TRPV1 Knockout Mice Research Articles

Glucosylsphingosine evokes pruritus via activation of 5-HT2A receptor and TRPV4 in sensory neurons.

Glucosylsphingosine (GS), an endogenous sphingolipid, is highly accumulated in the epidermis of patients with atopic dermatitis (AD) due to abnormal ceramide metabolism. More importantly, GS can evoke scratching behaviours. However, the precise molecular mechanism by which GS induces pruritus has been elusive. Thus, the present study aimed to elucidate the molecular signalling pathway of GS, especially at the peripheral sensory neuronal levels. Calcium imaging was used to investigate the responses of HEK293T cells or mouse dorsal root ganglion (DRG) neurons to application of GS. Scratching behaviour tests were also performed with wild-type and Trpv4 knockout mice. GS activated DRG neurons in a manner involving both the 5-HT2A receptor and TRPV4. Furthermore, GS-induced responses were significantly suppressed by various inhibitors, including ketanserin (5-HT2A receptor antagonist), YM254890 (Gαq/11 inhibitor), gallein (Gβγ complex inhibitor), U73122 (phospholipase C inhibitor), bisindolylmaleimide I (PKC inhibitor) and HC067047 (TRPV4 antagonist). Moreover, DRG neurons from Trpv4 knockout mice exhibited significantly reduced responses to GS. Additionally, GS-evoked scratching behaviours were greatly decreased by pretreatment with inhibitors of either 5-HT2A receptor or TRPV4. As expected, GS-evoked scratching behaviour was also significantly decreased in Trpv4 knockout mice. Overall, the present study provides evidence for a novel molecular signalling pathway for GS-evoked pruritus, which utilizes both 5-HT2A receptor and TRPV4 in mouse sensory neurons. Considering the high accumulation of GS in the epidermis of patients with AD, GS could be another pruritogen in patients with AD.

Berberine Alleviate Cisplatin-Induced Peripheral Neuropathy by Modulating Inflammation Signal via TRPV1.

Chemotherapy induced peripheral neuropathy (CIPN) is a severe neurodegenerative disorder caused by chemotherapy drugs. Berberine is a natural monomer compound of Coptis chinensis, which has anti-tumor effect and can improve neuropathy through anti-inflammatory mechanisms. Transient receptor potential vanilloid (TRPV1) can sense noxious thermal and chemical stimuli, which is an important target for the study of pathological pain. In both vivo and in vitro CIPN models, we found that berberine alleviated peripheral neuropathy associated with dorsal root ganglia inflammation induced by cisplatin. We confirmed that berberine mediated the neuroinflammatory reaction induced by cisplatin by inhibiting the overexpression of TRPV1 and NF-κB and activating the JNK/p38 MAPK pathways in early injury, which inhibited the expression of p-JNK and mediated the expression of p38 MAPK/ERK in late injury in vivo. Moreover, genetic deletion of TRPV1 significantly reduced the protective effects of berberine on mechanical and heat hyperalgesia in mice. In TRPV1 knockout mice, the expression of NF-κB increased in late stage, and berberine inhibited the overexpression of NF-κB and p-ERK in late injury. Our results support berberine can reverse neuropathic inflammatory pain response induced by cisplatin, TRPV1 may be involved in this process.

Lack of TRPV1 aggravates obesity-associated hypertension through the disturbance of mitochondrial Ca2+ homeostasis in brown adipose tissue

The combination of obesity and hypertension is associated with high morbidity and mortality; however, the mechanism underlying obesity-induced hypertension remains unclear. In this study, we detected the possible effects of TRPV1, a previously identified antihypertensive calcium (Ca2+) channel in adipose tissue, on the occurrence of obesity and hypertension in mice lacking UCP1, a spontaneously genetically manipulated obesity model, by generating TRPV1 and UCP1 double knockout mice. In these mice, obesity and hypertension appeared earlier and were more severe than in mice with the knockout of UCP1 or TRPV1 alone. The knockout of TRPV1 in UCP1 knockout mice further reduced functional brown adipose tissue (BAT) generation; decreased resting oxygen consumption, heat production, and locomotor activities; and was accompanied by severe mitochondrial respiratory dysfunction in BAT. Mechanistically, TRPV1, UCP1, and LETM1 acted as a complex to maintain an appropriate mitochondrial Ca2+ level, and TRPV1 knockout caused a compensatory increase in mitochondrial Ca2+ uptake via LETM1 activation. However, the compensatory response was blocked in UCP1−/− mice, resulting in dramatically reduced mitochondrial Ca2+ uptake and higher production of ATP and oxidative stress. This study provides in vivo evidence for the critical role of BAT mitochondrial Ca2+ homeostasis in obesity-associated hypertension and indicates that the TRPV1/UCP1/LETM1 complex may be an alternative intervention target.

