Transient Receptor Potential Vanilloid 1 Inhibition Research Articles

Transient receptor potential vanilloid 1 inhibition reduces brain damage by suppressing neuronal apoptosis after intracerebral hemorrhage.

Intracerebral hemorrhage (ICH) induces a complex sequence of apoptotic cascades and inflammatory responses, leading to neurological impairment. Transient receptor potential vanilloid 1 (TRPV1), a nonselective cation channel with high calcium permeability, has been implicated in neuronal apoptosis and inflammatory responses. This study used a mouse ICH model and neuronal cultures to examine whether TRPV1 activation exacerbates brain damage and neurological deficits by promoting neuronal apoptosis and neuroinflammation. ICH was induced by injecting collagenase in both wild-type (WT) C57BL/6 mice and TRPV1-/- mice. Capsaicin (CAP; a TRPV1 agonist) or capsazepine (a TRPV1 antagonist) was administered by intracerebroventricular injection 30 min before ICH induction in WT mice. The effects of genetic deletion or pharmacological inhibition of TRPV1 using CAP or capsazepine on motor deficits, histological damage, apoptotic responses, blood-brain barrier (BBB) permeability, and neuroinflammatory reactions were explored. The antiapoptotic mechanisms and calcium influx induced by TRPV1 inactivation were investigated in cultured hemin-stimulated neurons. TRPV1 expression was upregulated in the hemorrhagic brain, and TRPV1 was expressed in neurons, microglia, and astrocytes after ICH. Genetic deletion of TRPV1 significantly attenuated motor deficits and brain atrophy for up to 28 days. Deletion of TRPV1 also reduced brain damage, neurodegeneration, microglial activation, cytokine expression, and cell apoptosis at 1 day post-ICH. Similarly, the administration of CAP ameliorated brain damage, neurodegeneration, brain edema, BBB permeability, and cytokine expression at 1 day post-ICH. In primary neuronal cultures, pharmacological inactivation of TRPV1 by CAP attenuated neuronal vulnerability to hemin-induced injury, suppressed apoptosis, and preserved mitochondrial integrity in vitro. Mechanistically, CAP reduced hemin-stimulated calcium influx and prevented the phosphorylation of CaMKII in cultured neurons, which was associated with reduced activation of P38 and c-Jun NH2 -terminal kinase mitogen-activated protein kinase signaling. Our results suggest that TRPV1 inhibition may be a potential therapy for ICH by suppressing mitochondria-related neuronal apoptosis.

Apoptosis Induction Through Increased TRPV1 Activation by Synergic Effect of Melatonin and Doxorubicin in Human Osteosarcoma and Chondrosarcoma Cell Lines

Aim: We aimed to reveal the role of doxorubicin (Dox), melatonin (Mel) and transient receptor potential Vanilloid 1 (TRPV1) channels in bone and cartilage cancer cells during the treatment process. Human Bone Osteosarcoma (Saos-2/An1) and Human Chondrosarcoma (Hs 819.T) cell lines were used to prepare in-vitro experiment models. Methods: Both cell lines were cultured at 37°C. We have separated each cell line into five groups as follows: Controls, Dox, Dox+Capsazepine (Cpz), Dox+Melatonin (Mel), and combined Dox+Mel+Cpz given group. Capsaicin and capsazepine were added to cell culture mediums to activate or inactivate the TRPV1 channels, respectively. Cytosolic calcium, apoptosis, intracellular reactive oxygen, mitochondrial depolarization, caspase-3 and caspase-9 levels were measured. Results: Increased apoptotic activity was detected in doxorubicin given cell lines (Group II) when compared with the controls (p˂0.001). There was also a significantly higher apoptotic level in Dox+Mel group (Group IV), when compared with only Dox given group (p˂0.001). TRPV1 inhibition applied groups (Group III and V) have had lower apoptotic levels than other drug administered groups (p˂0.001). Conclusion: This study has indicated that apoptotic effects of Dox and Mel on both osteosarcoma and chondrosarcoma were strictly associated to TRPV1 channels, and that TRPV1 channels played an important role in whole mitochondria dependent pathways of apoptosis, which in turn may lead to increased intracellular Ca+2 levels and mitochondrial depolarization.

Zhenxin Anshen formula ameliorates atopic der-matitis-like skin dysfunction in mice and regulation of transient receptor potential vanilloid 1 and transient receptor potential ankyrin 1 in Neural pathways.

