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Neovascularization in corneal stroma in histology and evaluation of its length. (a) CD31 immunostaining was performed in cryosections of the mouse cornea. In WT mouse corneas, formation of CD31-labeled neovascularization (arrows) from the limbal vessels (arrowheads) in the corneal stroma was detected in the peripheral cornea at days 3 and 7. The length of the neovascularization was less in KO mice as compared with WT mice at day 3 and day 7 (b). p∗ < 0.05; n.s.: not significant. Bar, 100 μm.
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To investigate the effects of loss of transient receptor potential vanilloid receptor 1 (TRPV1) on the development of neovascularization in corneal stroma in mice. Blocking TRPV1 receptor did not affect VEGF-dependent neovascularization in cell culture. Lacking TRPV1 inhibited neovascularization in corneal stroma following cauterization. Immunohist...
Citations
... The structure of the corneal stromal layer plays a role in maintaining corneal transparency [12]. Primary cultured human corneal fibroblasts were found to express functional TRPV1 [13,[39][40][41]. Recent studies have shown that keratinocytes also express TRPV1 [42,43]. ...
... The loss or blocking of TRPV1 expression in mice with alkali burns inhibits the production of TGF1 and other proinflammatory factors, resulting in severe and longlasting corneal inflammation and scarring [39]. Inactivation of TRPV1 may therefore be a potential therapeutic target to improve inflammatory/fibrous wound healing [41]. ...
Dry eye disease (DED) is a prevalent ophthalmic ailment with intricate pathogenesis and that occurs primarily due to various factors which affect the ocular surface. DED is characterized by the disruption of tear film homeostasis, inflammatory reaction, and neuroparesthesia. Transient receptor potential vanilloid 1 (TRPV1) is a versatile receptor that can be stimulated by heat, acid, capsaicin (CAP), hyperosmolarity, and numerous inflammatory agents. There is accumulating evidence that implicates TRPV1 in the initiation and progression of DED through its detection of hypertonic conditions and modulation of inflammatory pathways. In this article, we present a comprehensive review of the expression and function of the TRPV1 channel in tissues and cells associated with DED. In addition, we outline the potential mechanisms that implicate TRPV1 in the pathophysiology of DED. The aim of this review is to establish a theoretical basis for TRPV1 as a possible therapeutic target in DED, thereby encouraging further investigations into its role in DED.
... Transforming growth factor β1 (TGFβ1) is one of the most effective cytokines that takes part in the healing process. Tomoyose et al., using a TRPV1 KO mice model, proved that lack of TRPV1 inhibited neovascularization in corneal stroma following cauterization and suppressed vascular endothelial growth factor (VEGF) and TGFβ1 mRNA expression in a mouse cornea [147]. Moreover, Nidegawa-Saitoh et al. demonstrated on TRPV1 −/− mice the implication of TRPV1 in corneal healing [148]. ...
Dry eye disease (DED) is a multifactorial disorder in which the eyes respond to minor stimuli with abnormal sensations, such as dryness, blurring, foreign body sensation, discomfort, irritation, and pain. Corneal pain, as one of DED’s main symptoms, has gained recognition due to its increasing prevalence, morbidity, and the resulting social burden. The cornea is the most innervated tissue in the body, and the maintenance of corneal integrity relies on a rich density of nociceptors, such as polymodal nociceptor neurons, cold thermoreceptor neurons, and mechano-nociceptor neurons. Their sensory responses to different stimulating forces are linked to the specific expression of transient receptor potential (TRP) channels. TRP channels are a group of unique ion channels that play important roles as cellular sensors for various stimuli. These channels are nonselective cation channels with variable Ca2+ selectivity. TRP homologs are a superfamily of 28 different members that are subdivided into 7 different subfamilies based on differences in sequence homology. Many of these subtypes are expressed in the eye on both neuronal and non-neuronal cells, where they affect various stress-induced regulatory responses essential for normal vision maintenance. This article reviews the current knowledge about the expression, function, and regulation of TRPs in ocular surface tissues. We also describe their implication in DED and ocular pain. These findings contribute to evidence suggesting that drug-targeting TRP channels may be of therapeutic benefit in the clinical setting of ocular pain.