Endothelial TRPV4 channels in lung edema and injury.

The alveolo-capillary barrier is relatively impermeable, and facilitates gas exchange via the large alveolar surface in the lung. Disruption of alveolo-capillary barrier leads to accumulation of edema fluid in lung injury. Studies in animal models of various forms of lung injury provide evidence that TRPV4 channels play a critical role in disruption of the alveolo-capillary barrier and pathogenesis of lung injury. TRPV4 channels from capillary endothelial cells, alveolar epithelial cells, and immune cells have been implicated in the pathogenesis of lung injury. Recent studies in endothelium-specific TRPV4 knockout mice point to a central role for endothelial TRPV4 channels in lung injury. In this chapter, we review the findings on the pathological roles of endothelial TRPV4 channels in different forms of lung injury and future directions for further investigation.

Cutaneous Neuroimmune Interactions of TSLP and TRPV4 Play Pivotal Roles in Dry Skin-Induced Pruritus.

Dry skin is a symptom of skin barrier dysfunction that evokes pruritus; however, the cutaneous neuroimmune interactions underlying dry skin-induced pruritus remain unclear. Therefore, we aimed to elucidate the mechanisms underlying dry skin-induced pruritus. To this end, an acetone/ethanol/water (AEW)-induced mouse model of dry skin was used in this study. We observed that the production of thymic stromal lymphopoietin (TSLP) significantly increased in the keratinocytes of AEW mice. Importantly, treatment with an antagonist of transient receptor potential cation channel subfamily V member 4 (TRPV4), HC067047, ameliorated dry skin conditions in AEW mice. The symptoms of dry skin were significantly reduced in Trpv4 knockout (KO) mice following treatment with AEW. The increase in the intracellular calcium levels by TSLP in the dorsal root ganglia (DRG) of Trpv4 KO mice was also significantly attenuated. The spontaneous scratching bouts were significantly decreased in both the HC067047-treated and Trpv4 KO AEW mice. Importantly, the TSLP-dependent release of tryptase from the mast cells was significantly reduced in both the HC067047-treated mice and Trpv4 KO AEW mice. Notably, inhibition of the TSLP-induced signaling pathway in DRG selectively reduced the spontaneous scratching bouts in AEW mice. Overall, the results demonstrated that the cutaneous neuroimmune interactions of TSLP and TRPV4 play pivotal roles in dry skin-induced pruritus.

The Roles of Transient Receptor Potential Vanilloid 1 and 4 in Pneumococcal Nasal Colonization and Subsequent Development of Invasive Disease.

Transient receptor potential (TRP) channels, neuronal stimulations widely known to be associated with thermal responses, pain induction, and osmoregulation, have been shown in recent studies to have underlying mechanisms associated with inflammatory responses. The role of TRP channels on inflammatory milieu during bacterial infections has been widely demonstrated. It may vary among types of channels/pathogens, however, and it is not known how TRP channels function during pneumococcal infections. Streptococcus pneumoniae can cause severe infections such as pneumonia, bacteremia, and meningitis, with systemic inflammatory responses. This study examines the role of TRP channels (TRPV1 and TRPV4) for pneumococcal nasal colonization and subsequent development of invasive pneumococcal disease in a mouse model. Both TRPV1 and TRPV4 channels were shown to be related to regulation of the development of pneumococcal diseases. In particular, the influx of neutrophils (polymorphonuclear cells) in the nasal cavity and the bactericidal activity were significantly suppressed among TRPV4 knockout mice. This may lead to severe pneumococcal pneumonia, resulting in dissemination of the bacteria to various organs and causing high mortality during influenza virus coinfection. Regulating host immune responses by TRP channels could be a novel strategy against pathogenic microorganisms causing strong local/systemic inflammation.