To investigate the efficacy of Zhenxin Anshen formula (, ZXAS) on atopic dermatitis (AD) by transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential ankyrin 1 (TRPA1) signalling pathway in mice and . AD-like lesions were induced by 1-chloro-2,4-dinitrobenzene (DNCB) to the shaved dorsal skin of BALB/c mice. BALB/c mice were divided into five groups: normal control, model control, cetirizine, low-, medium-, and high-dose of ZXAS. After ZXAS in-tervention, the skin lesions and blood samples were collected for hematoxylin and eosin-stained and measuring the concentrations of inflammatory cytokines. Immun-oglobulin E (IgE), interleukin (IL)-4, IL-5, IL-13, and thymic stromal lymphopoietin (TSLP) were de-tected by Enzyme-linked immunosorbent assay (ELISA). The spinal cords were collected for measuring the expression of gastrin-releasing peptide receptor (GRPR), TRPV1, and TRPA1 by using immunohistochemistry, western blotting, and quantitative real-time polymerase chain reaction (qRT-PCR) analyses. In addition, 3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay, flow cytometry, ELISA, and Western blotting were conducted for analysis of primary dorsal root ganglia (DRG) neurons . ZXAS treatment improved DNCB-induced AD-like lesions through reducing dermatitis score, number of scratching and epidermal thickness, accompanied by the de-creased IgE and Th2 inflammatory cytokines. ZXAS also supressed the mRNA and protein expression of GRPR, TRPV1, and TRPA1 in the spinal cord. The medicated sera of ZXAS decreased capsaicin-induced Ca influx and downregulated the expression of TRPV1, TRPA1, and phospholipase C in DRG neurons. The therapeutic effect of ZXAS on AD may be related to the regulation of TRPV1 and TRPA1 and inhibition of Ca2+ signals in neurons.

JI017 Attenuates Oxaliplatin-Induced Cold Allodynia via Spinal TRPV1 and Astrocytes Inhibition in Mice.

Oxaliplatin, a well-known chemotherapeutic agent, can induce severe neuropathic pain, which can seriously decrease the quality of life of patients. JI017 is an herb mixture composed of Aconitum carmichaelii, Angelica gigas, and Zingiber officinale. Its anti-tumor effect has been reported; however, the efficacy of JI017 against oxaliplatin-induced allodynia has never been explored. Single oxaliplatin injection [6 mg/kg, intraperitoneal, (i.p.)] induced both cold and mechanical allodynia, and oral administration of JI017 (500 mg/kg) alleviated cold but not mechanical allodynia in mice. Real-time polymerase chain reaction (PCR) analysis demonstrated that the upregulation of mRNA of spinal transient receptor potential vanilloid 1 (TRPV1) and astrocytes following oxaliplatin injection was downregulated after JI017 treatment. Moreover, TRPV1 expression and the activation of astrocytes were intensely increased in the superficial area of the spinal dorsal horn after oxaliplatin treatment, whereas JI017 suppressed both. The administration of TRPV1 antagonist [capsazepine, intrathecal (i.t.), 10 μg] attenuated the activation of astrocytes in the dorsal horn, demonstrating that the functions of spinal TRPV1 and astrocytes are closely related in oxaliplatin-induced neuropathic pain. Altogether, these results suggest that JI017 may be a potent candidate for the management of oxaliplatin-induced neuropathy as it decreases pain, spinal TRPV1, and astrocyte activation.

TRPV1 activation and internalization is part of the LPS-induced inflammation in human iPSC-derived cardiomyocytes

The non-selective cation channel transient receptor potential vanilloid 1 (TRPV1) is expressed throughout the cardiovascular system. Recent evidence shows a role for TRPV1 in inflammatory processes. The role of TRPV1 for myocardial inflammation has not been established yet. Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (hiPSC-CM) from 4 healthy donors were incubated with lipopolysaccharides (LPS, 6 h), TRPV1 agonist capsaicin (CAP, 20 min) or the antagonist capsazepine (CPZ, 20 min). TRPV1 expression was studied by PCR and western blotting. TRPV1 internalization was analyzed by immunofluorescence. Interleukin-6 (IL-6) secretion and phosphorylation of JNK, p38 and ERK were determined by ELISA. TRPV1-associated ion channel current was measured by patch clamp. TRPV1-mRNA and -protein were expressed in hiPSC-CM. TRPV1 was localized in the plasma membrane. LPS significantly increased secretion of IL-6 by 2.3-fold, which was prevented by pre-incubation with CPZ. LPS induced TRPV1 internalization. Phosphorylation levels of ERK, p38 or JNK were not altered by TRPV1 stimulation or inhibition. LPS and IL-6 significantly lowered TRPV1-mediated ion channel current. TRPV1 mediates the LPS-induced inflammation in cardiomyocytes, associated with changes of cellular electrophysiology. LPS-induced inflammation results in TRPV1 internalization. Further studies have to examine the underlying pathways and the clinical relevance of these findings.