... Immunohistochemistry and immunoreactivity detected the active forms of TGFb1 and VEGF in the stroma at the site of cauterization in mice lacking either TRPV1 or TRPA1 functional expression. However, the losses in TGFb1 and VEGF immunoreactivity seemed less marked in mice lacking TRPV1 (74). On the other hand, loss of TRPA1 expression suppressed more markedly both stromal neovascularization and inhibited macrophage infiltration. ...
The maintenance of normal vision is dependent on preserving corneal transparency. For this to occur, this tissue must remain avascular and its stromal architecture needs to be retained. Epithelial transparency is maintained provided the uppermost stratified layers of this tissue are composed of terminally differentiated non-keratinizing cells. In addition, it is essential that the underlying stromal connective tissue remains avascular and scar-free. Keratocytes are the source of fibroblasts that are interspersed within the collagenous framework and the extracellular matrix. In addition, there are sensory nerve fibers whose lineage is possibly either neural crest or mesenchymal. Corneal wound healing studies have been undertaken to delineate the underlying pathogenic responses that result in the development of opacification following chemical injury. An alkali burn is one type of injury that can result in severe and long- lasting losses in ocular transparency. During the subsequent wound healing process, numerous different proinflammatory cytokines and proteolytic enzymes undergo upregulation. Such increases in their expression levels induce maladaptive expression of sustained stromal inflammatory fibrosis, neovascularization, and losses in the smooth optical properties of the corneal outer surface. It is becoming apparent that different transient receptor potential channel (TRP) isoforms are important players in mediating these different events underlying the wound healing process since injury upregulates both their expression levels and functional involvement. In this review, we focus on the involvement of TRPV1, TRPA1 and TRPV4 in mediating some of the responses that underlie the control of anterior ocular tissue homeostasis under normal and pathological conditions. They are expressed on both different cell types throughout this tissue and also on corneal sensory nerve endings. Their roles have been extensively studied as sensors and transducers of environmental stimuli resulting from exposure to intrinsic modulators and extrinsic ligands. These triggers include alteration of the ambient temperature and mechanical stress, etc., that can induce pathophysiological responses underlying losses in tissue transparency activated by wound healing in mice losses in tissue transparency. In this article, experimental findings are reviewed about the role of injury-induced TRP channel activation in mediating inflammatory fibrotic responses during wound healing in mice.
... TRPV1 was shown to co-localize with the VEGFR in corneal fibroblasts [40]. Moreover, VEGF-dependent Ca 2+ influxes mediated by TRPV1 have been observed in pterygial cells (hPtEC), conjunctival epithelial cells (HCjEC), corneal keratinocytes and uveal melanoma cells [40][41][42][43]. Interestingly, activation of the cold-sensitive ion channel TRPM8 by 3-Iodothyronamine (3-T 1 AM) impeded these VEGF-mediated Ca 2+ surges [42]. ...
Calcium (Ca²⁺) is one of the most universal secondary messengers, owing its success to the immense concentration gradient across the plasma membrane. Dysregulation of Ca²⁺ homeostasis can result in severe cell dysfunction, thereby initiating several pathologies like tumorigenesis and fibrosis. Transient receptor potential (TRP) channels represent a superfamily of Ca²⁺-permeable ion channels that convey diverse physical and chemical stimuli into a physiological signal. Their broad expression pattern and gating promiscuity support their potential involvement in the cellular response to an altering environment. Growth factors (GF) are essential biochemical messengers that contribute to these environmental changes. Since Ca²⁺ is essential in GF signaling, altering TRP channel expression or function could be a valid strategy for GF to exert their effect onto their target. In this review, a comprehensive understanding of the current knowledge regarding the activation and/or modulation of TRP channels by GF is presented.
