Role of Piezo Channels in Joint Health and Injury.

Cartilage is an intrinsically mechanically sensitive tissue composed of chondrocytes as the only cell type. Chondrocyte mechanotransduction is not well understood, but recently we identified critical components of the mechanotransduction machinery demonstrating how mechanical stimulation of these cells can be converted into cellular calcium signals. Physiologic mechanical cues induce anabolic responses of (post-mitotic) chondrocytes via transient receptor potential vanilloid 4 ion channels, whereas injurious mechanical stress is transduced by Piezo1 jointly with Piezo2 ion channels. This chapter sheds light on the latter discovery and provides a rationale for follow-up questions, such as the nature of interaction between Piezo1 and Piezo2, and their tethering to the cytoskeleton. These recent insights can be leveraged toward translational medical progress to benefit diagnosis and treatment of osteoarthritis, representing a large and growing unmet medical need in the United States and large parts of the world.

Analysis of TRPV channel activation by stimulation of FCεRI and MRGPR receptors in mouse peritoneal mast cells.

The activation of mast cells (MC) is part of the innate and adaptive immune responses and depends on Ca2+ entry across the plasma membrane, leading to the release of preformed inflammatory mediators by degranulation or by de novo synthesis. The calcium conducting channels of the TRPV family, known by their thermo and osmotic sensitivity, have been proposed to be involved in the MC activation in murine, rat, and human mast cell models. So far, immortalized mast cell lines and nonspecific TRPV blockers have been employed to characterize the role of TRPV channels in MC. The aim of this work was to elucidate the physiological role of TRPV channels by using primary peritoneal mast cells (PMCs), a model of connective tissue type mast cells. Our RT-PCR and NanoString analysis identified the expression of TRPV1, TRPV2, and TRPV4 channels in PMCs. For determination of the functional role of the expressed TRPV channels we performed measurements of intracellular free Ca2+ concentrations and beta-hexosaminidase release in PMCs obtained from wild type and mice deficient for corresponding TRPV1, TRPV2 and TRPV4 in response to various receptor-mediated and physical stimuli. Furthermore, substances known as activators of corresponding TRPV-channels were also tested using these assays. Our results demonstrate that TRPV1, TRPV2, and TRPV4 do not participate in activation pathways triggered by activation of the high-affinity receptors for IgE (FcεRI), Mrgprb2 receptor, or Endothelin-1 receptor nor by heat or osmotic stimulation in mouse PMCs.

Transient receptor potential vanilloid 4 inhibits mouse colonic motility by activating NO-dependent enteric neurotransmission.

UNLABELLED: Recent studies implicate TRPV4 receptors in visceral pain signaling and intestinal inflammation. Our aim was to evaluate the role of TRPV4 in the control of gastrointestinal (GI) motility and to establish the underlying mechanisms. We used immunohistochemistry and PCR to study TRPV4 expression in the GI tract. The effect of TRPV4 activation on GI motility was characterized using in vitro and in vivo motility assays. Calcium and nitric oxide (NO) imaging were performed to study the intracellular signaling pathways. Finally, TRPV4 expression was examined in the colon of healthy human subjects. We demonstrated that TRPV4 can be found on myenteric neurons of the colon and is co-localized with NO synthase (NOS-1). In vitro, the TRPV4 agonist GSK1016790A reduced colonic contractility and increased inhibitory neurotransmission. In vivo, TRPV4 activation slowed GI motility and reduced stool production in mouse models mimicking pathophysiological conditions. We also showed that TRPV4 activation inhibited GI motility by reducing NO-dependent Ca(2+) release from enteric neurons. In conclusion, TRPV4 is involved in the regulation of GI motility in health and disease. KEY MESSAGES: • Recent studies implicate TRPV4 in pain signaling and intestinal inflammation. • Our aim was to characterize the role of TRPV4 in the control of GI motility. • We found that TRPV4 activation reduced colonic contractility. • Our studies also showed altered TRPV4 mRNA expression in IBS-C patients. • TRPV4 may be a novel pharmacological target in functional GI diseases.