Dichotomous effects of smooth muscle TRPV4 channels on vascular contractility

Ca2+ signaling mechanisms in smooth muscle cells (SMCs) determine vascular contractility. Transient receptor potential vanilloid 4 (TRPV4) ion channels have been described as a crucial Ca2+ influx pathway in SMCs from small, resistance-sized arteries. However, the impact of SMC TRPV4 (TRPV4SMC) channel activity on blood pressure remains unknown, mostly due to the lack of studies in SMC-specific knockout mice. We hypothesized that TRPV4SMC channels control the resting blood pressure. Radiotelemetric blood pressure recordings showed reduced daytime and nighttime blood pressures in the inducible, SMC-specific TRPV4 knockout (TRPV4SMC-/-) mice compared to the control mice. The effect of TRPV4SMC-/- on vascular contractility was assessed using two endogenous vasoconstrictor mechanisms- 1) SMC α1 adrenergic receptor (α1AR) stimulation and 2) increased intraluminal pressure. Phenylephrine (α1AR agonist)-induced constriction of pressurized mesenteric arteries (MAs, 80 mm Hg, 1 nM-10 mM phenylephrine) and blood pressure elevation (10 mg/kg phenylephrine, i.p.) were blunted in TRPV4SMC-/- mice, identifying a central role of TRPV4SMC channels in α1AR-induced vasoconstriction and blood pressure elevation. Individual Ca2+ influx signals through TRPV4SMC channels (TRPV4SMC sparklets) were recorded using spinning disk confocal imaging in fluo-4-loaded MAs pressurized to 80 mm Hg. Phenylephrine (1 mM) activated TRPV4SMC sparklets, an effect abolished by protein kinase C (PKC) inhibitor (Go6976, 1 mM). These results indicated that PKC-TRPV4SMC channel signaling is a major contributor to α1AR-induced vasoconstriction. In direct contrast with PE-induced constriction, intraluminal pressure-induced constriction was higher in MAs from TRPV4SMC-/- mice, suggesting that SMC TRPV4 channels limit pressure-induced vasoconstriction. Previous studies have shown that TRPV4SMC channels can activate large-conductance, Ca2+-sensitive K+ (BK) channels, thereby decreasing pressure-induced vasoconstriction. Consistent with these findings, vasoconstriction in response to BK channel inhibitor (paxilline, 1 mM) was reduced in MAs from TRPV4SMC-/- mice, supporting a dilatory TRPV4SMC channel-BK channel signaling under resting conditions. In situ proximity ligation assay revealed co-localization of TRPV4SMC channels with both α1ARs and BK channels. Overall, TRPV4SMC channels increase the resting blood pressure but have divergent effects on vascular contractility. TRPV4SMC channels contribute to α1AR-induced vasoconstriction but limit pressure-induced vasoconstriction. Therefore, selective vasoactive stimulus-TRPV4SMC channel coupling may determine the impact of TRPV4SMC channels on vascular resistance.

Studies on the Involvement of Integrin and the Tubulin Cytoskeleton in the Mechanism Responsible for TRPV1‐dependent NKCC1 Activation in Mouse Lens