The Synergistic Effect of TRPV1 on Oxidative Stress-Induced Autophagy and Apoptosis in Microglia.

Stroke mostly including ischemic stroke is the second leading mortality and disability worldwide. Oxidative stress injury occurred during ischemic stroke treatment generally. A high amount of reactive oxygen species (ROS) is involved in oxidative stress induction. Transient receptor potential vanilloid 1 (TRPV1) has been shown to regulate oxidative stress and apoptosis in microglia; however, the detailed mechanisms remain unclear. We aimed to explore whether autophagy-regulated oxidative stress and apoptosis are associated with TRPV1. The model of oxygen and glucose deprivation (OGD/R) in microglia was established. The siRNA of Atg5 and inhibitors and agonists of both autophagy and TRPV1 were involved in our study. Autophagy-related markers Atg5, LC3II/LC3I, and Beclin-1 were measured, and the autophagosome was observed under a transmission electron microscope (TEM). Caspase 3 was detected using ELISA. ROS and JC-1 were detected using flow cytometry. Apoptosis was observed by TUNEL. The results indicated that oxidative stress-induced injury and apoptosis may be impeded by the increasing autophagy, and TRPV1 inhibition could suppress the OGD/R-induced autophagy of microglia. However, the effect of TRPV1's inhibitor on oxidative stress and apoptosis was not obvious when the autophagy was blocked. These findings suggested that TRPV1 may exhibit antioxidative and antiapoptosis effect on OGD/R-induced microglia. However, the experimental results do not fully demonstrate that the TRPV1-mediated antioxidative and antiapoptosis effect is through the affecting autophagy entirely.

TRPV1 Hyperfunction Contributes to Renal Inflammation in Oxalate Nephropathy.

Inflammation worsens oxalate nephropathy by exacerbating tubular damage. The transient receptor potential vanilloid 1 (TRPV1) channel is present in kidney and has a polymodal sensing ability. Here, we tested whether TRPV1 plays a role in hyperoxaluria-induced renal inflammation. In TRPV1-expressed proximal tubular cells LLC-PK1, oxalate could induce cell damage in a time- and dose-dependent manner; this was associated with increased arachidonate 12-lipoxygenase (ALOX12) expression and synthesis of endovanilloid 12(S)-hydroxyeicosatetraenoic acid for TRPV1 activation. Inhibition of ALOX12 or TRPV1 attenuated oxalate-mediated cell damage. We further showed that increases in intracellular Ca2+ and protein kinase C α activation are downstream of TRPV1 for NADPH oxidase 4 upregulation and reactive oxygen species formation. These trigger tubular cell inflammation via increased NLR family pyrin domain-containing 3 expression, caspase-1 activation, and interleukin (IL)-1β release, and were alleviated by TRPV1 inhibition. Male hyperoxaluric rats demonstrated urinary supersaturation, tubular damage, and oxidative stress in a time-dependent manner. Chronic TRPV1 inhibition did not affect hyperoxaluria and urinary supersaturation, but markedly reduced tubular damage and calcium oxalate crystal deposition by lowering oxidative stress and inflammatory signaling. Taking all these results together, we conclude that TRPV1 hyperfunction contributes to oxalate-induced renal inflammation. Blunting TRPV1 function attenuates hyperoxaluric nephropathy.

Decursin Alleviates Mechanical Allodynia in a Paclitaxel-Induced Neuropathic Pain Mouse Model.

Chemotherapy-induced neuropathic pain (CINP) is a severe adverse effect of platinum- and taxane-derived anticancer drugs. The pathophysiology of CINP includes damage to neuronal networks and dysregulation of signal transduction due to abnormal Ca2+ levels. Therefore, methods that aid the recovery of neuronal networks could represent a potential treatment for CINP. We developed a mouse model of paclitaxel-induced peripheral neuropathy, representing CINP, to examine whether intrathecal injection of decursin could be effective in treating CINP. We found that decursin reduced capsaicin-induced intracellular Ca2+ levels in F11 cells and stimulated neurite outgrowth in a concentration-dependent manner. Decursin directly reduced mechanical allodynia, and this improvement was even greater with a higher frequency of injections. Subsequently, we investigated whether decursin interacts with the transient receptor potential vanilloid 1 (TRPV1). The web server SwissTargetPrediction predicted that TRPV1 is one of the target proteins that may enable the effective treatment of CINP. Furthermore, we discovered that decursin acts as a TRPV1 antagonist. Therefore, we demonstrated that decursin may be an important compound for the treatment of paclitaxel-induced neuropathic pain that functions via TRPV1 inhibition and recovery of damaged neuronal networks.

