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. Author manuscript; available in PMC: 2023 Jul 1.
Published in final edited form as: Can J Physiol Pharmacol. 2022 Apr 20;100(7):679–688. doi: 10.1139/cjpp-2022-0035

Porphyromonas gingivalis Infection Upregulates the Endothelin (ET) System in Brain Microvascular Endothelial Cells

Eda Karakaya 1,4, Yasir Abdul 1,4, Nityananda Chowdhury 2, Bridgette Wellslager 2, Sarah Jamil 1,4, Onder Albayram 1,3, Özlem Yilmaz 2, Adviye Ergul 1,3
PMCID: PMC9583200  NIHMSID: NIHMS1840731  PMID: 35442801

Abstract

Endothelin-1 (ET-1), the most potent vasoconstrictor identified to date, contributes to cerebrovascular dysfunction and brain ET-1 levels were shown to be related to ADRD progression. ET-1 also contributes to neuroinflammation, especially in infections of the central nervous system. Recent studies causally linked chronic periodontal infection with an opportunistic anaerobic bacterium Porphyromonas gingivalis (P. gingivalis) to AD development. Thus, the goal of the study was to determine the impact of P. gingivalis infection on the ET system and cell senescence in brain microvascular endothelial cells (BMVECs). Cells were infected with a multiplicity of infection (MOI) 50 P. gingivalis with and without extracellular ATP-induced oxidative stress for 24 hours. Cell lysates were collected for analysis of ETA/ETB receptor as well as senescence markers. ET-1 levels in cell culture media were measured with ELISA. P. gingivalis infection increased ET-1 (pg/ml) secretion, as well as the ETA receptor expression whereas decreased lamin A/C expression compared to control. Tight junction protein claudin 5 was also decreased under these conditions. ETA or ETB receptor blockade during infection did not affect ET-1 secretion or the expression of cell senescence markers. Current findings suggest that P. gingivalis infection may compromise endothelial integrity and activate the ET system.

Keywords: Porphyromonas gingivalis, ET-1, endothelial cells, Vascular Contributions to Cognitive Impairment

INTRODUCTION

Vascular contributions to cognitive impairment and dementia (VCID), a leading cause of ADRD, are characterized by the aging neurovascular unit being confronted and failing to cope with biological insults due to systemic and cerebral vascular disease (Corriveau et al. 2016; Zlokovic et al. 2020). While underlying etiologies are multifactorial, aging, microvascular dysfunction and inflammation appear to be common factors. Indeed, endothelial dysfunction and disruption of the blood-brain barrier (BBB) occur before neuropathologies and cognitive deficits can be detected in all dementias (Gorelick et al. 2017; Iadecola 2017). It is also recognized that systemic infection causes neuroinflammation and cerebral vascular dysfunction and chronic inflammation is associated with greater risk of cognitive impairment (Asby et al. 2021). However, mechanistic links between these commonalities are largely unknown.

Chronic periodontitis is a highly prevalent disease, especially in the aging population (Noble et al. 2009). Epidemiological studies including NHANES-III suggest that periodontitis can contribute to the development of cognitive deficits via amplified neuroinflammation as well as increased risk for stroke and cardiovascular diseases (Choi et al. 2019; Harding et al. 2017a; Harding et al. 2017b; Noble et al. 2009). Porphyromonas gingivalis (P. gingivalis), a major opportunistic Gram-negative bacterium, causes chronic periodontal disease (Lee et al. 2018a; Lee et al. 2020a; Yilmaz 2008). P. gingivalis lipopolysaccharide (LPS) as well as gingipains, a family of proteases secreted by P. gingivalis, have been detected in the brains of AD patients (Dominy et al. 2019). Moreover, oral infection with P. gingivalis worsens neuropathologies and cognitive function in a mouse model of AD (Dominy et al. 2019). Yet, the mechanisms by which systemic P. gingivalis infection promotes cognitive decline remain to be determined.

