variance (ANOVA) with Dunnett's post hoc test was employed; for nonparametric data, the Kruskal–Wallis test with Dunn's post hoc test was applied. A significance level of p < 0.05 was utilized to determine statistical significance, corresponding to a 95% confidence interval.

3. Results

3.1. Trypan Blue Experiments

Tenofovir is a widely used ARV for managing and reducing the risk of HIV infection. In this experiment, the concentrations of Tenofovir were determined based on its blood plasma levels in individuals undergoing treatment with this ARV. This approach was adopted to replicate the physiological concentration of Tenofovir in the bloodstream, and these concentrations were subsequently used for the treatment of bEnd.5 cells.

3.1.1. The Effects of Tenofovir Disoproxil Fumarate (TDF) on Cell Proliferation

TDF treatment (9.8–98 ng/mL) consistently suppressed the live cell count throughout the 96‐h experimental duration (Figure 2A–D). The reduction in dead cell count compared to the controls (Figure 2E–H) was proportional to the decrease in live cells. Consequently, as TDF treatment does not increase the number of dead cells, it does not contribute toward the increased toxicity even at the highest concentrations. Given that the decrease in cell numbers cannot be attributed to increased numbers of dead cells, TDF treatment must be suppressing bEnd.5 cell division. In Figure 3, this effect is presented as suppression of the cell division.

FIGURE 2.

The effects of TDF on bEnd.5 cell proliferation over 96 h. (A–D) The live cell count of bEnd.5 cells and (E–H) the dead cell count. The vehicle control (VC) group was employed as a reference for comparison, as no statistically significant difference was found between the control and VC groups. Statistical significance was performed using one‐way ANOVA with Dunnett and Dunn's post hoc test. All data are presented as mean ± SEM (n = 6), and statistical differences of TDF cell numbers are compared statistically to the VC (*p < 0.5, **p < 0.01, ***p < 0.001, ****p < 0.0001).

FIGURE 3.

The impact of TDF in suppressing cell division in bEnd.5 cells over 96 h is shown in A–D. These figures show that the number of dead cells alone cannot explain the decline in live cell numbers compared to control cell numbers. VC, vehicle control.

3.1.2. Tenofovir Disoproxil Fumarate (TDF) Suppression of Cell Division

It was initially expected that the reduction in total cell number would be due to an increase in cell death. However, the decrease in dead cells could not account for the decrease in live cell numbers when compared to controls. At 24 h of exposure, TDF (49 and 98 ng/mL) significantly suppressed cell proliferation (Figure 3A), and from 48 h onward, statistically significant suppression was escalated and evident across all concentrations and persisted through to 96 h. The decrease in cell numbers could not be explained by cell death alone, indicating that TDF primarily exerted its effect through inhibition of cell proliferation rather than cytotoxicity.

3.1.3. The Effects of Tenofovir Alafenamide (TAF) on Cell Proliferation

Given that TAF is a newer formulation of Tenofovir that clinically requires lower ARV doses, this underscores the necessity to explore the effects of TAF on brain endothelial cells. There was no statistical effect of TAF in bEnd.5 cell proliferation at 24 h, and the lower doses of TAF (1–2.5 ng/mL) did not elicit a statistical effect on cell proliferation throughout 96 h. However, the higher doses of TAF (5 and 10 ng/mL) consistently and statistically decreased live bEnd.5 cell counts from 48 h through the 96‐h experimental duration (Figure 4B–D). The statistically significant reduction in the dead cell count compared to the control at the 72‐h mark (Figure 4G) reflected a decrease in the total live bEnd.5 cell population, rather than indicating that TAF improves the mortality of bEnd.5 cells over the course of 72 h (Figure 4). At all other experimental timeframes, the number of dead cells in TAF‐treated cell cultures was statistically identical to the control (Figure 4E,G,H).

FIGURE 4.

The effects of TAF on bEnd.5 cell proliferation over 96 h. (A–D) The live cell count of bEnd.5 cells and (E–H) the dead cell count. The vehicle control (VC) group was employed as a reference for comparison, as no statistically significant difference was found between the control and VC groups. Statistical significance was performed using one‐way ANOVA with Dunnett and Dunn's post hoc test. All data are presented as mean ± SEM (n = 6), (*p < 0.5, **p < 0.01, ***p < 0.001, ****p < 0.0001).