Blocking of Transient Receptor Potential Vanilloid 1 (TRPV1) promotes terminal mitophagy in multiple myeloma, disturbing calcium homeostasis and targeting ubiquitin pathway and bortezomib-induced unfolded protein response

BackgroundChemoresistance remains a major treatment obstacle in multiple myeloma (MM). Novel new therapies are thus in need. Transient Receptor Potential Vanilloid type 1 (TRPV1) is a calcium-permeable ion channel that has been demonstrated to be expressed in solid tumors. Calcium channels have been shown to be involved in the regulation of cell proliferation, chemoresistance, migration and invasion. The aim of the current study was to evaluate its possible role in MM.MethodsPharmacological inhibitor was used to evaluate the role of TRPV1 in MM cell lines and primary MM cells. Flow cytometry, molecular analysis, fluorescent microscopy, proteomic analysis and xenograft in vivo model of MM with BM involvement were employed to assess the effect of TRPV1 inhibition and decipher its unique mechanism of action in MM.ResultsTRPV1 was found to be expressed by MM cell lines and primary MM cells. TRPV1 inhibition using the antagonist AMG9810-induced MM cell apoptosis and synergized with bortezomib, overcoming both CXCR4-dependent stroma-mediated and acquired resistance. In accordance, AMG9810 suppressed the expression and activation of CXCR4 in MM cells. TRPV1 inhibition increased mitochondrial calcium levels with subsequent mitochondrial ROS accumulation and depolarization. These effects were reversed by calcium chelation, suggesting the role of calcium perturbations in oxidative stress and mitochondrial destabilization. Furthermore, AMG9810 abolished bortezomib-induced accumulation of mitochondrial HSP70 and suppressed protective mitochondrial unfolded protein response. Proteomics revealed unique molecular signature related to the modification of ubiquitin signaling pathway. Consequently, 38 proteins related to the ubiquitylation machinery were downregulated upon combined bortezomib/AMG9810 treatment. Concomitantly, AMG9810 abolished bortezomib-induced ubiquitination of cytosolic and mitochondrial proteins. Furthermore, bortezomib/AMG9810 treatment induced mitochondrial accumulation of PINK1, significantly reduced the mitochondrial mass and promoted mitochondrial-lysosomal fusion, indicating massive mitophagy. Finally, in a recently developed xenograft model of systemic MM with BM involvement, bortezomib/AMG9810 treatment effectively reduced tumor burden in the BM of MM-bearing mice.ConclusionsAltogether, our results unravel the mechanism mediating the strong synergistic anti-MM activity of bortezomib in combination with TRPV1 inhibition which may be translated into the clinic.

Inhibition of transient receptor potential vanilloid 1 (TRPV1) channel regulates chikungunya virus infection in macrophages.

Chikungunya virus (CHIKV), a virus that induces pathogenic inflammatory host immune responses, is re-emerging worldwide, and there are currently no established antiviral control measures. Transient receptor potential vanilloid 1 (TRPV1), a non-selective Ca2+-permeable ion channel, has been found to regulate various host inflammatory responses including several viral infections. Immune responses to CHIKV infection in host macrophages have been reported recently. However, the possible involvement of TRPV1 during CHIKV infection in host macrophages has not been studied. Here, we investigated the possible role of TRPV1 in CHIKV infection of the macrophage cell line RAW 264.7. It was found that CHIKV infection upregulates TRPV1 expression in macrophages. To confirm this observation, the TRPV1-specific modulators 5'-iodoresiniferatoxin (5'-IRTX, a TRPV1 antagonist) and resiniferatoxin (RTX, a TRPV1 agonist) were used. Our results indicated that TRPV1 inhibition leads to a reduction in CHIKV infection, whereas TRPV1 activation significantly enhances CHIKV infection. Using a plaque assay and a time-of-addition assay, it was observed that functional modulation of TRPV1 affects the early stages of the viral lifecycle in RAW 264.7 cells. Moreover, CHIKV infection was found to induce of pNF-κB (p65) expression and nuclear localization. However, both activation and inhibition of TRPV1 were found to enhance the expression and nuclear localization of pNF-κB (p65) and production of pro-inflammatory TNF and IL-6 during CHIKV infection. In addition, it was demonstrated by Ca2+ imaging that TRPV1 regulates Ca2+ influx during CHIKV infection. Hence, the current findings highlight a potentially important regulatory role of TRPV1 during CHIKV infection in macrophages. This study might also have broad implications in the context of other viral infections as well.

