TRPV4 is a regulator in P. gingivalis lipopolysaccharide‐induced exacerbation of macrophage foam cell formation

Abstract Porphyromonas gingivalis (P.g), a major causative agent of periodontitis, has been linked to atherosclerosis, a chronic inflammatory vascular disease. Recent studies have suggested a link between periodontitis and arterial stiffness, a risk factor for atherosclerosis. However, the mechanisms by which P.g infection contributes to atherogenesis remain elusive. The formation of lipid‐laden macrophage “foam cells” is critically important to development and progression of atherosclerosis. We have obtained evidence that TRPV4 (transient receptor potential channel of the vanilloid subfamily 4), a mechanosensitive channel, is a regulator of macrophage foam cell formation both in response to P.g‐derived lipopolysaccharide (PgLPS) or to an increase in matrix stiffness. Importantly, we found that TRPV4 activity (Ca2+ influx) was increased in response to PgLPS. Genetic deletion or chemical antagonism of TRPV4 channels blocked PgLPS‐triggered exacerbation of oxidized LDL (oxLDL)‐mediated foam cell formation. Mechanistically, we found that (1) TRPV4 regulated oxLDL uptake but not its cell surface binding in macrophages; (2) reduced foam cell formation in TRPV4 null cells was independent of expression of CD36, a predominant receptor for oxLDL, and (3) co‐localization of TRPV4 and CD36 on the macrophage plasma membrane was sensitive to the increased level of matrix stiffness occurring in the presence of PgLPS. Altogether, our results suggest that TRPV4 channels play an essential role in P.g‐induced exacerbation of macrophage foam cell generation through a mechanism that modulates uptake of oxLDL.


Introduction
Atherosclerosis, a chronic inflammatory vascular disease, accounts for the majority of deaths linked to cardiovascular disease (CVD) (Lusis 2000;Moore and Tabas 2011;Falk et al. 2013). Tissue macrophages recognize and take up oxidized low-density lipoproteins (oxLDL) through various scavenger receptors (SR) such as CD36 and SR-A, and contribute to generation of lipid-loaded "foam cells," a critical early event in the development of artherosclerotic lesions (Lusis 2000;Collot-Teixeira et al. 2007;McLaren et al. 2011;Moore and Tabas 2011;Falk et al. 2013). Progressive generation and buildup of macrophage foam cells along with other inflammatory changes such as generation of cellular debris, lipids, expression of inflammatory cytokines, and deposition of calcium in the aortic intimal areas initiate the formation of atherosclerotic plaques, and consequently cause development of atherosclerosis and related pathologies (Lusis 2000;Bobryshev 2006;Libby 2008;Moore and Tabas 2011;Moore et al. 2013).
Approximately 50% of CVD patients lack traditional risk factors (Lusis 2000;McLaren et al. 2011;Moore and Tabas 2011;Moore et al. 2013;Ruparelia et al. 2017; Thomas and Lip 2017). Numerous clinical and experimental studies have shown that infection with various bacterial pathogens including Porphyromonas gingivalis may serve as an additional risk factor in the development and progression of atherosclerosis (Tonetti 2009;Kebschull et al. 2010;Hayashi et al. 2011Hayashi et al. , 2012Fukasawa et al. 2012;Teeuw et al. 2014;Chukkapalli et al. 2015;Hajishengallis 2015;Schmitt et al. 2015;Houcken et al. 2016). Emerging experimental and epidemiological studies suggest an association between periodontitis, a chronic infection of the periodontium, and atherosclerosis, even after controlling for traditional CVD-related risk factors (Tonetti 2009;Kebschull et al. 2010;Hayashi et al. 2011Hayashi et al. , 2012Fukasawa et al. 2012;Teeuw et al. 2014;Walters and Lai 2015). P. gingivalis, a predominant causative factor of periodontitis, has been reported to accelerate atherosclerosis in animal models (Hayashi et al. 2011(Hayashi et al. , 2012Fukasawa et al. 2012;Chukkapalli et al. 2015). However, the precise mechanism whereby P. gingivalis induces atherosclerosis is not well understood. Recent epidemiologic studies suggest a link between periodontal disease and the development of stiffness in arterial tissues (Schmitt et al. 2015;Houcken et al. 2016). Studies also suggest that arterial stiffness is an underappreciated risk factor for various cardiovascular diseases including atherosclerosis (Kothapalli et al. 2012;Hansen and Taylor 2016;Palombo and Kozakova 2016;Tedla et al. 2017). Interestingly, macrophages, a critical cell type in the development of atherosclerosis, have been shown to directly respond to changes in their internal and external biomechanical environment (Doherty et al. 1994;Blakney et al. 2012;Pi et al. 2014;Hind et al. 2015;Previtera and Sengupta 2015;Adlerz et al. 2016;Scheraga et al. 2016). Published reports by our group and others have shown that numerous proatherogenic macrophage functions including migration, phagocytosis, and proliferation are influenced by matrix stiffness, implying that stiffness may play a critical role in determining the proatherogenic response of macrophages in the context of periodontitis/ atherosclerosis (Doherty et al. 1994;Blakney et al. 2012;Pi et al. 2014;Hind et al. 2015;Previtera and Sengupta 2015;Adlerz et al. 2016;Scheraga et al. 2016). Additionally, it has been shown that bacterial LPS can increase macrophage rigidity and stiffening of vasculature in vivo (Doherty et al. 1994;Meng et al. 2015).

