The TRPC6 inhibitor, larixyl acetate, is effective in protecting against traumatic brain injury-induced systemic endothelial dysfunction
The incidence of traumatic brain injuries (TBIs) is on the rise in the USA. Concussions, or mild TBIs without skull fracture, account for about 75% of all TBIs. Mild TBIs (mTBIs) lead to memory and cognitive deficits, headaches, intraocular pressure rises, axonal degeneration, neuroinflammation, and an array of cerebrovascular dysfunctions, including increased vascular permeability and decreased cerebral blood flow. It has been recently reported that besides vascular dysfunction in the cerebral circulation, mTBI may also cause a significant impairment of endothelial function in the systemic circulation, at least within mesenteric microvessels. In this study, we investigated whether mTBI affects endothelial function in aortas and determined the contribution of transient receptor potential canonical (TRPC) channels to modulating mTBI-associated endothelial dysfunction.
We used a model of closed-head mTBI in C57BL/6, 129S, 129S-C57BL/6-F2 mice, and 129S-TRPC1 and 129S-C57BL/6-TRPC6 knockout mice to determine the effect of mTBI on endothelial function in mouse aortas employing ex vivo isometric tension measurements. Aortic tissue was also analyzed using immunofluorescence and qRT-PCR for TRPC6 expression following mTBI.
We show that in various strains of mice, mTBI induces a pronounced and long-lasting endothelial dysfunction in the aorta. Ablation of TRPC6 protects mice from mTBI-associated aortic endothelial dysfunction, while TRPC1 ablation does not impact brain injury-induced endothelial impairment in the aorta. Consistent with a role of TRPC6 activation following mTBI, we observed improved endothelial function in wild type control mice subjected to mTBI following 7-day in vivo treatment with larixyl acetate, an inhibitor of TRPC6 channels. Conversely, in vitro treatment with the pro-inflammatory endotoxin lipopolysaccharide, which activates endothelial TRPC6 in a Toll-like receptor type 4 (TLR4)-dependent manner, worsened aortic endothelial dysfunction in wild type mice. Lipopolysaccharide treatment in vitro failed to elicit endothelial dysfunction in TRPC6 knockout mice. No change in endothelial TRPC6 expression was observed 7 days following TBI.
These data suggest that TRPC6 activation may be critical for inducing endothelial dysfunction following closed-head mTBI and that pharmacological inhibition of the channel may be a feasible therapeutic strategy for preventing mTBI-associated systemic endothelial dysfunction.
KeywordsTraumatic brain injury TRPC channels Endothelial dysfunction Aorta Mice
Traumatic brain injury
Toll-like receptor type 4
Transient receptor potential canonical
Traumatic brain injuries (TBIs) are a major public health concern as they are a common cause of death and disability, both in the USA and worldwide. The incidence of TBIs is increasing in the USA [1, 2]. TBIs can occur after any sort of blow or injury to the head, which can happen in many different situations, including vehicle collisions, exercise- and sports-related injuries, military involvement, and violence. Virtually all individuals regardless of geographical location, socioeconomic status, occupation, or age are at risk of suffering a TBI in their lifetime. Worldwide TBI incidence is estimated to be approximately 69 million individuals per year .
TBIs can range from mild (Glasgow Coma Score 13–15, unsustained loss of consciousness, amnesia immediately before or after the incident, alterations in mental state at the time of the incidence, headache, or any focal neurological deficits following the incident) to severe (Glasgow Coma Score 3–8, coma, severe memory loss, permanent and disabling motor deficits) . Mild TBIs (mTBI) are caused by non-penetrating physical impacts without skull fracture. Up to 80% of all TBIs are categorized as mTBIs, which less frequently involved hospital stays and follow-up . Reports of mTBI are more prevalent in young children less than the age of 4, males between ages 15–24, and the elderly, particularly females over the age of 65 [1, 2]. There is also a growing body of evidence indicating that repetitive mTBIs can lead to permanent dysfunction, including increased risk of neurodegenerative disorders (amyotrophic lateral sclerosis, chronic traumatic encephalopathy, Parkinson’s, and Alzheimer’s diseases) [5, 6, 7] and cardiovascular complications [8, 9, 10, 11, 12, 13, 14, 15, 16].
TBI is a pro-inflammatory condition affecting predominantly the brain, but it also known to cause impairments in other organs, such as the eye, lungs, and mesenteric arteries [17, 18]. During closed-head mTBI, sterile immune reaction at the site of injury involves both the resident microglia and peripherally derived inflammatory cells that are recruited to the brain . The inflammatory reaction is important for clearing damaged cells and repair processes in the brain ; however, excessive activation of innate immunity produces a storm of cytokines that may leak through the impaired blood-brain barrier to the cerebral and systemic circulations and initiate collateral damage.