Protective effects of TRPV1 inhibition against sevoflurane-induced cell death

Sevoflurane is a widely used inhalational anesthetic that can induce developmental neurotoxicity, leading to cognitive dysfunction. In this study, we assessed the role of transient receptor potential vanilloid 1 (TRPV1) in mediating sevoflurane activation and whether the TRPV1 antagonist could prevent anesthesia-induced cell death. Here, we demonstrated that the expression of TRPV1 was increased after sevoflurane treatment, and pretreatment with TRPV1 antagonist SB366791 could attenuate the effect of sevoflurane on TRPV1 expression. Moreover, the inhibition of TRPV1 could prevent sevoflurane-induced cell death. The findings of this study provide novel insights into the treatment of general anesthesia-induced developmental neurotoxicity and even cognitive impairment.

TRPV1 inhibition supports cell survival during hypoxia/reoxygenation in the rat cardiomyoblast H9C2 cell line

Transient receptor potential vanilloid 1 (TRPV1) is a nonselective ion channel that gated calcium (Ca2 + ). Over the last decade, several TRPV1-targeted strategies based on remote, pre- and post-conditioning in order to decrease infarct size have been successful. In fact, during myocardial infarction, Ca2+ dysregulation occurs leading to cell death and causing irreversible myocardial injury. TRPV1 modulation would counteract these deadly effects. The aim of this study is to decipher molecular mechanisms implicated in TRPV1 modulation to better understand the role of TRPV1 in hypoxia-reoxygenation (H/R). Here, we used H9C2 cell line as an alternative model to adult cardiomyocytes in order to perform live imaging with genetic probes. First, using RT-PCR, Western blot and confocal microscopy, we demonstrated that TRPV1 is expressed in H9C2 and seems to be localized at endoplasmic reticular (ER) membrane. Secondly, thanks to cytosolic and reticular Ca2 + imaging (respectively with Fura-2 and ErGAP1), we showed that TRPV1 is a functional ER Ca2 + leak channel. Since ATP synthesis and cell fate are dependent on Ca2 + exchanges between ER and mitochondria, we then analyzed the role TRPV1 on the mitochondrial [Ca2 + ] using R-GECO probe. We observed that pharmacological TRPV1 modulation increases both cytosolic and mitochondrial Ca2 + contents by at least 20%. Finally, cell death was evaluated by flow cytometry after H/R sequences. In particular, we showed that TRPV1 inhibition improves cell survival (at least by 22%). Many physiological processes (including cell death/survival balance) rely on precise and spatiotemporal changes in the intracellular Ca2 + concentration. In this study, we show: – that TRPV1 could precisely regulate Ca2 + signals in a localization-specific way, (i.e. between ER and mitochondria); – that H9C2 is a valuable model to evaluate the role of TRPV1 in Ca2 + fluxes during H/R.

Inhibition of Transient Receptor Potential Vanilloid 1 Attenuates Blood-Brain Barrier Disruption after Traumatic Brain Injury in Mice.

Transient receptor potential vanilloid 1 (TRPV1) is expressed widely in the central nervous system and is activated by various stimuli. Inhibiting TRPV1 has neuroprotective effects in cerebral ischemia. The role of inhibiting TRPV1 to maintain blood-brain barrier (BBB) integrity after traumatic brain injury (TBI) remains unclear, however. Therefore, we investigated the effects of capsazepine-mediated TRPV1 inhibition on the BBB in a mouse model of TBI. Adult male C57BL/6 mice underwent controlled cortical impact injury and received capsazepine (1 μmol/kg body weight, twice daily, intraperitoneally) until sacrifice. Further, mouse brain microvascular endothelial (bEnd.3) cells were cultured and underwent biaxial stretch injury to investigate the mechanisms underlying the protective effects of capsazepine. The TRPV1 expression was upregulated in the pericontusional area after TBI, peaking at 24 h post-TBI. Capsazepine-treated mice demonstrated decreased brain edema (p = 0.010), Evans Blue extravasation (p = 0.001), tissue hemoglobin levels (p = 0.002), and loss of tight junction proteins (p = 0.016 ZO-1 expression; p = 0.013 occludin expression) after TBI compared with the vehicle-treated group. Capsazepine significantly alleviated early-stage apoptosis by attenuating activation of JNK, P38, and caspase-3, resulting in a protective effect on the level of ZO-1 in bEnd.3 cells after stretch injury. We conclude that the expression of TRPV1 is upregulated after TBI, and inhibition of TRPV1 attenuated disruption of the BBB in a mouse model of TBI, at least partly, through its antiapoptotic effects on brain endothelial cells. Blocking TRPV1 may be a promising pharmacotherapeutic intervention to protect against BBB disruption after TBI.