Functional transient receptor potential vanilloid 1 and transient receptor potential vanilloid 4 channels along different segments of the renal vasculature.

AIM: Transient receptor potential vanilloid 1 (TRPV1) and vanilloid 4 (TRPV4) cation channels have been recently identified to promote endothelium-dependent relaxation of mouse mesenteric arteries. However, the role of TRPV1 and TRPV4 in the renal vasculature is largely unknown. We hypothesized that TRPV1/4 plays a role in endothelium-dependent vasodilation of renal blood vessels. METHODS: We studied the distribution of functional TRPV1/4 along different segments of the renal vasculature. Mesenteric arteries were studied as control vessels. RESULTS: The TRPV1 agonist capsaicin relaxed mouse mesenteric arteries with an EC50 of 25 nm, but large mouse renal arteries or rat descending vasa recta only at >100-fold higher concentrations. The vasodilatory effect of capsaicin in the low-nanomolar concentration range was endothelium-dependent and absent in vessels of Trpv1 -/- mice. The TRPV4 agonist GSK1016790A relaxed large conducting renal arteries, mesenteric arteries and vasa recta with EC50 of 18, 63 nm and ~10 nm respectively. These effects were endothelium-dependent and inhibited by a TRPV4 antagonist, AB159908 (10 µm). Capsaicin and GSK1016790A produced vascular dilation in isolated mouse perfused kidneys with EC50 of 23 and 3 nm respectively. The capsaicin effects were largely reduced in Trpv1 -/- kidneys, and the effects of GSK1016790A were inhibited in Trpv4 -/- kidneys. CONCLUSION: Our results demonstrate that two TRPV channels have unique sites of vasoregulatory function in the kidney with functional TRPV1 having a narrow, discrete distribution in the resistance vasculature and TRPV4 having more universal, widespread distribution along different vascular segments. We suggest that TRPV1/4 channels are potent therapeutic targets for site-specific vasodilation in the kidney.

A role for transient receptor potential vanilloid 4 in tonicity-induced neurogenic inflammation.

BACKGROUND AND PURPOSE: Changes in extracellular fluid osmolarity, which occur after tissue damage and disease, cause inflammation and maintain chronic inflammatory states by unknown mechanisms. Here, we investigated whether the osmosensitive channel, transient receptor potential vanilloid 4 (TRPV4), mediates inflammation to hypotonic stimuli by a neurogenic mechanism. EXPERIMENTAL APPROACH: TRPV4 was localized in dorsal root ganglia (DRG) by immunofluorescence. The effects of TRPV4 agonists on release of pro-inflammatory neuropeptides from peripheral tissues and on inflammation were examined. KEY RESULTS: Immunoreactive TRPV4 was detected in DRG neurones innervating the mouse hindpaw, where it was co-expressed in some neurones with CGRP and substance P, mediators of neurogenic inflammation. Hypotonic solutions and 4alpha-phorbol 12,13-didecanoate, which activate TRPV4, stimulated neuropeptide release in urinary bladder and airways, sites of neurogenic inflammation. Intraplantar injection of hypotonic solutions and 4alpha-phorbol 12,13-didecanoate caused oedema and granulocyte recruitment. These effects were inhibited by a desensitizing dose of the neurotoxin capsaicin, antagonists of CGRP and substance P receptors, and TRPV4 gene knockdown or deletion. In contrast, antagonism of neuropeptide receptors and disruption of TRPV4 did not prevent this oedema. TRPV4 gene knockdown or deletion also markedly reduced oedema and granulocyte infiltration induced by intraplantar injection of formalin. CONCLUSIONS AND IMPLICATIONS: Activation of TRPV4 stimulates neuropeptide release from afferent nerves and induces neurogenic inflammation. This mechanism may mediate the generation and maintenance of inflammation after injury and during diseases, in which there are changes in extracellular osmolarity. Antagonism of TRPV4 may offer a therapeutic approach for inflammatory hyperalgesia and chronic inflammation.

TRPV channels' role in osmotransduction and mechanotransduction.