3.1.4. Tenofovir Alafenamide (TAF) Suppression of Cell Division

Figure 5A–D presents the effects of TAF statistically suppressing live bEnd.5 cell populations at higher concentrations (5 and 10 ng/mL). In contrast to suppression caused by TDF, no suppression of cell division was found after 24 h of TAF treatment. However, from 48 to 96 h, at 5 and 10 ng/mL, statistical significance in the suppression of cell division was achieved. Although suppression persisted through to 96 h, a slight recovery was noted at this latter time point.

FIGURE 5.

The impact of TAF in suppressing cell division in bEnd.5 cells over 96 h is shown in (A–D). These figures show that the number of dead cells by itself is unable to explain the decline in the number of live cells when compared to control cell numbers. VC, vehicle control.

3.2. Flow Cytometry

Flow cytometry reveals interrupted cell division by using a fluorescent dye to measure DNA content, revealing accumulations of cells in the G1, S, or G2/M phases. A shift in the DNA content distribution, with increased percentages of cells in a specific cell cycle phase, indicates an interruption. Our data showed a trend in both TDF and TAF treatment suppression of the BEC division. This trend in suppression demonstrated a more pronounced effect at the 96‐h treatment and therefore only the flow cytometry data for 96 h was presented. Flow cytometry was utilized to analyze the effects of TDF and TAF on bEnd.5 endothelial cell cycle distribution.

3.2.1. Effects of Tenofovir Disoproxil Fumarate (TDF) and Tenofovir Alafenamide (TAF) on Cell Cycle Progression

Figure 6A demonstrates a significant decrease in the proportion of cells in the G1‐phase at higher concentrations of TDF, accompanied by a corresponding increase in the S‐phase population, indicating that TDF disrupts the normal progression of the cell cycle and promotes cell cycle arrest in the S‐phase. Similar to TDF, TAF (Figure 6B) also demonstrated a dose‐dependent effect on cell cycle dynamics. Cells treated with TAF showed a decrease in the proportion of cells in the G1 phase as well as an accumulation of the cell population in the S‐phase. Results revealed differences in the cell cycle dynamics of treated cells, with a notable increase in the percentage of cells in the S phase compared to the control, suggesting a disruption in the progression from the S phase to the G2/M phase. This disruption in the cell cycle demonstrates Tenofovir's ability to affect cell division and proliferation (Figure 6).

FIGURE 6.

The effects of Tenofovir on the cell cycle dynamics of bEnd.5 cells using flow cytometry analysis. The treatment with (A) TDF (9.8–98 ng/mL) or (B) TAF (1–10 ng/mL) represents the 96 h only. Following treatment, cell cycle distribution was analyzed to assess changes in the proportion of cells within each phase (G1, S, and G2/M). The vehicle control (VC) group was employed as a reference for comparison, as no statistically significant difference was found between the control and VC groups. Statistical analysis, conducted using one‐way ANOVA with Dunnett and Dunn's post hoc tests, showed significant differences between the treated groups and controls, with p‐values indicating highly significant changes (*p < 0.05, ***p < 0.001, ****p < 0.0001). All data are expressed as mean ± SEM, with six replicates per concentration (n = 6). VC, vehicle control.

3.3. Trans Endothelial Electrical Resistance (TEER)

TEER measurements were conducted across bEnd.5 endothelial cell monolayers to assess the changes in integrity and permeability subsequent to treatment with TDF (9.8–98 ng/mL) and TAF (1–10 ng/mL). Cultured bEnd.5 cells were grown on inserts for 6 days in order to establish a confluent cellular monolayer, which was endorsed by the plateauing of TEER. Once the confluent monolayer was formed, treatment with TDF or TAF was administered over the course of the next 4 days. Decreased TEER values indicated increased permeability across the confluent monolayers of bEnd.5 cells, while elevated TEER values signified enhanced barrier integrity and decreased monolayer permeability.