Differences in the effects of TRPV1 antagonists on energy metabolism in mice.

Transient receptor potential vanilloid 1 (TRPV1) is a nociceptive cation channel that is activated by heat, protons and chemical ligands such as capsaicin. We investigated the roles of the capsaicin receptor, TRPV1, in controlling the energy metabolism of the whole body. It has been reported that the activation of TRPV1 by its agonists enhances energy metabolism. In this study, we used a respiratory gas analysis system to examine whether the inhibition of TRPV1 changes energy metabolism in mice. In addition, we examined the contributions of different modes of TRPV1 activation (heat, protons and capsaicin) to determine the influence of 3 different TRPV1 antagonists on energy metabolism. Here, we showed that intragastric administration of AMG517, a nonselective antagonist of TRPV1 (for heat, protons and capsaicin), enhanced energy metabolism as much as did intraperitoneal administration. On the other hand, intraperitoneal administration of AMG9810, a nonselective antagonist like AMG517, enhanced energy expenditure more than intragastric administration. However, the administration of JYL1421, a TRPV1 antagonist that very strongly inhibits TRPV1 activated by capsaicin, did not change energy metabolism. Taken together, these results suggest that the type of TRPV1 antagonists and the routes of its administration have different effects on energy metabolism in a normal body. Surprisingly, co-administration of JYL1421 and capsaicin significantly enhanced the energy metabolism more than administration of capsaicin alone. These results support the possibility that an unconventional mechanism is responsible for the increase in energy metabolism that occurs via TRPV1 inhibition.

Blocking of Transient Receptor Potential Vanilloid1 (TRPV1) Promotes Lysosomal Destabilization and Enhances Bortezomib-Induced ER Stress and Cell Death Via HSP70 and LAMP3 Down-Regulation: Novel Therapeutic Target for Multiple Myeloma

Introduction: Multiple myeloma (MM) is a neoplastic disorder that is characterized by clonal proliferation of plasma cells in the bone marrow (BM). Acquired or de novo resistance to current anti-MM therapy remains a major treatment obstacle. Novel new therapies are thus in need. Recent data have highlighted the contribution of Ca2+channels in the regulation of cell proliferation, chemo-resistance, migration and invasion. Transient Receptor Potential Vanilloid type-1 (TRPV1) is a calcium-permeable ion channel that has been demonstrated to be expressed in solid tumors. As no data is available evaluating TRPV1 in MM, the aim of the current study was to evaluate its possible role in MM.Results: Elevated levels of TRPV1 transcript was detected in MM cell lines (n=8) and BM aspirates from MM patients (n=24) in comparison to normal BM (n=5). AMG9810 a specific antagonist of TRPV1, significantly reduced the viability of MM cell lines (n=8) and primary CD138+ cells (n=6),in a time- and dose-dependent manner (p<0.01) and induced apoptosis manifested by phosphatidylserine externalization, loss of mitochondrial membrane potential (ψm), caspase 3 cleavage and DNA fragmentation. AMG9810-triggered apoptosis could be partially blocked by inhibition of calpains and cathepsins, indicating the role of lysosomal rapture in AMG9810-mediated cell death. Indeed, treatment with TRPV1 antagonist induced rapid lysosomal acidification and increased the number of acidic vesicles (detected by acridine orange stain). The acidic vesicles appeared as early as 1 hour post exposure to AMG9810 preceding the mitochondrial destabilization and apoptosis, thus suggesting that TRPV1 blockade induces lysosomal-induced cell death in MM. Furthermore, TRPV1 inhibition with AMG9810 completely suppressed the pro-survival AKT/mTOR pathway and significantly reduced the levels of anti-apoptotic factors BCL-2 and BCL-XL. Combining AMG9810 with the proteasome inhibitor bortezomib (Bort) induced synergistic cell death in both native and Bort-resistant cells (CI<0.4). Moreover, TRPV1 inhibition successfully overcame the CXCR4-mediated protection from Bort provided by BM stromal cells. This finding suggests that the TRPV1 channel may regulate the activity of CXCR4 chemokine receptor in MM cells affecting the MM-microenvironment interactions. In accordance, the TRPV1 antagonist AMG8910 prevented the responsiveness of CXCR4-expressing MM cells to CXCL12 stimulation, decreased the phosphorylation of signaling mediators like Erk1/2 and AKT and suppressed cell migration, while TRPV1 activator capsaicin promoted the CXCR4-mediated signaling and migration. Gene and protein expression analysis were next performed to delineate the molecular mechanisms underlying the observed synergism between Bort and AMG9810. Bort treatment resulted in robust induction of endoplasmic reticulum (ER) stress genes including the increase in pro-apoptotic factors ATF4, CHOP and GADD34. Compensatory unfolded protein response (UPR) was activated as well, with increase in chaperons HSP27, HSP70, HSP90, and lysosomal chaperon LAMP3 known to stabilize lysosome, protecting cells against lysosomal membrane permeabilization (LMP) and subsequent cell death. AMG9810 further increased ER stress, elevating CHOP and GADD34 expression, while significantly reducing both basal and Bort-increased levels of HSP70 and LAMP3, thus overcoming the protective response to Bort treatment and prompting lethal LMP. Finally, combining Bort with AMG9810 resulted in significantly reduced ROS that was correlated with impaired mitochondria and increased MM apoptosis, suggesting that dissipation of intracellular ROS may be involved in AMG9810-promoted cytotoxicity.Conclusions: Altogether, our data indicate that TRPV1 is implicated in MM cell survival, proliferation, migration, microenvironment interactions and stress response. TRPV1 inhibition by AMG9810 inhibits CXCR4-mediated migration and stromal protection, synergizes with Bort, amplifies ER stress, targets cytoprotective HSP70 and LAMP3, destabilizes lysosome, impairs mitochondria and promotes MM cell death. These results unravel the mechanism mediating the strong synergistic anti-MM activity of Bort in combination with TRPV1 inhibition which may be translated into the clinic. [Display omitted] DisclosuresPeled:Biokine: Consultancy; Biosight: Consultancy.