Cerebrovascular dysfunction has long been associated with TBI. In addition, there is evidence indicating that TBIs also likely have significant effects on the systemic vasculature . A recent study found that open-head TBI causes significant microvascular endothelial dysfunction in the mesenteric bed, lasting at least 24 h post-injury . Endothelial dysfunction is an established risk factor for cardiovascular disease [20, 21, 22], specifically associated with essential hypertension , cerebral ischemia , and may precipitate vasospasm.
Transient receptor potential canonical (TRPC) proteins are a family of proteins implicated in modulating smooth muscle and endothelial function [25, 26, 27]. TRPC proteins form receptor- and store-operated Ca2+ permeable, nonspecific cation channels in the plasma membrane. The seven members in the TRPC subfamily (TRPC1-TRPC7) can be stimulated via G-protein-coupled receptors, such as the histamine H1 receptor, the muscarinic M1 receptor, or the α1 adrenoceptor [25, 26, 28, 29, 30]. In mouse endothelial cells, the TRPC6 channel can be activated by bacterial lipopolysaccharides (LPS) in a Toll-like receptor type 4 (TLR4)-dependent manner .
In this study, we investigated the long-term effects of mTBI on the systemic vasculature. We used a mouse closed-head mTBI model to determine whether mTBI leads to endothelial dysfunction in the aorta utilizing isometric tension measurements and determined the role of the TRPC1 and TRPC6 channels in the pathogenesis of mTBI-induced aortic endothelial dysfunction using commercially available TRPC1 and TRPC6 knockout mouse strains.
C57BL/6, 129S, 129S-C57BL/6-F2, 129S-TRPC1-KO (Stock # 37347-JAX), and 129S-C57BL/6-TRPC6-KO (Stock # 37345-JAX) mouse strains were purchased from The Jackson Laboratory and/or were bred in house. Both male and female mice were used. All animal procedures were performed in accordance to the NIH guide and were approved by the Indiana University IACUC. The mice were euthanized under isoflurane anesthesia by decapitations.
Closed-head mild traumatic brain injury
Assessment of endothelial-dependent vasodilation
Mice were euthanized by decapitations under isoflurane anesthesia. Mouse aortas were isolated and cleaned from the fat and connective tissue in a calcium and magnesium-free phosphate buffer solution (PBS, Lonza). A wire myograph from GlobalTown Microtech., Inc. (Sarasota, FL) was used to monitor the force generated by aortic arch rings. The isometric tension measurements were performed as described elsewhere [32, 33, 34, 35]. Briefly, aortic arches were hung on the wires of the wire myograph and were placed into the 5-ml tissue baths filled with the standard Krebs buffer maintained at 37 °C and continuously oxygenated by bubbling a gas mixture of 95% O2 and 5% CO2 during all of the performed experiments. The preload in all experiments was set to 0.7–1 g. Increasing concentrations of phenylephrine and then acetylcholine was added directly into the tissue baths while the contraction force was measured. SNAP (S-Nitroso-N-acetyl-DL-penicillamine, a nitric oxide donor) was used to assess the maximal receptor-independent aortic ring dilations. The analog ring tension data were digitized with a frequency of 20 Hz and recorded on a computer’s hard drive.
Mouse aortas were prepared as described above. Isolated mouse aortas were fixed in 4% paraformaldehyde for 2 h on ice. Tissue was then washed in phosphate buffered saline, and frozen in Tissue-Tek O.C.T Compound (Sakura Finete) using a mixture of dry ice and isopentane. After cryosections (~ 8 μm) were obtained, O.C.T. was removed from tissue samples by a 5–10 min wash in Tris buffered saline (TBS). Samples were permeabilized in 0.2% Triton X-100 in TBS for 5 min and were then washed three times in TBS. After blocking in 5% horse serum in TBS at room temperature for 1 h, sections were incubated at 37° with the TRPC6 antibody (1:100, Alomone lab, ACC-017) and an anti-smooth muscle actin antibody conjugated to Cy3 (1:250; C1698, Sigma). Sections without the TRPC6 primary antibody served as a negative control. TRPC6 immunoreactivity was detected with anti-rabbit Alexa Fluor 647 (1:4000; Jackson ImmunoResearch). Stained samples were mounted in ProLong Gold with DAPI (Invitrogen) and visualized by confocal microscopy (Olympus Fluoview FV1000).