In signal transduction of metazoan cells, transient receptor potential (TRP) ion channels have been identified that respond to diverse external and internal stimuli, among them osmotic and mechanical stimuli. This chapter will summarize findings on the TRPV subfamily, both its vertebrate and invertebrate members. Of the six mammalian TRPV channels, TRPV1, -V2, and -V4 were demonstrated to function in transduction of osmotic and/or mechanical stimuli. TRPV channels have been found to function in cellular as well as systemic osmotic homeostasis in vertebrates. Invertebrate TRPV channels, five in Caenorhabditis elegans and two in Drosophila, have been shown to play a role in mechanosensation, such as hearing and proprioception in Drosophila and nose touch in C. elegans, and in the response to osmotic stimuli in C. elegans. In a striking example of evolutionary conservation of function, mammalian TRPV4 has been found to rescue mechanosensory and osmosensory deficits of the TRPV mutant line osm-9 in C. elegans, despite no more than 26% orthology of the respective amino acid sequences.

Functionality of the TRPV subfamily of TRP ion channels: add mechano-TRP and osmo-TRP to the lexicon!

Transient receptor potential (TRP) ion channels have been identified as cellular sensors responding to diverse external and internal stimuli. This review will cover the TRPV subfamily that comprises vertebrate and invertebrate members. The six mammalian TRPV channels were demonstrated to function in thermosensation, mechanosensation, osmosensation and Ca(2+) uptake. Invertebrate TRPV channels, five in Caenorhabditis elegans and two in Drosophila, have been shown to play a role in mechanosensation, such as hearing and proprioception in Drosophila and nose touch in C. elegans, and in the response to osmotic and chemical stimuli in C. elegans. We will focus here on the role that TRPV ion channels play in mechanosensation and a related sensory (sub-)modality, osmosensation.

Vanilloid receptor-related osmotically activated channel (VR-OAC), a candidate vertebrate osmoreceptor.

The detection of osmotic stimuli is essential for all organisms, yet few osmoreceptive proteins are known, none of them in vertebrates. By employing a candidate-gene approach based on genes encoding members of the TRP superfamily of ion channels, we cloned cDNAs encoding the vanilloid receptor-related osmotically activated channel (VR-OAC) from the rat, mouse, human, and chicken. This novel cation-selective channel is gated by exposure to hypotonicity within the physiological range. In the central nervous system, the channel is expressed in neurons of the circumventricular organs, neurosensory cells responsive to systemic osmotic pressure. The channel also occurs in other neurosensory cells, including inner-ear hair cells, sensory neurons, and Merkel cells.

Mouse keratin 4 is necessary for internal epithelial integrity.

Keratins are intermediate filaments of epithelial cells. Mutations in keratin genes expressed in skin lead to human disorders, including epidermolysis bullosa simplex and epidermolytic hyperkeratosis. We examined the role of keratin 4 (K4) in maintaining the integrity of internal epithelial linings by using gene targeting to generate mice containing a null mutation in the epithelial K4 gene. Homozygous mice that do not express K4 develop a spectrum of phenotypes that affect several organs which express K4 including the esophagus, tongue, and cornea. The cellular phenotypes include basal hyperplasia, lack of maturation, hyperkeratosis, atypical nuclei, perinuclear clearing, and cell degeneration. These results are consistent with the notion that K4 is required for internal epithelial cell integrity. As mutations in K4 in humans lead to a disorder called white sponge nevus, the K4-deficient mice may serve as models for white sponge nevus and for understanding the role of K4 in cellular proliferation and differentiation.

Effective treatment of models of multiple sclerosis by matrix metalloproteinase inhibitors.