Previously we showed shrinkage of the mouse lens by hyperosmotic solution causes TRPV1-dependent activation of NKCC1. In various tissues the cytoskeleton has been shown to play a role in responses to osmotic stress. Here we present evidence for the involvement of integrin and tubulin in the activation of NKCC1 in mouse lens epithelium. Studies were carried out on lenses from wild-type (WT) and TRPV1 knockout (KO) mice. Rubidium (Rb) uptake, measured using an atomic absorption spectrophotometer, was used as a measure of NKCC activity. ERK1/2 phosphorylation was measured by quantitative Western blot analysis. Intracellular calcium was measured in Fura-2 loaded cultured lens epithelium by a ratiometric imaging technique. In WT mice, the integrin agonist leukadherin 1 (LA-1, 25 μM) increased Rb uptake by 33% in intact lenses and increased intracellular calcium concentration in cultured lens epithelium from 197±18 to 341±25 nM in (n=5, P<0.001). In contrast, LA-1 failed to produce any significant change of Rb uptake in TRPV1 KO lenses and did not increase calcium in cultured lens epithelium from TRPV1 KO animals. The increase of Rb uptake elicited by LA-1 in WT lenses was abolished by the putative tubulin stabilizer, paclitaxel (100 nm). The TRPV1 antagonist, A889425 (1.0 μM) also abolished the Rb uptake response to LA-1. LA-1, the TRPV1 agonist capsaicin, and hyperosmotic solution (350 mOsm) all caused ERK1/2 activation in WT cultured lens epithelium. In each case, the ERK1/2 response was transient with a peak response at ~5 min. The ERK1/2 activation responses to hyperosmotic stress and LA-1 induced were markedly suppressed by A889425 and by paclitaxel. Taken together, the results point to a link between integrin, the tubulin cytoskeleton, and TRPV1-dependent activation of NKCC1 in the mouse lens. It is noteworthy that the integrin agonist LA-1 as well as hyperosmotic stress both were found to activate ERK1/2, the upstream kinase that was shown previously associated with activation of NKCC1 in the lens.

Cardiomyocyte TRPV4 deletion preserves cardiac function following pressure overload‐induced pathological hypertrophy independent of cardiac fibrosis

Left ventricular hypertrophy is compensatory adaptive response towards physical stimuli. However, prolonged changes in hemodynamic pressure due to pathological insults disrupts the electrical and bio-mechanical coupling between cardiomyocytes which leads to heart failure. Previous studies have demonstrated the role of soluble factors in the regulation of cardiomyocyte function, however, mechanosensor and mechanotransduction pathways in cardiomyocytes are remain unknown. We have recently shown that global deletion of mechanosensitive, transient receptor potential vanilloid 4 ion channel (TRPV4) preserves cardiac function and structure via reducing cardiac fibrosis after MI. Here we investigated cardiomyocyte specific role of TRPV4 following pressure overload induced hypertrophy. First, we found that TRPV4 expression is increased following pressure overload induced hypertrophy in wildtype (WT) mice. Global deletion of TRPV4 preserved myocardial structure and function compared to WT after 28 days of TAC. To understand the cardiomyocyte TRPV4 role, we have generated cardiomyocyte specific TRPV4 knockout mice (TRPV4CMKO) by crossing TRPV4lox/lox with Myh6cre mice. The littermates were confirmed for TRPV4 deletion in cardiomyocytes through genotyping and Real time PCR in tail snips and isolated cardiomyocytes. Next, we subjected the TRPV4lox/lox and TRPV4CMKO to TAC and then measured cardiac function. After 28 days of TAC, absence of TRPV4 in cardiomyocyte preserved the cardiac function compared to TRPV4lox/lox animals. Surprisingly, histological analysis revealed increased cardiac hypertrophy (cardiomyocyte cross sectional area) and cardiac fibrosis in TRPV4CMKO mice compared to TRPV4lox/lox mice or sham controls. Taken together our results suggest that cardiomyocyte specific deletion of TRPV4 preserves cardiac function despite of increased pathological remodeling of the heart and identify TRPV4 as new therapeutic target for the heart failure.

Elucidation of the Role of Transient Receptor Potential Vanilloid-1 (TRPV1) Channels in Epileptogenesis

The transient receptor potential vanilloid-1 (TRPV1), previously known as the capsaicin receptor or vanilloid receptor 1 (VR1) is a nonselective cation channel that acts as an integrator of nociceptive information in sensory neurons and their sensory nerve endings with unmyelinated (C) or thin myelinated (Aδ) fibers. It is activated by capsaicin, resiniferatoxin, piperine, noxious heat (&gt; 43ºC), protons, lipoxygenase products, and some endogenous cannabinoids. TRPV1 receptors are also expressed in the brain on neurons, glia cells and pericytes and might be involved in the modulation of epileptogenesis. TRPV1 modulates synaptic plasticity and neurotransmission, mediates long-term depression of glutamate release in the hippocampus and suppress excitatory transmission in dentate gyrus. TRPV1-knockout mice have altered susceptibility to hyperthermic seizures. Studies in vitro showed that capsaicin reduced epileptiform activity but increased neuronal discharge in excitable cells. Capsaicin given via systemic routes at low doses was shown to reduce seizures induced by kainic acid and pentylenetetrazole and to afford neuroprotection of hippocampus in vivo. These effects were associated with reduced oxidative stress and inflammation in brain. In contrast, high doses of capsaicin either elicited or enhanced seizures in animals. In addition, piperine, a TRPV1 agonist, demonstrated anti-epileptic activity in several animal models via a multiplicity of mechanisms. Moreover, non-psychotropic cannabinoids such as cannabidiol and cannabidivarin, the endocannabinoid anandamide, and acetaminophen demonstrated anti-epileptiform activity in vivo and in vitro via mechanisms that might involve TRPV1 receptors. By surveying recent research findings, this review article is intended to present the current research status on the involvement of TRPV1 receptors in epileptogenesis so as to stimulate further investigations into the detailed molecular mechanisms by which capsaicin as well as other chemical modalities impact epileptogenesis via modulating TRPV1 channels. (First online: Apr 12, 2021)