Total mRNA was isolated from freshly collected mouse aortas using the PureLink mRNA isolation kit (Thermo Fisher Scientific). Five hundred nanograms of RNA was used in reverse transcription reactions (High-Capacity RT-cDNA Kit, Life Technologies). Real-time PCR was performed using Syber Green (Roche). Levels of mRNA expression were normalized to expression of Hprt as an internal control and are expressed relative to the mean values seen in samples from sham control mice. Primers used for PCR were TRPC1 Forward: TCC CAA AGA GCA GAA GGA CTG, TRPC1 Reverse: CAA AGC AGG TGC CAA TGA A; mTRPC6, RT2 qPCR assay (PPM04056A, Qiagen).
Drugs and solutions
All drugs were purchased from Sigma-Aldrich or Cayman Biochemical. The solution composition was as follows. The standard Krebs buffer contained (in millimolar) 130 NaCl, 5 KCl, 2 CaCl2, 1.2 NaH2PO4, 0.56 MgCl2, 25 NaHCO3, and 5 glucose. The 70 KCl solution contained (in millimolar) 65 NaCl, 70 KCl, 2 CaCl2, 1.2 NaH2PO4, 0.56 MgCl2, 25 NaHCO3, and 5 glucose.
Sigma plot 12 was used to analyze the aortic ring tension data. Two-way ANOVA followed by a Student-Newman-Keuls post hoc pairwise multiple comparison test was used to compare the tested experimental groups affected by two different factors when the data sets were normally distributed populations with equal variances. The t test was used to compare two tested groups. The data sets were considered significantly different if the p value was less than 0.05. All data were presented as mean ± standard error (S.E.).
Aortas from TBI mice exhibit a pronounced endothelial dysfunction
TRPC1 genetic ablation does not affect TBI-associated endothelial dysfunction
TRPC6 genetic ablation prevents TBI-associated endothelial dysfunction
TRPC6 activation is critical for mediating TBI-associated endothelial dysfunction
TLR4 activation is also essential for mediating TBI-associated endothelial dysfunction
TBI does not affect TRPC6 expression in the endothelial layer of the aortic arch
The growing incidence of TBIs, in addition to increasing evidence of long-term and systemic sequalae, highlights the importance of developing new approaches and treatment paradigms. In this study, we demonstrated that closed-head mild TBI causes long-lasting systemic endothelial dysfunction, which is resolved between 7 and 21 day post-injury, in mice. We found that TRPC6 activation may underlie the development of this systemic endothelial dysfunction after TBI. Furthermore, we established that larixyl acetate, an inhibitor of TRPC6, is effective in preventing TBI-associated systemic endothelial dysfunction when it was administered intraperitoneally for 7 days following TBI.
Our finding that closed-head mild TBI-induced aortic endothelial dysfunction in mice of three different genetic backgrounds (C57BL/6, 129S, and 129S-C57BL/6-F2) indicates that this is not a limited phenomenon and is consistent with a recent study demonstrating that severe open-head TBI may cause microvascular endothelial dysfunction in the mesenteric bed . In this previous study, it was proposed that vessels of the systemic circulation may have a “molecular memory” of neurotrauma that may continue for 24 h. We have extended these studies and shown that a mild closed-head TBI also causes endothelial dysfunction of systemic conduit vessels, such as the aorta and we provide evidence that the TBI-associated systemic vascular dysfunction lasts at least 7 days post-injury.
The open-head TBI-associated endothelial dysfunction in mesenteric arteries previously reported, was suggested to occur due to upregulation of arginase, a Mn2+-containing metalloenzyme that converts L-arginine to urea and ornithine [37, 38]. As L-arginine is the substrate for endothelial NO synthase (eNOS), arginase-induced degradation of L-arginine limits eNOS-dependent NO production and subsequent vasodilation. In our studies in the aorta following mild closed-head TBI, we found that TRPC6 genetic ablation prevents TBI-associated aortic endothelial dysfunction. As TRPC6 is a Ca2+ permeable channel, and intracellular Ca2+ rises may rapidly increase arginase expression , it is possible that TRPC6 is an upstream element that contributes to increasing arginase expression. However, we cannot rule out that different molecular mechanisms may underlie endothelial dysfunction in the aorta and mesenteric arteries. Future experiments will be required to determine whether there is a difference between the aortic and mesenteric beds in regard to arginase expression and activity.