3.3.1. Effects of Tenofovir Disoproxil Fumarate (TDF) on TEER

Significant decrease in TEER occurred two days after the commencement of TDF treatment. Thereafter, during the last 2 days of the experiment, a trend was observed in the monolayer's integrity: as the concentration of Tenofovir increased, there was a corresponding decrease in TEER (Figure 7). TDF caused a 55% increase in permeability over the time course of the experiment.

FIGURE 7.

The effects of TDF on TEER in confluent monolayers of bEnd.5 cells over 9 days. (A) The establishment of a confluent monolayer up to day 6 followed by the effects of TDF treatment on TEER in confluent monolayers of bEnd.5 cells, ranging from 9.8 to 98 ng/mL, with the lilac box (in A) representing the TEER values measured before the initiation of the TDF treatment. The dashed black line (B–D) represents the TEER values measured before the initiation of TDF treatment, and solid red line serves as a reference for TEER changes during TDF treatment. The VC group was employed as a reference for comparison, as no statistically significant difference was found between the control and VC groups. Statistical analysis, performed using one‐way ANOVA with Dunnett and Dunn's post hoc tests, confirmed the significance of these findings, with p‐values indicating highly significant differences (**p < 0.01, ***p < 0.001, ****p < 0.0001). All data are presented as mean ± SEM, with six replicates per concentration (n = 6). VC, vehicle control.

3.3.2. Effects of Tenofovir Alafenamide (TAF) on TEER

TAF induced a significant reduction in TEER by days 7–9, with greater effects observed at higher concentrations (Figure 8; 5 and 10 ng/mL). On average, treatment across the bEnd.5 monolayers (Figure 8A) indicated a substantial loss of barrier integrity. In comparison with TDF, which resulted in a 55% increase in permeability, treatment with TAF produced a comparatively smaller, though still notable, 38% increase in permeability over the course of the experiment. These findings show that while both prodrugs compromise endothelial monolayer integrity, TDF exerts a more pronounced disruptive effect on the monolayer permeability function than TAF.

FIGURE 8.

(A) The effects of TAF on TEER accross confluent monolayers of bEnd.5 cells over 9 days. By day 6, the monolayers had reached confluency (lilac box in A), and TAF treatment was administered from day 6 onwards at concentrations ranging from 1 to 10 ng/mL. The dashed black line (B–D), corresponds to the lilac box (A) and represents the TEER values before TAF treatment. Graphs E–H represents the effects of TAF on TEER across bEnd.5 monolayers (indicated by a solid red line). The VC group was employed as a reference for comparison, as no statistically significant difference was found between the control and VC groups. Statistical analysis, conducted using one‐way ANOVA with Dunnett and Dunn's post hoc tests, confirmed the significance of these decreases, with p‐values highlighting varying degrees of statistical significance when compared to the VC (*p < 0.5, **p < 0.01, ***p < 0.001). All data are presented as mean ± SEM, with six replicates per concentration (n = 6). VC, vehicle control.

4. Discussion

Recent reports approximated 40.8 million people were living with HIV, with an estimated 31.6 million individuals receiving ARV therapy [10]. Given the widespread use of ARVs, TDF remains one of the most commonly prescribed drugs in HIV treatment. Although the exact global number of TDF users remains uncertain, it is estimated that more than 4 million people take TDF daily for the treatment of HIV/AIDS or chronic hepatitis B [20]. The majority of these individuals—over 3 million—reside in developing countries, where TDF is distributed either free of charge or at substantially reduced costs, while fewer than 1 million patients in developed regions receive the drug [21]. As treatment strategies continued to evolve, efforts were made to develop formulations with enhanced efficacy and tolerability. In 2015, a newer formulation of Tenofovir, known as TAF, was approved by the U.S. Food and Drug Administration, offering an alternative with improved safety and pharmacokinetic profiles [20]. Despite advances in therapy, neurological complications remain a major concern among individuals living with HIV, and globally, between 25% and 60% of people living with HIV are affected by HIV‐associated neurocognitive disorders (HAND) [22]. Despite the wide use of ARVs globally, little to no information is available on the effects of these ARVs on the endothelial cells of brain capillaries, which are responsible for protecting and regulating substances into and out of the brain.