At the level of the microvascular network, specialized cells work together in the neurovascular unit (NVU) to maintain cerebral blood flow (CBF), oxygen delivery and energy supply (Schaeffer and Iadecola 2021). The NVU consists of vascular cells (endothelial cells and pericytes), glial cells (astrocytes, microglia and oligodendrocytes) and neurons (Schaeffer and Iadecola 2021). The endothelial cells are connected by tight junctions and covered by astrocytic end feet to create a strong barrier forming the basis of the BBB. Impaired endothelial cell integrity is one of the cerebral microvascular alterations occurring over time and contributing to BBB dysfunction (Schaeffer and Iadecola 2021). Endothelin-1 (ET-1), a potent vasoconstrictor with proliferative, prooxidative and proinflammatory properties, is a likely candidate that may be involved in many facets of the cerebrovascular disease (Li et al. 2018). The endothelin system is widely distributed throughout the central nervous system (CNS). Brain microvascular endothelial cells, neurons, and glial cells synthesize ET-1 and express receptors for the ligand (Davenport et al. 2016; Li et al. 2018). In addition to its vasoactive actions, upregulated ET-1 has been shown to cause an increase in BBB permeability in various CNS infections (D’Orleans-Juste et al. 2019). Elevated tissue and circulating ET-1 levels correlate with the degree of brain hypoperfusion in patients with AD and VCID (Barker et al. 2014; Thomas et al. 2015). In light of these observations, the goal of this study was to determine the impact of P. gingivalis infection on the ET system in brain microvascular endothelial cells (BMVECs). We hypothesized that P. gingivalis infection upregulates the expression of ET-1 and its receptors leading to endothelial senescence and loss of barrier function.

MATERIALS AND METHODS

BMVEC culture

The male-derived human BMVEC line HBEC-5i (American Type Culture Collection-ATCC, CRL 3245) was cultured in 75 cm2 culture flasks that were coated with 0.2% w/v gelatin (porcine Type A; Sigma-Aldrich) prior to cell seeding (Eigenmann et al. 2013; Poller et al. 2008; Puech et al. 2018; Wassmer et al. 2006; Weksler et al. 2013). A 1:1 ratio of endothelial growth media (VEC Technologies, Rensselaer, NY, USA) and Medium 199 (Corning, Manassas, VA, USA) was used for cell culture. The VEC media includes serum and antibiotics, while 10% FBS and 1% penicillin-streptomycin were added to the M199. Cells were seeded with 80% confluency on 100 mm plates which were previously coated with 0.2% w/v gelatin and left in 1:1 ratio VEC:M199 media overnight. On the infection day, the media was changed to Modification of Eagle’s Medium (DMEM; Corning, Manassas, VA, USA) containing no penicillin-streptomycin but 1% FBS serum. After a 15 min period, 3mM ATP stimulation was induced. After 1 hour of ATP stimulation, cells were infected with P. gingivalis strain ATCC 33277 at 50 MOI (multiplicity of infection). Cells and supernatants were collected for western blotting and ET-1 ELISA, respectively. For some experiments with ET receptor blockers, after 6 hours of starvation, the media was switched to 1% FBS containing DMEM (without antibiotics). 1uM BQ123 and BQ788 were added to corresponding plates. 30 min after the addition of inhibitor and before the P. gingivalis infection, 3mM ATP stimulation was induced. After 1 hour of ATP stimulation, 50 MOI P. gingivalis infection was done. Cells and supernatants were collected for western blotting and ET-1 ELISA, respectively.

Western Blot Analysis

Expression of ETA and ETB receptors was measured by immunoblotting. Briefly, equivalent amounts of cell lysates (15 μg protein/lane) were loaded onto 10% SDS-PAGE, proteins separated, and proteins transferred to nitrocellulose membranes. The membranes were blocked with 5% bovine serum albumin followed by incubation for 12 hours at 4°C with ETA receptor primary antibody (ab85163, Abcam), ETB receptor primary antibody (AER002, Alomone labs), p16INK4a polyclonal antibody (PA1–30670), p21 monoclonal antibody (HJ21) (AHZ0422), Occludin1 monoclonal antibody (OC-3F10) (33–1500), Claudin 5 monoclonal antibody (4C3C2) (35–2500), Cyclin D1 (92G2) Rabbit monoclonal antibody (MT10014CV), Lamin A/C rabbit polyclonal antibody (2032) at 1:1000 dilution or anti β-actin at 1:10.000 dilution. After washing, membranes were incubated for 1 hour at 20°C with appropriate secondary antibodies (horseradish peroxidase [HRP]-conjugated; dilution 1:5000). Pre-stained molecular weight markers were run in parallel to identify the molecular weight of proteins of interest. For chemiluminescent detection, the membranes were treated with an enhanced chemiluminescent reagent and the signals were monitored on Amersham imager 680 (GE Healthcare Bio-Sciences Corp., Marlborough, MA). Relative band intensity was determined by densitometry on Image-J and normalized with β-actin protein.