Animal and cell culture
We acquired the TRPV4 knockout (TRPV4 KO) mouse line from Zhang (Medical College of Wisconsin, Milwaukee, WI). The original creator of these mice on a C57BL/ 6 background was Suzuki (Jichi Medical University, Tochigi, Japan) (Suzuki et al. 2003). Congenic wild type (WT) mice were purchased from Charles River Laboratories (Wilmington, Massachusetts, USA). All animal experiments were performed following Institutional Animal Care and Use Committee guidelines approved by the University of Maryland review committee. Murine resident macrophages (MRMs) and bone marrow-derived macrophages (BMDMs) were isolated as we described previously (Rahaman et al. 2006(Rahaman et al. , 2013. Briefly, thioglycollate-elicited peritoneal MRMs from backgroundmatched control WT and TRPV4 KO mice were plated on coverslips in 12-well plates in RPMI-1640 medium containing 10% FCS. After 2 h of incubation, nonadherent MRMs were washed out, fresh medium was added, and incubation was continued for 24 h. For BMDM culture, femurs and femur heads from 6 to 7 week old WT and TRPV4 KO mice were collected, and bone marrow was flushed out with RPMI-1640. The suspended bone marrow cells were filtered through a 70 lm strainer. The single cell suspension was centrifuged, plated in RPMI-1640 medium containing M-CSF (20 ng/mL), and incubated for 7-8 days to differentiate into macrophages.

Measurement of intracellular calcium
Calcium responses in BMDMs were recorded on a FlexStation system (Molecular Devices, Sunnyvale, CA) using the FLIPR Calcium 6 Assay Kit. Since TRPV4-elicited Ca 2+ influx was greater in BMDMs than MRMs, we used BMDMs for Ca 2+ influx studies. Cells were seeded into collagen coated (10 lg/mL) 96-well plastic plates with RPMI containing 10% serum and 25 ng/mL M-CSF. BMDMs required 7-8 days for complete differentiation and adherence to the surface. After cellular adhesion the medium was replaced with 0.5% serum containing RPMI and 500 ng/mL pgLPS to select wells. Adhered BMDMs were incubated for 90 min with FLIPR Calcium 6 dye containing a buffer system (HBSS and 20 mmol/L HEPES) and 2.5 mmol/L probenecid. The selective TRPV4 agonist, GSK101, was used to induce cytosolic Ca 2+ influx. To block TRPV4-generated Ca 2+ influx, BMDMs were pretreated with vehicle or TRPV4 antagonist, GSK219, for 45 min. Cytosolic Ca 2+ influx was measured as relative fluorescence units (RFU), and was recorded by measuring DF/F (Max-Min) as we described previously (Rahaman et al. 2014;Goswami et al. 2017;Sharma et al. 2017).

Foam cell assays
MRMs from TRPV4 KO and WT mice were seeded either on collagen-coated (10 lg/mL) glass coverslips or on polyacrylamide gels of varying stiffness (0.5 and 8 kPa) in RPMI 1640. After the initial 48 h incubation, 50 lg/mL of control native LDL (nLDL) or oxLDL with or without 500 ng/mL PgLPS were added, and incubation was continued for 20 h, as we published previously (Rahaman et al. 2006(Rahaman et al. , 2013. To identify foam cells, MRMs were stained with Oil Red O following our published method (Rahaman et al. 2006(Rahaman et al. , 2013.