The study first aimed to examine the long‐term comparative effects of TDF and TAF on the proliferation and toxicology of the bEnd.5 cell line; thereafter, we investigated the effect of these ARVs on the permeability across monolayers of bEnd5 cells. The Tenofovir concentrations chosen for this study were based on blood plasma levels observed in individuals undergoing daily ARV therapy, as reported by Podany et al. [14]. These concentrations were selected to approximate the physiologically relevant range of drug exposure achieved in vivo following standard dosing regimens of both TDF and TAF. Therefore, the concentration range used in this study was selected to reflect both the upper and lower plasma limits of Tenofovir exposure associated with these formulations, allowing for a comparative assessment of their potential differential effects on BBB endothelial cell integrity.

4.1. Effects on Cell Proliferation

Cultured bEnd.5 cell proliferation was decreased by the TDF in a dose‐dependent manner across all time points (Figure 2). Interrogating the data showed that the decrease in live cell numbers, relative to controls, could not be accounted for by the paralleled decrease in dead cell numbers. Furthermore, the decrease in dead cells with increased ARV concentrations did not reflect an anti‐toxicity effect but rather reflected the normal dead cell numbers for lower cell culture populations. Treating bEnd.5 cells with increasing concentrations of TAF also caused a similar, but less significant, decrease in live cell numbers and dead cell numbers (Figure 4).

For both the TDF and TAF‐treated cell groups, the number of dead cells in the cell cultures could not explain the decrease in live cell numbers. The logical deduction is that both TDF and TAF had the ability to suppress bEnd.5 cell division. By comparing the control live cell numbers to both the treated live cell numbers and dead cell numbers, we were able to show the level of suppression of cell division for both TDF (Figure 3) and TAF (Figure 5). Our analyses showed that TDF was able to suppress cell division within 24 h of treatment and also showed that the suppression was dose‐related and occurred across the concentration range.

Our novel findings indicate that ARV drugs, specifically TDF, have a detrimental effect on the rate of cell division in brain endothelial cells (Figures 2 and 3). These findings are supported by a study conducted by Cohen et al. [23], which demonstrated that treating HUVECs with TDF resulted in a significant reduction in cell proliferative capacity. However, it is important to note that the HUVEC model is not considered a reliable model for studying the BBB, as HUVECs lack the barrier function typical of the BBB [24]. Our results are further supported by previous observations of Ferrer and Rakhmanina [25] and Dejesus et al. [26], who showed that TDF use caused drug‐related toxicities; however, these studies were related to renal and bone toxicity. Together with these findings, our results suggest that long‐term use of TDF causes cell suppression of the BECs.

After observing the effects of TDF on the in vitro BBB model, we investigated the impact of TAF on bEnd.5 cells, as TAF, being a co‐formulation of TDF, is reported to have improved renal and bone density outcomes compared to TDF [14, 26].

When comparing TDF and TAF, TDF had a stronger suppressive effect on bEnd.5 cell division than TAF. Exposure to TDF caused a consistent reduction in live cell numbers across all time points, accompanied by a decrease in dead cells at higher concentrations, indicating a clear suppressive effect. In contrast, TAF only affected cell division after 48 h of treatment and only statistically affected the highest two doses in the concentration range. Thus, TAF produced a milder and delayed response, with no significant impact on cell proliferation at 24 h and only moderate suppression observed between 48 and 96 h at higher concentrations (Figures 3 and 5). In both cases, the reduction in total cell numbers was not due to increased cell death but rather a slowing of cell proliferation, suggesting that both drugs inhibit cell division without inducing cytotoxic effects [25].

4.2. Effects on Cell Cycle Progression

Endothelial cells form the primary regulating endothelium, which constitutes the BBB, and are characterized by their ability to undergo cell division to maintain the integrity of the BBB capillaries. Brain endothelial cell division is crucial to maintain homeostasis in response to cerebrovascular physiological and pathological challenges [27]. Therefore, uninterrupted progression of the cell cycle is essential for preserving endothelial integrity, as disruptions in cell cycle progression could potentially impair brain capillary endothelium function and lead to BBB dysfunction. Flow cytometry is an analytical technique widely used to assess the effects of treatments on cell cycle dynamics, allowing for precise quantification of cells within different phases of the cell cycle [28]. This technique is particularly valuable in understanding how therapeutic agents, such as ARV drugs, impact cellular proliferation and division [29]. By quantifying changes in the proportion of cells within each phase of the cell cycle, we aimed to find out whether treatment with these ARVs suppresses cell division or alters cell cycle progression.