Immunostaining for P. gingivalis Infection

BMVECs were seeded upon four-well plates at a density of 8×104 cells. BMVECs were then incubated with P. gingivalis ATCC 33277 at an MOI of 50 for 24 hours. BMVECs were then fixed utilizing 10% neutral buffered formalin (NBF) for 30 min, permeabilized with 0.1% TritonX-100 in PBS for 15 min, and blocked with 3% BSA in PBS/0.05% Tween for 30 min. Cells were then incubated with a custom-made rabbit anti-P. gingivalis ATCC 33277 antibody (1:1,000; Pacific Immunology, Ramona, CA) for 1 hour. Cells were then washed and incubated with Alexa Fluor 488 conjugated secondary goat anti-rabbit antibody (1:2000; Invitrogen) for 1 hour. Following antibody incubation, BMVECs were then washed and their actin cytoskeletons were stained via incubation with Alexa Fluor 568 Phalloidin (1:3000; Invitrogen) for 1 hour. The cells were mounted utilizing glass coverslips and Vectashield Vibrance mounting media with DAPI. BMVECs were then examined and representative images were obtained via wide-field fluorescence microscope (Zeiss Axio Imager A1) at 40x magnification.

16S RNA-based Antibiotic Protection Assay for Quantitative P. gingivalis Invasion of BMVECs

The intracellular invasion and survival of P. gingivalis in BMVECs was determined as we previously detailed in (Lee et al. 2020a). In short, BMVECs were incubated with P. gingivalis ATCC 33277 at MOI 50 for 1, 3, 6, or 24 hours. After the designated incubation periods, BMVECs were washed in PBS and were incubated in gentamicin (300 μg/mL) and metronidazole (200 μg/mL) for 1 hour to remove any extracellular bacteria (Yilmaz et al. 2002). Following antibiotic treatment, the total RNA of each condition was isolated and collected utilizing Trizol Reagent (Invitrogen). Any potential genomic DNA contamination was then removed via DNase treatment (Ambion). Following DNase digestion, cDNA was synthesized utilizing 1 μg of each condition’s total isolated RNA via a High Capacity cDNA Reverse Transcriptase Kit (Applied Biosystems). A dilution of 1:10 cDNA was utilized to detect P. gingivalis-specific 16s rRNA via SYBR Green Real-time qPCR (Forward: 5′-TGTAGATGACTGATGGTGAAAACC-3′;Reverse:5′-ACGTCATCCCCACCTTCCTC-3′) (Harding et al.). qPCR was then conducted with the CFX96 real-time system (Bio-Rad), with an initial cycle of 98°C for 3 min followed by 40 cycles of 9 5°C for 15 s, 60.7°C for 30 s, and 72°C for 30 s. A standard curve was also created, as we previously described in (Lee et al.). This standard curve was utilized to determine the number of colony forming units (CFU) of corresponding Ct (Threshold cycle) values obtained via qPCR performed with the cDNA of each infected BMVEC condition.

ET-1 ELISA

The concentration of ET-1 in the BMVEC culture supernatants was measured by enzyme-linked immunosorbent assay (ELISA) from R&D Systems (QuantiGlo ELISA) following the manufacturer’s instructions. The results calculated on duplicate wells for each sample were expressed as pg/ml for cell culture supernatants.

Cell Senescence Detection

Four groups of cells were grown on slides for the following conditions: No infection, ATP, P. gingivalis and P. gingivalis + ATP. Following infection for 24 h, cells were fixed with 2% PFA and using The CellEvent Senescence Green Detection Kit (C10851), β-galactosidase levels in cells were visualized with Green FITC Filter via Keyence All-in-One Fluorescence Microscope BZ-X800. For each condition, 3–5 random images were acquired and staining pattern and cell morphology were assessed qualitatively. The experiment was repeated on three times.