TRPV4 Regulates Soman-Induced Status Epilepticus and Secondary Brain Injury via NMDA Receptor and NLRP3 Inflammasome.

Nerve agents are used in civil wars and terrorist attacks, posing a threat to public safety. Acute exposure to nerve agents such as soman (GD) causes serious brain damage, leading to death due to intense seizures induced by acetylcholinesterase inhibition and neuronal injury resulting from increased excitatory amino-acid levels and neuroinflammation. However, data on the anticonvulsant and neuroprotective efficacies of currently-used countermeasures are limited. Here, we evaluated the potential effects of transient receptor vanilloid 4 (TRPV4) in the treatment of soman-induced status epilepticus (SE) and secondary brain injury. We demonstrated that TRPV4 expression was markedly up-regulated in rat hippocampus after soman-induced seizures. Administration of the TRPV4 antagonist GSK2193874 prior to soman exposure significantly decreased the mortality rate in rats and reduced SE intensity. TRPV4-knockout mice also showed lower incidence of seizures and higher survival rates than wild-type mice following soman exposure. Further in vivo and in vitro experiments demonstrated that blocking TRPV4 prevented NMDA receptor-mediated glutamate excitotoxicity. The protein levels of the NLRP3 inflammasome complex and its downstream cytokines IL-1β and IL-18 increased in soman-exposed rat hippocampus. However, TRPV4 inhibition or deletion markedly reversed the activation of the NLRP3 inflammasome pathway. In conclusion, our study suggests that the blockade of TRPV4 protects against soman exposure and reduces brain injury following SE by decreasing NMDA receptor-mediated excitotoxicity and NLRP3-mediated neuroinflammation. To our knowledge, this is the first study regarding the "dual-switch" function of TRPV4 in the treatment of soman intoxication.

Selective blockade of transient receptor potential vanilloid 4 reduces cyclophosphamide-induced bladder pain in mice

Transient receptor potential vanilloid 4 (TRPV4) is a non-selective cation channel activated by various physical stimuli such as cell swelling and shear stress. TRPV4 is expressed in bladder sensory nerves and epithelium, and its activation produces urinary dysfunction in rodents. However, there have been few reports regarding its involvement in bladder pain. Therefore, we investigated whether TRPV4 is involved in bladder pain in mouse cystitis model. Intraperitoneal injection of cyclophosphamide (CYP; 300 mg/kg) produced mechanical hypersensitivity in the lower abdomen associated with a severe inflammatory bladder in mice. The mechanical threshold was reversed significantly in Trpv4-knockout (KO) mice. Repeated injections of CYP (150 mg/kg) daily for 4 days provoked mild bladder inflammation and persistent mechanical hypersensitivity in mice. Trpv4-KO mice prevented a reduction of the mechanical threshold without an alteration in bladder inflammation. A selective TRPV4 antagonist also reversed the mechanical threshold in chronic cystitis mice. Although expression of Trpv4 was unchanged in the bladders of chronic cystitis mice, the level of phosphorylated TRPV4 was increased significantly. These results suggest involvement of TRPV4 in bladder pain of cystitis mice. A TRPV4 antagonist might be useful for patients with irritable bladder pain such as those with interstitial cystitis/painful bladder syndrome.