Abstract 17443: Inhibition of the Calcineurin Interaction Site on TRPV1 Reduces Myocardial Infarct Size in Rats

Introduction: With recent public concerns of opioid overuse, misuse and addiction along with the heightened FDA restrictions on opioid prescribing, non-opioid cardiac-safe pain therapeutics for those with cardiovascular disease is needed. The transient receptor potential vanilloid 1 (TRPV1), a mediator of heat, pain and noxious stimuli, mediates nociception. While blocking TRPV1 reduces pain, the finding preconditioning-induced cardioprotection is lost in TRPV1 knockout mice or by TRPV1 inhibition indicates a dual role for TRPV1 both in protection from cardiac injury and in pain signaling. Hypothesis: We tested whether a peptide corresponding to a calcineurin interaction site on TRPV1 could separate the functions of TRPV1 by reducing myocardial injury while providing pain control. Methods: The peptide was synthesized using a Liberty peptide synthesizer and conjugated to TAT for intracellular entry. Male Sprague-Dawley rats 8 weeks of age were used for both isolated heart and in vivo studies consisting of 30 minutes of ischemia followed by reperfusion. Infarct size was assessed by triphenyltetrazolium staining. Results: Testing the peptide using a calcineurin activity assay kit in vitro showed the peptide inhibited calcineurin activity vs. TAT control (0.46±0.03* vs. 0.77±0.02, nmol phosphate, n=3/group, *P&lt;0.05). Further, peptide treatment of isolated hearts (1μM, infused over 10 minutes prior to ischemia) reduced infarct size and creatine phosphokinase (CPK) release when compared to TAT-treated hearts (Infarct size: 19±3%* vs. 53±4%, %infarct/left ventricle; CPK: 69±22* vs. 459±132, n=6/group, *P&lt;0.01). In an in vivo myocardial ischemia-reperfusion model, the peptide (1mg/kg), given subcutaneously to the abdominal skin prior to ischemia, reduced infarct size compared to control (50±2*% vs. 66±1%, *P&lt;0.001). Initial evidence also suggests that the peptide provides analgesia in pain models. Conclusions: Our results suggest a peptide blocking the calcineurin interaction site of TRPV1 reduces myocardial infarct size. Therefore, disrupting specific protein-protein interactions between calcineurin and TRPV1 may provide a novel strategy to design non-opioid therapeutics for cardioprotection, while also providing pain relief.