Statistical analyses

Infection of BMVECs with Pg over time (Fig. 1) was analyzed with one-way ANOVA followed by Tukey’s post hoc comparisons. Data on Figures 2, 3, and 4 were analyzed by two-way ANOVA (no infection x infection) X (control x ATP) followed by Tukey’s post hoc comparisons. Data on Figures 5 and 6 were analyzed with one-way ANOVA followed by Tukey’s post hoc comparisons. ANOVA results are shown on the graphs which are plotted scatter plots with mean ± sem of 3–6 individual experiments. GraphPad Prism8 software was used for all analyses.

Fig. 1. P. gingivalis invades and survives within human BMVECs.

Fig. 1.

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BMVECs were infected with P. gingivalis at 50 MOI for 1, 3, 6, and 24 h. A) Following infection of BMVECs with P. gingivalis for 24 h, cells were fixed, permeabilized, and stained with anti-P. gingivalis antibody followed by Alexa Fluor-488-conjugated secondary antibody to visualize the intracellular P. gingivalis (green), Rhodamine Phalloidin to visualize actin filaments (red), and DAPI to visualize nuclei (Blue). Representative images were obtained via Zeiss Axio Imager A1 at 40x magnification. B) P. gingivalis infected BMVECs (MOI 50) at all time-points were assayed by in situ quantitative antibiotic-protection-assay to determine the level of intracellular and metabolically active bacteria as described in methods in detail. Data is normalized to 1 h post-infection. Experiments were performed on three separate occasions in triplicate. One-way ANOVA was utilized to assign significance (p<0.005). Tukey’s post hoc comparisons are indicated by *p<0.05, ***p<0.001.

Fig. 2. P. gingivalis stimulates the ET system in BMVECs.

Fig. 2.

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A. ET-1 levels in the cell culture supernatant were greater in cells infected with P. gingivalis in the absence of ATP treatment (n=4–6) while it was lower in cells infected with P. gingivalis in the presence of ATP treatment. B. Representative images of westerns blots for ETA and ETB receptor expression (n=3–6 experiments in duplicate). C. ETA receptor expression was also increased with P. gingivalis infection. D. There was no change in ETB receptor levels. Two-way ANOVA tables are shown on the graphs and Tukey’s post hoc comparisons are indicated by *p<0.05, ** p<0.01 and *** p<0.001.

Fig. 3. P. gingivalis infection mediates senescence-like changes in BMVECs.

Fig. 3.

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There was no change in p21 (A), p16 (B), or Cyclin D1 (D) levels 24 after infection with P. gingivalis in the absence or presence of ATP (n=3–6 experiments in duplicate). (C) Lamin A/C levels were lower in infected groups. (E) Representative images of β-galactosidase staining in infected cells (n=3). Following infection for 24 h, cells were fixed with 2% PFA and using the CellEvent Senescence Green Detection Kit (C10851). Images were captured via Keyence All-in-One Fluorescence Microscope BZ-X800. P. gingivalis + ATP infected cells showed ameboid and round morphology and a higher percentage of the cells showed staining whereas there were unstained cells in other conditions. Two-way ANOVA tables are shown on the graphs.

Fig. 4. P. gingivalis decreases membrane integrity protein.

Fig. 4.

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Following infection for 24 h, cells were collected and lysed. Western blot was conducted with occludin1 and claudin-5 antibodies. Both Occludin 1 (A) and Claudin-5 (B) levels were lower when cells were infected with P. gingivalis (n=3–6 experiments in duplicate). C. Representative images of westerns blots for occludin1 and claudin5 expression (n=3–6 experiments in duplicate). Two-way ANOVA tables are shown on the graphs and Tukey’s post hoc comparisons are indicated by *p<0.05.

Fig. 5. ET receptor blockade did not affect ET-1 secretion or levels of ETA or ETB receptors.

Fig. 5.