Endothelial TRPV4 channels prevent tumor growth and metastasis via modulation of tumor angiogenesis and vascular integrity.

Transient receptor potential vanilloid 4 (TRPV4) is a ubiquitously expressed polymodally activated ion channel. TRPV4 has been implicated in tumor progression; however, the cell-specific role ofTRPV4in tumor growth, angiogenesis, and metastasis is unknown. Here, we generated endothelial-specific TRPV4 knockout (TRPV4ECKO) mice by crossingTRPV4lox/loxmice with Tie2-Cre mice. Tumor growth and metastasis were significantly increased in a syngeneic Lewis lung carcinoma tumor model of TRPV4ECKO mice compared to TRPV4lox/lox mice. Multiphoton microscopy, dextran leakage, and immunohistochemical analysis revealed increased tumor angiogenesis and metastasis that were correlated with aberrant leaky vessels (increased width and reduced pericyte and VE-cadherin coverage). Mechanistically, increases in VEGFR2, p-ERK, and MMP-9 expression and DQ gelatinase activity were observed in the TRPV4ECKOmouse tumors. Our results demonstrated that endothelial TRPV4 is a critical modulator of vascular integrity and tumor angiogenesis and that deletion of TRPV4 promotes tumor angiogenesis, growth, and metastasis.

Heat hypersensitivity is attenuated with altered expression level of spinal astrocytes after sciatic nerve injury in TRPV1 knockout mice

Transient receptor potential vanilloid 1 (TRPV1) modulates pain. Studies have indicated that TRPV1 is upregulated in the spinal dorsal horn in the neuropathic pain model, but its mechanism is unknown. Here, we examined the mechanism by which TRPV1 modulates neuropathic pain by employing partial sciatic nerve ligation (pSNL) in adult male C57BL/6 J (wild-type: WT) and TRPV1 knockout (Trpv1-/-) mice. We analyzed mechanical/heat sensitivities (von Frey test/hot plate test) and glial/neuronal activities (Iba-1/GFAP/FosB by immunofluorescence) in laminae I and II in the L5 ipsilateral dorsal horn of the spinal cord. Mechanical/heat sensitivities, expression levels of microglial Iba-1 and astrocytic GFAP, and the number of FosB-positive neurons were significantly increased on days 7 and 14 in the pSNL group compared with the sham-operated and non-operated groups of both WT and Trpv1-/- mice. While mechanical sensitivity was comparable between WT and Trpv1-/- mice, the threshold against heat sensitivity was markedly prolonged in Trpv1-/- than WT mice on day 14 after pSNL. Conversely, the increment of FosB positive neurons was significantly attenuated in Trpv1-/- than WT mice on days 7 and 14 after pSNL. These results suggest that TRPV1 may modulate thermal perception via increased astrocytes in the dorsal horn of the spinal cord.

The Mysteries of Capsaicin-Sensitive Afferents

A fundamental subdivision of nociceptive sensory neurons is named after their unique sensitivity to capsaicin, the pungent ingredient in hot chili peppers: these are the capsaicin-sensitive afferents. The initial excitation by capsaicin of these neurons manifested as burning pain sensation is followed by a lasting refractory state, traditionally referred to as “capsaicin desensitization,” during which the previously excited neurons are unresponsive not only to capsaicin but a variety of unrelated stimuli including noxious heat. The long sought-after capsaicin receptor, now known as TRPV1 (transient receptor potential cation channel, subfamily V member 1), was cloned more than two decades ago. The substantial reduction of the inflammatory phenotype of Trpv1 knockout mice has spurred extensive efforts in the pharmaceutical industry to develop small molecule TRPV1 antagonists. However, adverse effects, most importantly hyperthermia and burn injuries, have so far prevented any compounds from progressing beyond Phase 2. There is increasing evidence that these limitations can be at least partially overcome by approaches outside of the mainstream pharmaceutical development, providing novel therapeutic options through TRPV1. Although ablation of the whole TRPV1-expressing nerve population by high dose capsaicin, or more selectively by intersectional genetics, has allowed researchers to investigate the functions of capsaicin-sensitive afferents in health and disease, several “mysteries” remain unsolved to date, including the molecular underpinnings of “capsaicin desensitization,” and the exact role these nerves play in thermoregulation and heat sensation. This review tries to shed some light on these capsaicin mechanisms.