Binding and uptake of oxLDL
To examine binding, MRMs seeded on glass coverslips with or without 500 ng/mL pgLPS were incubated with DiI-labeled oxLDL (DiI-oxLDL) (5 lg/mL) for 60 min at 4°C (Rahaman et al. 2006(Rahaman et al. , 2013. To assess uptake, cells were treated similarly following DiI-oxLDL treatment, but were incubated at 37°C, and were imaged at 30 min, as we previously published (Rahaman et al. 2006(Rahaman et al. , 2013. For both binding and uptake assay, fluorescence intensity was examined by Zeiss Axio Observer microscope (63x), and quantified by NIH ImageJ software.

Immunofluorescence analysis
MRMs were seeded on polyacrylamide gels (0.5, 8, and 50 kPa) for 48 h, fixed with 3% paraformaldehyde, and incubated with antibodies specific to TRPV4 (1:100) or CD36 (1:100) protein. Goat-anti rabbit Alexa Fluor 488 (1:300) or goat-anti Mouse Alexa Flour 594 (1:300) was used as the secondary antibody. We used prolong diamond antifade reagent (Life technologies) with DAPI as the mounting reagent. We quantified immunofluorescence intensity of stained cells by ImageJ software (NIH), and the results are presented as Integrated Density (Int. Density: the product of Area and Mean Gray Value).

Quantitative real-time polymerase chain reaction (qRT-PCR)
We used RNeasy Micro kit (Qiagen) to harvest total RNA from WT and TRPV4 knockout MRMs. We performed one-step qRT-PCR analysis using QuantiNova SYBR Green RT-PCR Kit (Qiagen) according to the manufacturer's instructions. CD36, TRPV4, TNF-a, IL-6, IL-1b, and control GAPDH primers were purchased from Thermofisher (USA), and qRT-PCR was carried out per the manufacturer's instructions using TaqMan gene Expression Assay (Applied Biosystems). Normalized mRNA expression of CD36 or TRPV4 was determined using mRNA for GAPDH as the internal control. We used the comparative C T method described in the ABI 7900 HT sequence detection system user bulletin.

Data analysis
All data are reported as mean AE SEM. Statistical analysis between control and experimental groups was performed using the Student's t-test or ANOVA using Prism software; p ≤ 0.05 was considered to indicate significance.

Results
P. gingivalis lipopolysaccharide-triggered exacerbation of oxidized LDL-induced macrophage foam cell formation is reliant on TRPV4.
We compared oxLDL-induced foam cell formation in WT and TRPV4 KO (TRPV4À/À) MRMs in the presence or absence of stimulation with P. gingivalis -derived LPS (PgLPS). As expected, we found a fourfold increase in foam cell generation in oxLDL treated WT MRMs compared to control native LDL (nLDL) treated cells ( Fig. 1A and B). The combined treatment with PgLPS and oxLDL further increased macrophage foam cells in WT MRMs compared to untreated controls ( Fig. 1A and B). The results showed that the deficiency of TRPV4 function in TRPV4À/À MRMs abrogated (by more than twofold) macrophage foam cell formation regardless of treatment with oxLDL alone or oxLDL plus PgLPS ( Fig. 1A and B). Similarly, we observed that TRPV4 antagonism by pharmacologic inhibition (GSK219 treatment) in MRMs abrogated PgLPS-induced exacerbation of oxLDL-mediated macrophage foam cell formation ( Fig. 1C and D). We assessed expression levels of TNF-a, IL-6, and IL-1b in WT and TRPV4 KO MRMs by qRT-PCR analysis. We detected reduced expression levels of all three mRNAs in WT cells compared to TRPV4 KO treated with oxLDL for 24 h (Fig. 1E). Taken together, these findings indicate that TRPV4 plays a role in oxLDL-induced inflammatory protein expression and in PgLPS-induced exacerbation of oxLDL-mediated macrophage foam cell generation.
TRPV4 deficiency prevents P. gingivalis lipopolysaccharide-induced exacerbation of foam cell formation in response to augmented matrix stiffness We tested whether PgLPS treatment would cause enhanced macrophage foam cell formation in response to increased matrix stiffness in the presence of TRPV4, a matrix stiffness sensitive channel. In cells growing on a stiff matrix (8 kPa), we found a twofold enhancement in oxLDLinduced foam cell formation in WT cells in response to PgLPS compared to oxLDL alone ( Fig. 2A and B). The results showed that a deficiency of TRPV4 function (TRPV4À/À MRMs) abrogated (by fourfold) oxLDLinduced macrophage foam cell formation in response to PgLPS compared to WT cells. Taken together, these results suggest that biomechanical stimuli such as matrix stiffness may modulate exacerbation of PgLPS-induced foam cell formation in a TRPV4-dependent manner.
TRPV4-dependent Ca 2+ influx is increased in response to P. gingivalis lipopolysaccharide We tested whether a physiological inflammatory stimulus mediated by PgLPS affected TRPV4-dependent Ca 2+ influx. To record the presence of TRPV4-mediated Ca 2+ influx in BMDMs, we detected Ca 2+ influx induced by a selective TRPV4 channel agonist, GSK101, with or without PgLPS pretreatment (Thorneloe et al. 2012;Goswami et al. 2017). Results indicated that cytosolic Ca 2+ influx was potentiated in the PgLPS-treated cells compared to the unstimulated group ( Fig. 3A and B). As we expected, results showed that Ca 2+ influx was undetectable in similarly treated TRPV4 KO BMDMs (Fig. 3A and B). Furthermore, GSK101-induced Ca 2+ influx was inhibited by the selective TRPV4 channel antagonist, GSK219, in PgLPS pretreated BMDMs compared to antagonist- untreated controls (Thorneloe et al. 2012;Goswami et al. 2017) (Fig. 3C). These results confirmed that loss of TRPV4 function by genetic deficiency or by pharmacologic antagonism abrogated PgLPS-induced Ca 2+ influx.
In addition, we observed that PgLPS-induced TRPV4elicited Ca 2+ influx was augmented in a matrix stiffness-dependent manner when macrophages were grown on stiffer matrices (8 and 25 kPa) compared to macrophages grown on soft matrix (1 kPa) (Fig. 3D). Furthermore, genetic deletion of TRPV4 in BMDMs, specifically reduced matrix stiffness-induced Ca 2+ influx (Fig. 3D). Altogether, these findings showed that TRPV4-dependent