Following exposure, a significant decrease in the proportion of cells in the G1 phase and an increase in the proportion of cells in the S phase were observed for both TDF and TAF treatments (Figure 6). This indicates that TDF disrupts the normal cell cycle progression by potentially preventing cells from transitioning out of the S‐phase, resulting in S‐phase accumulation. The extent of these effects increased with higher concentrations of TDF, which suggests that there is a direct relationship between TDF dosage and its impact on cell division. Similarly, higher concentrations of TAF led to a decrease in the cell population in the G1 phase as well as an increased accumulation of cells in the S‐phase, which indicates that TAF also disrupts normal cell cycle progression. While the overall pattern of the cell cycle disruption by TDF and TAF is similar, the extent of the effects varied depending on the concentration used.

Although these are novel findings for brain capillary endothelium, the suppression in cells within the G1 phase aligns with findings of Milián et al. [30], who observed a similar effect when treating renal cells (NRK‐52E) with 50 μM of TDF. Both TDF and TAF show a significant effect on the G1 phase, with higher concentrations correlating with a greater shift of cells into the S phase at 96 h. This suggests that cells are arrested in the S phase, leading to S‐phase accumulation, preventing progress to the G2 phase, thereby disrupting normal cell cycle progression. This observation is consistent with the findings of Brüning A, Burger P, and Gingelmaier A, who showed that Truvada, a combination of the ARVs Emtricitabine and Tenofovir, induces cell cycle arrest in ovarian cancer cells, either at the S‐phase or G2/M‐phase, depending on the specific cell line and the duration of Emtricitabine/Tenofovir treatment [31]. Their study further concluded that Tenofovir was the main antiproliferative component of Truvada.

4.3. Effects on Monolayer Permeability

Microvascular endothelial cells form a monolayer that constitutes the BBB capillaries, linked by tight junction proteins that maintain the CNS microenvironment by selectively regulating the transcellular uptake of molecules into the brain [7]. It has long been established that there is an inverse relationship between electrical resistance and endothelial cell permeability. As a result, in vitro assays measuring TEER are often used as an electrical resistance indicator of permeability to indicate or monitor the formation or the alteration of the endothelial monolayer [32]. The two‐dimensional monolayer mimics a three‐dimensional capillary endothelium that has been transected apically along its longitudinal plane when folded into a flat sheet of endothelium, which indeed perfectly mimics the experimental monolayer, with its apical surface representing the luminal surface of the capillary and the basal surface representing the outer surface of the capillary. In our study, we assessed TEER across confluent endothelial monolayers as a measure of transendothelial permeability following treatment with TDF or TAF. To ensure that these monolayers mimicked the capillary endothelium, we only treated monolayers with TDF/TAF once TEER plateaued, which indicated that the monolayer was confluent and had formed paracellular tight junctions [18, 33].

We observed that treatment of bEnd.5 cell confluent monolayers to all concentrations of TDF (9.8–98 ng/mL) increased monolayer permeability by up to 55%, as evidenced by the significant decrease in TEER after 3 days of treatment (Figure 7). Similarly, TAF (1 to 10 ng/mL) also increased monolayer permeability, however, to a lesser degree (38%), with a significant dose‐dependent decrease in TEER after one day of treatment (Figure 8). This increase in permeability may be attributed to Tenofovir‐induced increase in paracellular permeability due to the compromise of tight junctions, and may potentially be further aggravated by the suppression of cell proliferation, which may compromise the ability of cells in the monolayer to replace the normal turnover of apoptotic cells at a fast enough rate, compromising the impermeability of the monolayer (Figures 3 and 5). Although studies on Tenofovir's impact on tight junction regulation are limited, evidence suggests that ARVs can enhance endothelial cell paracellular permeability. For instance, a study by Bertrand et al. [34], demonstrated that exposure to Efavirenz, an ARV drug used for HIV treatment, significantly weakened the integrity of the blood–brain barrier by decreasing the levels of key tight junction proteins, including claudins‐1/5, occludin, ZO‐1, and JAM‐1. Similarly, an in vivo study by Kanmogne [35] demonstrated that treatment with the ARVs Efavirenz, Dolutegravir, or Bictegravir—often used in combination with Tenofovir to enhance HIV treatment efficacy—in C57/BL6 mice led to decreased expression of the endothelial tight junction protein occludin and increased BBB permeability. These findings are consistent with our permeability data, which indicates that TDF or TAF treatment results in increased permeability in brain endothelial cells [34].