Abstract 16955: Contrasting Effects of Smooth Muscle TRPV4 Channels on Vascular Function

Introduction: Transient receptor potential vanilloid 4 (TRPV4) channels are a major Ca 2+ entry pathway in vascular endothelial cells. TRPV4 channels are also expressed in vascular smooth muscle cells (SMC). However, the effect of SMC TRPV4 (TRPV4 SMC ) channels on vascular resistance and blood pressure remains controversial. Hypothesis: We hypothesized that TRPV4 SMC channels are essential regulators of vascular function and blood pressure. Methods: Inducible, SMC-specific TRPV4 knockout (TRPV4 SMC -/- ) mice were used. Blood pressure was monitored using radiotelemetry catheters. SMC Ca 2+ signals were recorded with a spinning disk confocal imaging system. Pressure myography studies assessed vascular reactivity in MAs. Results: Resting diastolic blood pressure and mean arterial pressure were lower in TRPV4 SMC -/- mice when compared to control mice. Selective TRPV4 channel agonist, GSK1016790A (GSK101), induced Ca 2+ influx signals in SMCs that were inhibited by TRPV4 channel inhibitor GSK2193874 and were absent in MAs from TRPV4 SMC -/- mice. In pressure myography studies, GSK101 constricted the MAs in control mice, but not in TRPV4 SMC -/- mice. Moreover, MAs from TRPV4 SMC -/- showed lower vasoconstriction in response to phenylephrine, a α1 adrenergic receptor agonist. These results indicated that Ca 2+ influx through TRPV4 SMC channels contributes to vasoconstriction and increased vascular resistance. However, studies of pressure-induced constriction or myogenic tone indicated that MAs from TRPV4 SMC -/- mice exhibit a higher myogenic constriction at pressures ≥ 60 mm Hg. Moreover, membrane depolarization-induced vasoconstriction (KCl, 33 and 60 mM) was also increased in TRPV4 SMC -/- mice when compared to the control mice, indicating that TRPV4 SMC channels oppose pressure- or membrane depolarization-induced vasoconstriction. Conclusions: TRPV4 SMC channels are essential regulators of resting blood pressure. While TRPV4 SMC channels contribute to α1 adrenergic receptor-induced vasoconstriction, they negatively regulate pressure-induced vasoconstriction. The divergent effects of TRPV4 SMC channels on vascular function may be determined by the selective stimulus-TRPV4 SMC channel coupling and downstream signaling mechanisms.

Cytokine activin C ameliorates chronic neuropathic pain in peripheral nerve injury rodents by modulating the TRPV1 channel.

The cytokine activin C is mainly expressed in small-diameter dorsal root ganglion (DRG) neurons and suppresses inflammatory pain. However, the effects of activin C in neuropathic pain remain elusive. Male rats and wild-type and TRPV1 knockout mice with peripheral nerve injury - sciatic nerve axotomy and spinal nerve ligation in rats; chronic constriction injury (CCI) in mice - provided models of chronic neuropathic pain. Ipsilateral lumbar (L)4-5 DRGs were assayed for activin C expression. Chronic neuropathic pain animals were treated with intrathecal or locally pre-administered activin C or the vehicle. Nociceptive behaviours and pain-related markers in L4-5 DRGs and spinal cord were evaluated. TRPV1 channel modulation by activin C was measured. Following peripheral nerve injury, expression of activin βC subunit mRNA and activin C protein was markedly up-regulated in L4-5 DRGs of animals with axotomy, SNL or CCI. [Correction added on 26 November 2020, after first online publication: The preceding sentence has been corrected in this current version.] Intrathecal activin C dose-dependently inhibited neuropathic pain in spinal nerve ligated rats. Local pre-administration of activin C decreased neuropathic pain, macrophage infiltration into ipsilateral L4-5 DRGs and microglial reaction in L4-5 spinal cords of mice with CCI. In rat DRG neurons, activin C enhanced capsaicin-induced TRPV1 currents. Pre-treatment with activin C reduced capsaicin-evoked acute hyperalgesia and normalized capsaicin-evoked persistent hypothermia in mice. Finally, the analgesic effect of activin C was abolished in TRPV1 knockout mice with CCI. Activin C inhibits neuropathic pain by modulating TRPV1 channels, revealing potential analgesic applications in chronic neuropathic pain therapy.