Reduction in macrophage foam cell formation in TRPV4 deficient cells is independent of expression of CD36
We assessed expression levels of CD36 in WT and TRPV4 KO MRMs by qRT-PCR, immunoblot, and immunofluorescence analysis. We detected similar expression levels of CD36 mRNA and CD36 protein in both WT and TRPV4 KO macrophages treated or not with PgLPS for 24 h ( Fig. 4A and B). Since we found that increasing matrix stiffness upregulated the ability of TRPV4 to augment PgLPS-induced Ca 2+ influx (Fig. 3D), we evaluated whether changes in matrix stiffness would cause enhanced expression levels of CD36 protein in MRMs by immunofluorescence analysis. We found that culture of both WT and TRPV4 KO macrophages on a stiffer matrix (8 or 50 kPa) compared to a softer matrix (0.5 kPa) for 24 h resulted in similar levels of CD36 protein expression (Fig. 4C). Altogether, these results indicated that reduced foam cell formation in TRPV4 deficient macrophages is independent of CD36 expression.

Plasma membrane colocalization of TRPV4 and CD36 is sensitive to changes in matrix stiffness in PgLPS-treated MRMs
To determine whether TRPV4 plays a role in macrophage foam cell formation, we first detected the expression levels of TRPV4 with or without PgLPS stimulation in WT MRMs. Our results showed similar expression levels of TRPV4 mRNA with or without PgLPS treatment (Fig. 5A). Interestingly, immunoblot data showed that treatment with PgLPS for 24 h increased TRPV4 protein expression in a dose-dependent manner ( Fig. 5B and C). Numerous factors have been reported to modulate plasma membrane accumulation of TRPV4 and CD36 (Cuajungco et al. 2006;Ring et al. 2006;Yamada et al. 2009). We assessed changes in plasma membrane accumulation and possible colocalization of TRPV4 and CD36 in response to increasing matrix stiffness in MRMs simulated with PgLPS. Immunofluorescence data showed that exposure of MRMs to a stiff matrix (8 kPa) compared to a soft matrix (0.5 kPa) for 24 h promoted increased plasma membrane enrichment and colocalization of TRPV4 and CD36. These data suggest that changes in matrix stiffness may provide a potential mechanism for functional crosstalk between TRPV4 and CD36 (Fig. 5D). We also noticed that exposure of MRMs to a stiff matrix (8 kPa) compared to a soft matrix (0.5 kPa) for 24 h resulted in an increase in cell surface area.