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The levels of ET-1 in the cell culture supernatant (A) or ET receptors in cell lysates (B and C) did not change after 24 hours infection with P. gingivalis and ATP stimulation in the presence of ETA and ETB receptor antagonists, BQ123 and BQ788 respectively (n=3–6 experiments in duplicate).

Fig. 6. ET receptor antagonists did not affect the senescence response or membrane integrity proteins in BMVECs.

Fig. 6.

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There was no change in p21 (A), p16 (B), Lamin A/C (C) or Cyclin D1 (D) levels 24 hours after infection with P. gingivalis and ATP stimulation in the presence of ETA and ETB receptor antagonists BQ123 and BQ788 respectively (n=3–6 experiments in duplicate). There was also no change in membrane integrity proteins Occludin1 (E) and Claudin5 (F) levels in the presence of ETA and ETB receptor antagonists (n=3–6 experiments in duplicate).

RESULTS

Infection of BMVECs with P. gingivalis upregulates the ET system

Presence of P. gingivalis was detected in the cytoplasm suggesting that this obligate anaerobic bacterium can invade, replicate and survive in BMVECs as it has been shown in gingival epithelial cells (Fig.1). Cell culture media collected at the end of the experiment (24 h post-infection) showed significant increase in ET-1 levels in the infected cells and presence of ATP lowered ET-1 levels as compared to control conditions in both infected and uninfected cells (Fig. 2A). Post hoc analyses indicated that ET-1 levels are higher in control infected cells than in uninfected control group, and ATP lowers ET-1 under infection conditions. In BMVECs, there appeared to be more ETA receptors with P. gingivalis infection (Fig. 2B and C). There was no change in ETB receptors (Fig. 2B and D).

Infection of BMVECs with P. gingivalis alters the expression of senescence markers

To determine the effect of P. gingivalis infection on cellular senescence, multiple markers were measured in the same homogenates used for ET receptor expression. There was no difference in p21, p16, and Cyclin D protein levels among the experimental conditions (Fig. 3 A, B, and D), in cells infected with P. gingivalis with or without ATP treatment. However, Lamin A/C was lower in infected cells (Fig. 3C). Qualitative assessment of β-galactosidase staining suggested that % of positively stained cells was greater in cells infected with P. gingivalis + ATP which also showed ameboid morphology (Fig. 3E).

Infection of BMVECs with P. gingivalis alters the expression of tight junction proteins

Assessment of tight junction proteins showed that both Occludin 1 and Claudin 5 levels are lower in infected cells (Fig. 4A and C). Post hoc analysis did not indicate further differences in individual comparisons.

ET receptor inhibition does not affect P. gingivalis effects on BMVECs

Since initial experiments showed a dramatic increase in ET-1 secretion and ETA receptor expression along with changes in senescence markers and tight junction protein Claudin 5 within the 24-h period of P. gingivalis infection with ATP treatment, next set of experiments determined the impact of ET receptor blockade on the autocrine regulation of the ET system as well as on cellular integrity. Inhibition of ETA and ETB receptors with BQ123 or BQ788, respectively, did not lead to a significant change in ET-1, ETA or ETB levels but there was high variability (Fig. 5A–C). ET receptor blockade had no effect on cellular senescence markers or tight junction proteins (Fig. 6A–E).

DISCUSSION

A long line of research has indicated close associations between chronic periodontal disease and cognitive impairment (Choi et al. 2019; Harding et al. 2017a; Harding et al. 2017b; Noble et al. 2009). P. gingivalis, an opportunistic Gram (−) microorganism, is a major contributor to the etiology of chronic severe periodontitis, and it was recently identified in the brains of patients suffering from AD (Dominy et al. 2019). Moreover, chronic P. gingivalis infection was reported to lead to cognitive decline in a preclinical model establishing causality (Dominy et al. 2019). Based on this recent development and the following lines of evidence in the ADRD research that 1) increased BBB permeability and cerebrovascular dysfunction are early biomarkers, 2) microvascular degeneration is common, and 3) changes in the ET system are associated with infectious and non-infectious inflammatory diseases of the central nervous system including ADRD, we asked i) Can P. gingivalis invade/infect BMVECs?, ii) Does P. gingivalis infection affect cellular senescence and integrity?, iii) Does it affect the ET system?, and iv) Are P. gingivalis effects on BMVECs are mediated by the ET system?