During mild TBI, blood-brain barrier disruption allows the release of a plethora of pro-inflammatory and damage-associated molecules into the systemic circulation . Increased levels of chemokine CCL2; TNF-α; and interleukins (IL-) 6, 8, and 10 have previously been found in blood serum several days after severe TBI [41, 42]. However, it remains unclear which molecule, or factor, triggers TRPC6 activity after mild TBI in our mouse model. Notably, we did not observe any increase of TRPC6 expression levels in the aortic wall after TBI indicating that changes in TRPC6 activity rather than expression may be mediating aortic endothelial dysfunction. Consistent with this, we found that activation of TLR4 signaling via LPS caused pronounced aortic endothelial dysfunction without TBI, in a TRPC6-dependent manner. We propose that a TLR4 agonist may be present in the systemic circulation after TBI that triggers TRPC6 activity. This hypothesis is supported by a previous report that TLR4 genetic ablation reduces tissues injury events associated with brain trauma  and by our data indicating that 7-day treatment with TAK-242, a specific inhibitor of TLR4, significantly improved endothelial function in TBI mice (Fig. 8).
A recent study demonstrated that TBI is also associated with significant cardiac dysfunction identified by decreased left ventricular ejection fraction and fractional shortening, which was observed up to 30 days post-TBI . The authors established that splenectomy significantly decreased cardiac dysfunction, but not neurological or cognitive function, after TBI. Thus, it appears that the TBI-induced neuroinflammation may trigger systemic inflammation and immune cell infiltration in the cardiac tissue, further implicating systemic immune response as a factor underlying TBI-mediated cardiovascular dysfunction.
In this study, we found that the genetic ablation of TRPC1 in mice does not protect from TBI-associated vascular dysfunction. These data are consistent with the observation by Peters et al.  that TRPC1−/− mice exhibited similar neurological deficits as wild type mice up to 21 days after closed-head mild TBI. Interestingly, Peters et al.  found that carvacrol, 5-isopropyl-2-methylphenol, isolated from the essential oil of Origanum vulgare, was effective in improving neurological severity score in TRPC1−/− mice but not in wild type mice, implicating TRPC1 elimination as a sensitizer for the carvacrol beneficial effect after TBI. However, Peters et al.  did not investigate the effect of closed-head TBI on the vasculature.
Importantly, we found that pharmacological inhibition of TRPC6 with larixyl acetate was effective at preventing TBI-induced systemic vascular dysfunction (Fig. 4). Larixyl acetate-dependent inhibition of TRPC6 was first described in the elegant paper by Dr. Michael Schaefer’s research team . Urban et al.  demonstrated that the compound’s apparent IC50 value was approximately 0.65 μM for TRPC6, which is 10–100× lower than that for other members of the TRPC channel subfamily. Importantly, the authors showed that larixyl acetate’s cytotoxicity was very low and that the compound did not lose its bioactivity even after a 24-h incubation in citrate-supplemented human whole blood at 37 °C. Together, these data suggest that larixyl acetate treatment may be an effective therapeutic strategy for limiting the damaging effects on TBI on the systemic vasculature.
We show for the first time that TRPC6 genetic ablation or pharmacological inhibition with larixyl acetate prevents TBI-associated systemic endothelial dysfunction in mice. These findings identify TRPC6 as a promising target for developing new therapeutic drugs to treat endothelial dysfunction after TBI.
We thank Dr. Minghua Zhong for technical assistance during some of described ex vivo isometric tension studies.
This study was supported by the grants from the NIH to A.G.O. (R01 HL115140), the Indiana State Department of Health Trauma and Injury Prevention: the Indiana Spinal Cord and Brain Injury Research Fund (A.G.O. and F.A.W.), the St. Vincent Research Foundation (F.A.W.), and the Indiana Clinical and Translational Sciences Institute’s “The Concepts to Clinic Project Development Team funding-ICReATE Program” funded by the NIH-National Center for Advancing Translational Sciences, Clinical and Translational Sciences Award (# UL1TR001108).
Availability of data and materials
All data generated or analyzed during this study are included in this published article.
FAW and AOG conceived the study. XC, FAW, and AOG were involved in the design of the described experiments. XC, AOG, BHP, and AMR analyzed and interpreted the data. XC performed all of the ex vivo experiments. BPH performed all of the qPCR and immunofluorescence experiments. NT performed the traumatic brain injury experiments and long-term treatments with larixyl acetate and TAK-242. XC, AMR, BPH, FAW, and AOG contributed to writing the manuscript. All authors read and approved the final version of the manuscript.
This study was approved by the Indiana University School of Medicine Institutional Animal Care and Use Committee (protocol # 11284).
Consent for publication
The authors declare that they have no competing interests.
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