TRPV4 regulates PgLPS-induced oxLDL internalization but not its cell surface binding in macrophages
We analyzed binding and internalization of oxLDL in macrophages to investigate whether TRPV4 influenced macrophage foam cell formation in response to PgLPS by modulating these responses. WT and TRPV4 KO MRMs were incubated with fluorescent dye-labeled LDL (DiI-oxLDL) at 4°C followed by 37°C (see Methods) to determine whether TRPV4 played a role in binding and uptake of oxLDL. Our results indicated similar binding of DiI-oxLDL in TRPV4 KO cells compared to WT cells ( Fig. 6A-B). Interestingly, TRPV4 KO MRMs exhibited significantly reduced DiI-oxLDL uptake after 1 h compared with WT MRMs with or without PgLPS stimulation ( Fig. 6C-D). Furthermore, we evaluated whether inhibition of TRPV4 channel activity in BMDMs by a selective small chemical inhibitor, GSK219, would influence internalization of DiI-oxLDL. Our data showed that TRPV4 antagonism by GSK219 made no difference in oxLDL binding in WT MRMs stimulated with PgLPS (Fig. 6A). However, internalization of DiI-oxLDL was significantly higher in untreated WT macrophages compared to GSK219-treated cells after 1 h incubation ( Fig. 6C-D). Taken together, these results suggest that TRPV4 activity regulates PgLPS-induced oxLDL uptake but not it's binding at the macrophage surface.