P. gingivalis is a major pathogen that causes periodontitis, a highly prevalent chronic disease (Harding et al. 2017b; Yilmaz 2008). P. gingivalis colonizes in the oral mucosa and can invade, replicate intracellularly, and spread from one cell to other in the gingival epithelial layer (Lee et al. 2017; Lee et al. 2018b; Yilmaz et al. 2006). Studies by us and others have shown that intracellular invasion of P. gingivalis does not induce apoptosis or necrosis and can render these cells resistant to cell death induced by proapoptotic molecules later in infection (Yao et al. 2010; Yilmaz et al. 2004). This chronic infection has long been postulated to lead to bacteremia and cause systemic diseases such as atherosclerosis and ADRD (Lee et al. 2020b; Velsko et al. 2014). In a recent study, we showed the presence of live P. gingivalis in deep epithelial layers, lamina propria, and the gingival epithelial microvasculature in biopsies from periodontitis patients for the first time (Lee et al. 2020b). As discussed above, more recently gingipains, P. gingivalis virulence factors, have been detected in the brains of AD patients (Dominy et al. 2019). The level of the gingipains in the brain were reported to correlate with tau pathology in these patients. In preclinical studies, the chronic administration of P. gingivalis-LPS, a main pathogenic factor, has been reported to lead to cognitive decline (Zhang et al. 2018). Another study showed that oral infection by P. gingivalis worsens cognitive function and the deposition of plaques in transgenic human amyloid precursor protein (hAPP-J20) mouse model (Ilievski et al. 2018). Oral P. gingivalis infection or injection of gingipains in BALB/c led to neuronal death and augmented Aβ1–42 production, which is a critical component of amyloid plaques (Dominy et al. 2019). However, how P. gingivalis gains access to the brain is not known. Since BMVECs form the BBB that protects the brain, direct infection and damage to these cells may be one point of entry. Our data show that P. gingivalis enters BMVECs and shows a perinuclear localization by 24 h. We have reported that P. gingivalis infection modulates survival mechanisms and renders gingival epithelial cells resistant to cell death prolonging its own intracellular survival in the host (Lee et al. 2018a). Whether the microorganism can survive long term, replicate and spread from one BMVEC to other remains to be established.

Senescence is a biological process is which a cell is irreversibly arrested in the cell cycle and therefore, no longer able to replicate, but remains metabolically active (Burton and Faragher 2015; Ohtani 2019). Cellular senescence can be a favorable physiological process such as in wound healing and embryonic development, and in other instances deleterious to the homeostasis of the surrounding cellular environment (Ohtani 2019; Rodier and Campisi 2011). Accumulation of senescent cells in various tissues is believed to contribute to progressive functional impairments that come with chronological aging as well as age-related disorders such as ADRD (Shimizu and Minamino 2020). Given that cerebrovascular dysfunction is an early finding in ADRD, a recent study focused on the microvasculature and identified senescent cerebromicrovascular endothelial cells in the aged mouse brain using single-cell RNA sequencing. Since P. gingivalis infection confers an antiapoptotic phenotype to gingival epithelial cells, we next looked at senescence markers in BMVECs infected with P. gingivalis in the absence and presence of a danger molecule, extracellular ATP because P. gingivalis can specifically inhibit both oxidative stress and cell death in human gingival epithelial cells induced by the ATP stimulation (Choi et al. 2013; Roberts et al. 2017; Yilmaz et al. 2008). In the 24 h time frame, there was no change in proteins involved in cell cycle including p16, p21 and Cyclin D1. However, Lamin A/C, a filamentous protein found in the inner nuclear membrane architecture, was decreased with P. gingivalis infection, suggesting initiation of the senescence process. We also noted that the morphology of BMVECs was different, i.e. more ameboid, and a greater percentage of the cells showed β-gal staining, especially in the co-presence of P. gingivalis and ATP. Tight junction protein Claudin 5 was also decreased in these cells, which may impact the integrity and barrier function of BMVECs. While it needs to be further tested, we speculate that these changes in cellular phenotype may allow P. gingivalis to survive and access the brain parenchyma.