The proinflammatory Th1 cytokine, tumor necrosis factor-alpha (TNF alpha), the cell death signaling molecule FasL, and several extracellular matrix degrading metalloproteinases have been implicated in the pathogenesis of multiple sclerosis (MS). The latter enzymes, as well as TNF alpha-converting enzyme and FasL-converting enzyme, can be blocked by matrix metalloproteinase inhibitors (MMPIs). In this study, we show that a potent MMPI was clinically effective in an animal model for MS, experimental autoimmune encephalomyelitis (EAE) in the SJL/J mouse. Efficacy was remarkable, as indicated by blocking and reversal of acute disease and reduced number of relapses and diminished mean cumulative disease score in chronic relapsing animals. Also, demyelination and glial scarring were significantly decreased in MMPI-treated mice with chronic relapsing EAE, as was central nervous system gene expression for TNF alpha and fasL. It is interesting that expression of the beneficial cytokine interleukin-4 (IL-4) was increased, and IL-4 was expressed on glial cells. The relevance of these compounds for MS was underscored by their ability to specifically inhibit TNF alpha shedding and cytotoxicity of myelin-autoreactive human cytotoxic CD4+ T-cell clones. This is the first report to show a positive effect by MMPIs on chronic relapsing EAE, its central nervous system cytokine profile, and on TNF alpha shedding by human myelin-autoreactive T cells.

Experimental autoimmune encephalomyelitis in mice lacking glial fibrillary acidic protein is characterized by a more severe clinical course and an infiltrative central nervous system lesion.

Insights into the role of the astrocyte intermediate filament protein, glial fibrillary acidic protein (GFAP), have only recently emerged with reports on subtle abnormalities in GFAP-deficient mice, including the documentation of defective long-term maintenance of central nervous system myelination. Here, we extend these observations by examining the astroglial response in GFAP-/- mice with autoimmune encephalomyelitis (EAE), a model for multiple sclerosis. Clinically, the monophasic disease was more severe in GFAP-/- mice than in wild-type littermates despite increased remyelination in the former. More in keeping with the clinical course was the observation of an infiltrative EAE lesion in GFAP-/- mice. GFAP-/- astrocytes had a reduced cytoarchitectural stability as evidenced by less abundant and irregularly spaced hemidesmosomes. The blunt GFAP-/- astrocyte processes possessed intermediate filaments consisting mainly of vimentin, though to a lesser degree than in the wild-type. In contrast, in wild-type littermates, GFAP was most abundant and nestin occurred at lower levels. Taken together, the present study introduces the novel concepts that GFAP plays an important role in the control of clinical disease associated with formation of a clearly defined edge to the EAE lesion and that GFAP is operative in the regulation of the intermediate filament components in reactive fibrillary astrogliosis.

Mutation in the mismatch repair gene Msh6 causes cancer susceptibility.

Mice carrying a null mutation in the mismatch repair gene Msh6 were generated by gene targeting. Cells that were homozygous for the mutation did not produce any detectable MSH6 protein, and extracts prepared from these cells were defective for repair of single nucleotide mismatches. Repair of 1, 2, and 4 nucleotide insertion/deletion mismatches was unaffected. Mice that were homozygous for the mutation had a reduced life span. The mice developed a spectrum of tumors, the most predominant of which were gastrointestinal tumors and B- as well as T-cell lymphomas. The tumors did not show any microsatellite instability. We conclude that MSH6 mutations, like those in some other members of the family of mismatch repair genes, lead to cancer susceptibility, and germline mutations in this gene may be associated with a cancer predisposition syndrome that does not show microsatellite instability.

Clonal expansion and decreased occurrence of peripheral blood gamma delta T cells of the V delta 2J delta 3 lineage in multiple sclerosis patients.

gamma delta T cells are implicated in autoimmune diseases but their precise function remains unclear. In multiple sclerosis (MS) brain tissue, gamma delta T cells co-localize with heat shock protein (hsp)65+ oligodendrocytes and are oligoclonally restricted in the V delta 2J delta 3 lineage. To investigate the homing properties and the degree of heterogeneity of V delta 2J delta 3+ gamma delta T cells in MS, peripheral blood lymphocytes (PBL) from 34 MS patients, 42 controls (14 autoimmune control patients, 28 healthy volunteers), and 11 lymphatic tissues of MS patients and controls were studied by RT-PCR and sequencing. V delta 2J delta 3 TCR rearrangement was detected in 27 out of 28 healthy controls, and was significantly less frequent in MS patients (24 out of 34) and autoimmune control patients (seven out of 14). It was present only in five out of 11 tissue specimens, none of them from MS patients. Direct sequencing and cloning/automated sequencing of the V delta 2J delta 3 PCR products indicated heterogeneity in healthy controls and oligoclonality in MS patients, but also in autoimmune control patients. Differences between MS patients and healthy controls were more accentuated in exacerbating hospitalized patients than in clinically stable outpatients participating in a clinical trial. Only one V delta 2J delta 3 sequence of a total of 85 different sequences obtained was shared between two MS patients. Taken together, evidence for clonal expansion of V delta 2J delta 3+ gamma delta T cells was found in PBL of MS patients. This T cell subset, previously shown to be present in 100% of chronic-active MS plaques, might home to the CNS in MS, resulting in its under-representation in the blood.