TRPV4 KO WT
It has been reported that exposure to Porphyromonas gingivalis lipopolysaccharide, an immunomodulatory molecule commonly found in the blood stream of patients with chronic periodontal disease, enhances binding and internalization of modified/oxidized LDL, induces macrophage foam cell formation, and aggravates M1 macrophage infiltration and macrophage-mediated inflammation in infarcted tissue (Qi et al. 2003;Hayashi et al. 2011;Fukasawa et al. 2012;Teeuw et al. 2014;Chukkapalli et al. 2015;Schmitt et al. 2015;Houcken et al. 2016;Goswami et al. 2017). We have sought to determine the cellular and molecular mechanisms that regulate the binding and internalization of oxLDL within macrophages that may be responsible for generation of PgLPS-induced foam cells. Our present data support the notion that TRPV4-mediated Ca 2+ influx integrates PgLPS-induced signals to bolster macrophage foam cell generation. Considerable evidence suggests that oxLDL promotes Ca 2+ influx and macrophage foam cell formation (Yang et al. 2000;Rahaman et al. 2011b). Interestingly, these oxLDL-mediated effects were shown to be abrogated by nonspecific Ca 2+ channel blockers (Yang et al. 2000;Rahaman et al. 2011b). Published reports from our laboratory and others have shown that TRPV4induced Ca 2+ influx has diverse roles in different cell types including macrophages (Rahaman et al. 2014;Scheraga et al. 2016;Goswami et al. 2017;Sharma et al. 2017). Our current results show that TRPV4 augments oxLDL-induced foam cell formation in response to PgLPS stimulation. We also showed that oxLDL-induced expression of inflammatory cytokines was reduced in TRPV4 null cells. These results are consistent with our previous report that TRPV4 plays a role in oxLDL-induced macrophage foam cell formation .
Recent evidence documents an atheroprotective role of TRPV4 in which TRPV4 function in endothelial cells is linked to activation of eNOS and suppression of monocyte adhesion to endothelial cells (Xu et al. 2016). In contrast, impairment of TRPV4 channels has been linked to endothelial dysfunction, reduced macrophage foam cell generation, and vascular diseases (Zhang et al. 2009;Ye et al. 2012;Du et al. 2016;Goswami et al. 2017). In this study, we found that treatment with PgLPS results in upregulation of TRPV4 protein expression and TRPV4induced Ca 2+ influx in macrophages. The kinetics of Ca 2+ influx in BMDMs in response to TRPV4 agonist, GSK101, was much more gradual than the originally reported steep kinetics for GSK101 in HeLa cells transiently transfected with TRPV4 (Jin et al. 2011). It is possible that the differential kinetics pattern (steep vs. gradual) may be related to the origin of cells (overexpression of transfected TRPV4 in Hela cells vs. primary BMDMs). Recently, Scheraga et al. showed that TRPV4 activation is required for LPS-induced macrophage phagocytosis and stimulation of inflammatory cytokines (Scheraga et al. 2016). We found that matrix stiffness altered PgLPS-induced pro-atherogenic responses such as oxLDL internalization and foam cell formation in a TRPV4-dependent manner. In addition, we demonstrated that PgLPS exposure to macrophages on a stiff matrix induced increased TRPV4 Ca 2+ influx activity. Collectively, our current results showed that TRPV4-elicited Ca 2+ influx integrates PgLPS-and matrix stiffnessinduced signals to mediate macrophage oxLDL uptake and foam cell formation. Furthermore, our data demonstrate that TRPV4 regulates the development of foam cells, possibly by regulating the internalization of oxLDL, but does not regulate the binding of oxLDL to the cell surface. Loss of TRPV4 function, either by genetic deletion or pharmacologic antagonism (by GSK219), abrogates PgLPS-stimulated Ca 2+ influx and uptake of oxLDL by macrophages growing on a stiff substrate. Accumulating data support the notion that both a biochemical factor, for example, lipopolysaccharide, and a biomechanical factor, for example, matrix stiffness, may  Figure 5B. Results are expressed as mean AE SEM. *P < 0.05 for cells with 250 ng/mL PgLPS versus without PgLPS; **P < 0.01 for cells with 1000 ng/mL PgLPS versus without PgLPS; Student's t-test. (D) WT MRMs were maintained on various stiffness collagen-coated (10 lg/mL) polyacrylamide gels (0.5 and 8 kPa) with 1000 ng/mL PgLPS for 48 h, and then immunostained with anti-CD36 and TRPV4 IgG. Representative immunofluorescence images from three independent experiments are shown (original magnification, 409). promote athero-inflammatory macrophage function and atherosclerosis (Doherty et al. 1994;Shi et al. 1996;Yang et al. 2000  Binding of DiI-oxLDL GSK219 Figure 6. TRPV4 is required for oxLDL uptake but not its cell surface binding to macrophages. (A). WT and TRPV4 KO MRMs were incubated with or without DiI-labeled oxLDL (2.5 lg/mL) for 60 min at 4°C to assess oxLDL binding. Representative images of DiI-oxLDL binding on the macrophage surface are shown (n = 5 fields/condition). (B) Quantification of results in Figure 6A using NIH ImageJ software. (C) WT and TRPV4 KO MRMs were incubated with or without DiI-labeled oxLDL (5 lg/mL) for 30 min at 37°C, and oxLDL uptake was assessed. Representative images from five different fields per condition are shown (original magnification, 409). DiI-oxLDL uptake indicated by red fluorescence. (D) Bar graphs show mean DiI fluorescence intensity (mean AE SEM) (NIH ImageJ software). **P < 0.01 for WT cells versus TRPV4À/À without PgLPS; ## P < 0.01 for WT with PgLPS versus WT without PgLPS; ***P < 0.001 for WT with PgLPS versus TRPV4À/À with PgLPS; n = 20 cells/condition. migration, adhesion, apoptosis, and survival during atherogenesis. Efforts have been made to elucidate the mechanisms underlying foam cell formation with the goal of preventing atherosclerosis. Here, we identified a novel role of TRPV4 channels in PgLPS-triggered exacerbation of macrophage foam cell formation, indicating an association of TRPV4 in proatherogenic processes in macrophages.
Our current data appear to have identified a specific plasma membrane receptor/channel, TRPV4, as a potential mediator of inflammatory/proatherogenic responses associated with pathogenesis of periodontitis-induced atherosclerosis. Previous reports from our laboratory and others have shown a link between CD36-mediated uptake of oxLDL and macrophage foam cell formation (Rahaman et al. 2006(Rahaman et al. , 2011bMoore and Tabas 2011;Moore et al. 2013). Since CD36 is the major scavenger receptor for oxLDL-induced macrophage foam cell formation, we examined expression levels of CD36 in WT and TRPV4 KO cells. We found similar expression levels of CD36 protein in both WT and TRPV4 KO cells stimulated by PgLPS, suggesting that reduced foam cell formation in the absence of TRPV4 is not due to lack of CD36 expression. Thus, we postulate that augmented colocalization of TRPV4 and CD36 in response to increasing matrix stiffness in PgLPS-treated macrophages may be linked to increased foam cell formation. A precise understanding of the mechanisms coupling periodontitis and atherosclerosis will be important to provide a rationale for long-term longitudinal human studies required to assess causality, and to develop novel therapeutic interventions.