When comparing the results of the TDF‐treated cells to those treated with TAF, a similar increase in monolayer permeability was observed over the 4‐day period. However, there was a notably greater permeability in the bEnd.5 cells exposed to TDF compared to those exposed to TAF for the same duration (Figures 6 and 7). This increased permeability could be attributed to a reduction in the number of live cells following TDF‐induced suppression of bEnd.5 cell division, which led to a 55% increase in monolayer permeability. In contrast, the decreased cell proliferation induced by TAF (Figures 3 and 5) may have contributed to the lower reduction in monolayer permeability (38%). This postulation is supported by neither TDF nor TAF demonstrating toxicity to bEnd.5 cells (Figures 2E–H and 4E–H). There is limited literature specifically on the effects of Tenofovir on the toxicology of bEnd.5 cells; nevertheless, it is well‐documented that TDF has greater renal and bone toxicities compared to TAF in clinical settings [14, 26]. These observations align with our permeability data, which suggest that long‐term TAF treatment results in a lesser degree of compromising the permeability of our in vitro BBB model compared to TDF.

4.4. Study Limitations

While the bEnd.5 murine endothelial model provides a robust and reproducible platform for in vitro investigation, we acknowledge that it does not fully recapitulate the complexity and physiological relevance of the human BBB. Therefore, future studies should aim to validate these findings using human‐based models, such as primary human brain microvascular endothelial cells, to enhance translational relevance and better reflect human‐specific responses to ARV exposure.

5. Perspectives

Our study shows that both TDF and TAF suppressed bEnd.5 brain endothelial cell proliferation in a dose‐dependent manner without inducing cytotoxicity. TDF exerted an earlier and stronger suppressive effect on cell division when compared with TAF. TAF produced a milder and delayed suppressive response, becoming significant only at higher concentrations after 48 h. Flow cytometry analysis revealed that both TDF and TAF disrupted normal cell cycle progression, which likely contributed to the observed suppression of proliferation. Neither TDF nor TAF demonstrated overt toxicity toward bEnd.5 cells. TDF increased monolayer permeability by up to 55%, while TAF produced a lesser increase of approximately 38%. The increased permeability is likely due to Tenofovir‐induced impairment of tight junction integrity and reduced proliferative capacity. The observed effects on permeability and proliferation are therefore attributed to inhibition of cell division rather than cell death. Together, these findings suggest that long‐term exposure to TDF may contribute to BBB dysfunction. These results provide important new insights into how these drugs might affect the BBB and highlight the importance of further investigation into their potential impacts on CNS health. Accordingly, the enhanced efficacy of TAF over TDF may represent a meaningful therapeutic advantage in the context of HIV‐associated BBB compromise.

Author Contributions

Conceptualization, D.F. and S.B.; methodology, D.F., C.W.; software, D.F.; validation, D.F., K.B.M., C.W. and S.B.; formal analysis, S.B., D.F., C.W. and K.B.M.; investigation, S.B.; resources, D.F.; data curation, S.B. and D.F.; original draft preparation, S.B.; review and editing, D.F.; visualization, D.F.; supervision, D.F., C.W. and K.B.M.; project administration, D.F.; funding acquisition, D.F. All authors have read and agreed to the published version of the manuscript.

Funding

UWC Senate research funds (20/5/9) and the Thuthuka National Research Funding (NRF: 138277) were received for the consumables of the research project.

Consent

The authors have nothing to report.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

All experimental data collected are archived within the University of the Western Cape (UWC) archives and are available as per UWC data and intellectual property policy guidelines and their associated copyright protection.