TRPV1 inhibition does not prevent cold dry air-elicited symptoms in non-allergic rhinitis

The transient receptor potential vanilloid 1 (TRPV1)-expressing sensory C-fibers may play a role in the development of nasal hyper-responsiveness and symptoms of non-allergic rhinitis (NAR). To evaluate the effects of a TRPV1-antagonist, SB-705498, on cold dry air (CDA)-induced symptoms in patients with NAR. This randomized, double-blind, placebo-controlled, crossover study evaluated 14 days of once daily, topical intranasal SB-705498 12 mg in 40 patients with NAR using a CDA challenge experimental model in an environmental exposure chamber (EEC, Cetero Research, Mississauga, Ontario). The primary endpoint was total symptom score (TSS), expressed as weighted mean over 60 minutes (WM0-60) or maximum TSS at 1 hour and 24 hours postdosing. Treatment with SB-705498, relative to placebo, did not improve WM0-60 or maximum TSS at 1 hour and 24 hours post-dosing on days 1 or 14. Mean (95% CI) treatment differences (SB-705498 - placebo) on day 14 were, for WM0-60 at 1 hour: -0.12 (-0.60, 0.36); for maximum TSS at 1 hour: -0.03 (-0.58, 0.51). SB-705498 had no impact on any other efficacy parameters. SB-705498 was well tolerated and pharmacokinetics analysis supported the dosing regimen. SB-705498 12 mg for 14 days did not alleviate the CDA-induced symptoms of NAR. Despite engagement of the TRPV1 receptor, there was no translation to clinical efficacy, suggesting redundancy in symptom pathways.

Effect of Mucosal TRPV1 Inhibition in Allergic Rhinitis

Transient receptor potential vanilloid-1 (TRPV1) has been implicated as a mediator of itch in allergic rhinitis. To address this possibility, we synthesized a TRPV1 blocker (SB-705498) for nasal administration in patients with seasonal allergic rhinitis. The pharmacological activity of SB-705498 was confirmed on human TRPV1-expressing HEK293 cells, using fluorometric calcium imaging, and in patients with allergic rhinitis subjected to nasal capsaicin challenges. The effect of SB-705498 was studied in patients with seasonal allergic rhinitis subjected to daily allergen challenges for 7 days, using a double-blind, placebo-controlled, randomized and cross-over design. SB-705498 was delivered by nasal lavage 2 min. before each allergen challenge. Primary end-point was total nasal symptom score on days 5-7. Nasal peak inspiratory flow (nPIF) and eosinophil cationic protein (ECP) content in nasal lavages were also monitored. Daily topical applications of SB-705498 at a concentration that inhibited capsaicin-induced nasal symptoms had no effect on total symptom score, nPIF and ECP levels in allergen-challenged patients with seasonal allergic rhinitis. The individual symptoms, nasal itch or sneezes, were also not affected. These findings may indicate that TRPV1 is not a key mediator of the symptoms in allergic rhinitis. However, additional studies, using drug formulations with a prolonged duration of action, should be conducted before TRPV1 is ruled out as a drug target in allergic rhinitis.

Cardioprotection by ischemic postconditioning is lost in isolated perfused heart from diabetic rats: Involvement of transient receptor potential vanilloid 1, calcitonin gene-related peptide and substance P

We previously found that the expression of transient receptor potential vanilloid 1 (TRPV1) and contents of calcitonin gene-related peptide (CGRP) and substance P (SP), two main neuropeptides released from TRPV1, were decreased in diabetic hearts. This study aimed to test whether decreased TRPV1, CGRP and SP levels were responsible for the loss of cardioprotection by ischemic postconditioning (IPostC) in isolated perfused heart from streptozotocin-induced diabetic rats. IPostC effectively protected non-diabetic hearts against ischemia/reperfusion injury by improving cardiac function and lowering creatine kinase (CK) and cardiac troponin I (cTnI) release, which could be abolished by inhibiting TRPV1, CGRP receptor or SP receptor. However, IPostC had no effect on cardiac function and the release of CK and cTnI in diabetic hearts regardless of whether TRPV1, CGRP receptor or SP receptor were inhibited. CGRP or SP-induced postconditioning significantly prevented both non-diabetic and diabetic hearts from ischemia/reperfusion injury by improving cardiac function and lowering CK and cTnI release. Additionally, IPostC markedly increased CGRP and SP release in non-diabetic hearts, which could be reversed with TRPV1 inhibition, but not CGRP receptor or SP receptor inhibition. However, IPostC failed to affect CGRP and SP release in diabetic hearts in the presence or absence of TRPV1, CGRP receptor or SP receptor inhibition. These results indicate that the loss of cardioprotection by IPostC during diabetes is partly associated with a failure to increase CGRP and SP release, likely due to decreased TRPV1 expression and CGRP and SP contents in diabetic hearts.