GFAP is necessary for the integrity of CNS white matter architecture and long-term maintenance of myelination.

To investigate the structural role of glial fibrillary acidic protein (GFAP) in vivo, mice carrying a null mutation in GFAP were generated. In 7/14 mutant animals older than 18 months of age, hydrocephalus associated with white matter loss was detected. Mutant mice displayed abnormal myelination including the presence of actively myelinating oligodendrocytes in adults, nonmyelinated axons in optic nerve, and reduced myelin thickness in spinal cord. White matter was poorly vascularized and the blood-brain barrier was structurally and functionally impaired. Astrocytic structure and function were abnormal, consisting of shortened astrocytic cell processes, decreased septation of white matter, and increased CNS extracellular space. Thus, GFAP expression is essential for normal white matter architecture and blood-brain barrier integrity, and its absence leads to late-onset CNS dysmyelination.

On the role of human herpesvirus 6 in viral latency in nervous tissue and in cerebral lymphoma.

Latent infections by human herpesvirus 6 (HHV6) in nervous tissue and its role in human disease are poorly understood. For the present study, an improved PCR method has been applied to brain tissue samples from 5 different brain regions from 20 forensic post-mortem cases without neurologic involvement. Spleen tissue from these cases as well as 5 cerebral lymphoma tissue samples were also examined. HHV6 DNA was detected in 3 of 20 brains. The viral sequences could be amplified from cortical brain tissue from these 3 cases. In one of these cases, HHV6 DNA was detectable in two separate tissue samples. PCR was negative in brain lymphoma and spleen tissue. These findings point toward HHV6 latency in brain tissue and might thus support the reported glial tropism of this virus. No role could be found for HHV6 in the pathogenesis of cerebral lymphoma.

Human herpesvirus 6 polymerase chain reaction findings in human immunodeficiency virus associated neurological disease and multiple sclerosis.

A role for human herpesvirus 6 (HHV6) in the neurological complications associated with infection by human immunodeficiency virus (neuro-AIDS) and during multiple sclerosis (MS) is not known. For the present study, an improved PCR and immunofluorescence serology method were applied to sera and cerebrospinal fluid (CSF) from 27 neuro-AIDS, 36 MS and 24 non-inflammatory control patients. HHV6 DNA was present in 30-40% of the cellular CSF from all groups. In the acellular CSF, HHV6 could be detected in four of 36 MS, 2 of 27 neuro-AIDS and none of the control patients. HHV6 IgG was present in one of 27 neuro-AIDS, and one of 36 MS patients. HHV6 IgG was present in all patients. There was no correlation between clinical features and HHV6 PCR findings or HHV6 antibodies. The significance of the present documentation of HHV6 DNA in the acellular CSF from a minority of MS and neuro-AIDS patients remains to be determined.

Recurrent acute inflammatory demyelinating polyradiculitis after allogeneic bone marrow transplantation.

We report a patient who developed recurrent acute inflammatory demyelinating polyradiculitis (AIDP) receiving immunosuppressive treatment with cyclosporin and prednisone for secondary chronic graft versus host disease (GVHD) following allogeneic bone marrow transplantation (BMT) from an unrelated donor for chronic myelogenous leukemia (CML). After the second relapse of AIDP, cyclosporin was discontinued and a rapid, sustained improvement of his neurological deficits occurred. The role of cyclosporin and systemic CMV infection in the pathogenesis of AIDP in this patient are discussed.