ET-1 is the most potent vasoconstrictor identified to date with proinflammatory and proliferative properties. As briefly alluded to above, cerebrovascular compromise and changes in cerebral blood flow occur early in ADRD. As such, a possible role of ET-1 in the development and progression of ADRD has long been debated (Li et al. 2018). Clinically, ET-1 and endothelin converting enzymes (ECE-1 and 2) activities are upregulated in postmortem temporal cortex specimens from AD patients. Decreased ratio of myelin-associated glycoprotein (MAG), which is highly susceptible to ischemia, and proteolipid protein 1 (PLP1), which is more resistant to ischemia, is a surrogate marker of hypoperfusion. Increased ET-1 levels correlate positively with MAG/PLP ratio in the postmortem brains of individuals diagnosed with ADRD. Using the same approach for assessment of perfusion status, a recent study showed that systemic infection exacerbates cerebrovascular dysfunction in dementia patients (Asby et al. 2021). Moreover, an intriguing study showed that ET-1 secreted from endothelial cells constrict capillaries via the activation of ETA receptors on pericytes in human brain cortical slices (Nortley et al. 2019). As highlighted in a recent review, ET-1 contributes to the progression of neuroinflammatory processes within the brain (D’Orleans-Juste et al. 2019). A highly significant finding of the current study is that P. gingivalis infection mediates a dramatic increase in ET-1 secretion from BMVECs and also upregulates the ETA receptor subtype. ET-1 mediates its diverse effects via two distinct G protein-coupled receptor subtypes, ETA and ETB (Davenport et al. 2016). ETA receptor, localized mainly on vascular smooth muscle cells of arteries and arterioles as well as on pericytes surrounding capillaries, is responsible for the contractile and proliferative responses to ET-1 (Davenport et al. 2016). While endothelial cells throughout the body express only the ETB receptor subtype, BMVECs are unique in their expression of ETA receptors (Abdul et al. 2020). Another possible mechanism by which ET-1 can impact cerebrovascular function is the disruption of BBB integrity. Since we observed decreases in Claudin 5 and senescence-like changes in infected BMVECs along with parallel increases in ET-1 and ETA expression, we next investigated the autocrine effects of ET-1 on BMVECs by repeating the infection experiments with P. gingivalis plus ATP in the presence of BQ123 or BQ788, ETA and ETB receptor antagonists, respectively. ET receptor blockade did not affect ET-1 secretion or levels of ETA or ETB receptors. There were no changes in senescence markers or tight junction proteins, either. These findings suggest that within the time frame of the study, ET-1 does not contribute to endothelial senescence but warrant future studies for time course experiments with more functional outcomes.

We recognize the limitations of this initial study. We took a reductionist approach to determine P. gingivalis effects on BMVECs and used only one time point of infection (24 h). We also used only 50 MOI for infection based on our extensive experience in human gingival epithelial cells. Longer infection times are needed to evaluate the impact of P. gingivalis on cellular senescence and integrity over a time course. Even with this limited infection, we saw a robust increase in ET-1 levels secreted into medium as well as in ETA receptor expression, which are novel findings. We assessed only autocrine effects of ET-1 on BMVECs by using ET receptor antagonists. Paracrine effects of secreted ET-1 and other possible inflammatory mediators on NVU cells remain to be determined. Lastly, the mechanisms by which P. gingivalis increases ET-1 and ETA levels are yet to be investigated. Nevertheless, our findings pinpoint novel mechanisms by which chronic P. gingivalis infection may contribute to compromised BBB and possibly ADRD.

ACKNOWLEDGEMENTS

Funding:

This study was supported by Veterans Affairs (VA) Merit Review (BX000347), VA Senior Research Career Scientist Award (IK6 BX004471), National Institute of Health (NIH) RF1NS083559 and R01 NS104573 (multi-PI, Susan C. Fagan as co-PI) to Adviye Ergul; R01 DE030313-S1 to Ozlem Yilmaz; UL1TR001450/SCTR2201 to Yasir Abdul; MUSC’s Specialized Center of Research Excellence (SCORE) Career Enhancement Core (CEC) Scholarship to Onder Albayram.

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