Transient receptor potential vanilloid 4 channel deletion regulates pathological but not developmental retinal angiogenesis.

Transient receptor potential vanilloid 4 (TRPV4) channels are mechanosensitive ion channels that regulate systemic endothelial cell (EC) functions such as vasodilation, permeability, and angiogenesis. TRPV4 is expressed in retinal ganglion cells, Müller glia, pigment epithelium, microvascular ECs, and modulates cell volume regulation, calcium homeostasis, and survival. TRPV4-mediated physiological or pathological retinal angiogenesis remains poorly understood. Here, we demonstrate that TRPV4 is expressed, functional, and mechanosensitive in retinal ECs. The genetic deletion of TRPV4 did not affect postnatal developmental angiogenesis but increased pathological neovascularization in response to oxygen-induced retinopathy (OIR). Retinal vessels from TRPV4 knockout mice subjected to OIR exhibited neovascular tufts that projected into the vitreous humor and displayed reduced pericyte coverage compared with wild-typemice. These results suggest that TRPV4 is a regulator of retinal angiogenesis, its deletion augments pathological retinal angiogenesis, and that TRPV4 could be a novel target for the development of therapies against neovascular ocular diseases.

The Contractile Phenotype of Skeletal Muscle in TRPV1 Knockout Mice is Gender-Specific and Exercise-Dependent.

The transient receptor potential vanilloid 1 (TRPV1) belongs to the transient receptor potential superfamily of sensory receptors. TRPV1 is a non-selective cation channel permeable to Ca2+ that is capable of detecting noxious heat temperature and acidosis. In skeletal muscles, TRPV1 operates as a reticular Ca2+-leak channel and several TRPV1 mutations have been associated with two muscle disorders: malignant hyperthermia (MH) and exertional heat stroke (EHS). Although TRPV1−/− mice have been available since the 2000s, TRPV1’s role in muscle physiology has not been thoroughly studied. Therefore, the focus of this work was to characterize the contractile phenotype of skeletal muscles of TRPV1-deficient mice at rest and after four weeks of exercise. As MS and EHS have a higher incidence in men than in women, we also investigated sex-related phenotype differences. Our results indicated that, without exercise, TRPV1−/− mice improved in vivo muscle strength with an impairment of skeletal muscle in vitro twitch features, i.e., delayed contraction and relaxation. Additionally, exercise appeared detrimental to TRPV1−/− slow-twitch muscles, especially in female animals.

12(S)-HETE mediates diabetes-induced endothelial dysfunction by activating intracellular endothelial cell TRPV1

Patients with diabetes develop endothelial dysfunction shortly after diabetes onset that progresses to vascular disease underlying the majority of diabetes-associated comorbidities. Increased lipid peroxidation, mitochondrial calcium overload, and mitochondrial dysfunction are characteristics of dysfunctional endothelial cells in diabetic patients. We here identified that targeting the lipid peroxidation product 12(S)-hydroxyeicosatetraenoic acid-induced [12(S)-HETE-induced] activation of the intracellularly located cation channel transient receptor potential vanilloid 1 (TRPV1) in endothelial cells is a means to causally control early-stage vascular disease in type I diabetic mice. Mice with an inducible, endothelium-specific 12/15-lipoxygenase (12/15Lo) knockout were protected similarly to TRPV1-knockout mice from type 1 diabetes-induced endothelial dysfunction and impaired vascular regeneration following arterial injury. Both 12(S)-HETE in concentrations found in diabetic patients and TRPV1 agonists triggered mitochondrial calcium influx and mitochondrial dysfunction in endothelial cells, and 12(S)-HETE effects were absent in endothelial cells from TRPV1-knockout mice. As a therapeutic consequence, we found that a peptide targeting 12(S)-HETE-induced TRPV1 interaction at the TRPV1 TRP box ameliorated diabetes-induced endothelial dysfunction and augmented vascular regeneration in diabetic mice. Our findings suggest that pharmacological targeting of increased endothelial lipid peroxidation can attenuate diabetes-induced comorbidities related to vascular disease.