... Suppression of in vivo neovascularization was demonstrated by Tomoyose et al., in a Trpv1 gene knock out mice. After the induction of a cauterization injury at the central cornea, they showed that, in the absence of TRPV1, stromal neovascularization was inhibited, probably secondary to lower levels of both TGFβ-1 and vascular endothelial growth factor [43]. ...
The endocannabinoid system (ECS) is a complex regulatory system, highly conserved among vertebrates. It has been widely described in nearly all human tissues. In the conjunctiva and cornea, the ECS is believed to play a pivotal role in the modulation of the local inflammatory state as well as in the regulation of tissue repair and fibrosis, neo-angiogenesis and pain perception. This review aims to summarize all the available data on ECS expression and its function in ocular surface structures to provide a specific insight concerning its modulation in dry eye disease, and to propose directions for future research.
... Two recently described mechanisms help to explain this response: The first one involves activation of the transient receptor potential vanilloid 1 (TRPV1) in epithelial cell cultures by osmolarity, temperature, and chemical challenges, which induce secretion of inflammatory cytokines (IL-6 and IL-8) through the Mitogen-Activated Protein Kinase signaling pathway, but also induces corneal epithelial cell migration through epidermal growth factor receptor transactivation (72)(73)(74)(75) . In addition, the activation of TRPV channels in keratocytes present in a deeper corneal layer (the stroma) promotes the secretion of transforming growth factor-beta and induces the production of collagen, which is responsible for stromal scar formation (70,(76)(77)(78)(79) . Taken together, these findings indicate that superficial damage to the epithelium prompts fast wound healing and the preservation of transparency despite a painful and inflammatory process and deeper injuries to the corneal stroma, which destroys the nerve network and jeopardizes the eye globe integrity or triggers a mechanism of new vessel growth, and strengthens the globe wall (corneal stroma). ...
The burden of corneal blindness and visual deficiency can be felt worldwide. Its association with several endemic diseases such as childhood blindness, trauma, infectious keratitis (including variants caused by herpes, hanseniasis, and fungi), vitamin A deficiency, diabetes mellitus, and other dry eye syndromes reflects its poorly understood underlying mechanisms and suggests that the actual frequency of the disease is underestimated. The low effectiveness of preventive and therapeutic strategies against corneal scarring or deformity predicts a high frequency of patients with corneal blindness in the future. Corneal blindness is associated with environmental factors and socioeconomic limitations that restrain health assistance and maintain a modest efficiency of the current therapeutic strategies for resolving corneal diseases in large-scale programs. We present here a critical review of the concepts associated with corneal blindness that need to be considered when planning strategies to prevent and treat corneal blindness worldwide (to be able to leave Plato’s cave, where corneal blindness is encaged.
... In agreement with this observation, blocking TRPV1 with capsazepine and A784168 inhibited neovascularization in a mouse model of oxygen-induced retinopathy [89]. Moreover, while pharmacological inhibition of TRPV1 with the selective antagonist SBB366791 did not prevent VEGF-induced tube formation in HUVEC, genetic deletion of TRPV1 abrogated stromal neovascularization in an in vivo cornea after a cauterization injury at the central cornea [158]. Consistently, the expression of pro-inflammatory/angiogenic factors (i.e., transforming growth factor β1 (TGFβ1) and VEGF) was suppressed in TRPV1-knockout (TRPV1 −/− ) mice [158]. ...
... Moreover, while pharmacological inhibition of TRPV1 with the selective antagonist SBB366791 did not prevent VEGF-induced tube formation in HUVEC, genetic deletion of TRPV1 abrogated stromal neovascularization in an in vivo cornea after a cauterization injury at the central cornea [158]. Consistently, the expression of pro-inflammatory/angiogenic factors (i.e., transforming growth factor β1 (TGFβ1) and VEGF) was suppressed in TRPV1-knockout (TRPV1 −/− ) mice [158]. Thus, targeting TRPV1 could provide an effective strategy to treat severe ischemic disorders. ...
Therapeutic angiogenesis represents an emerging strategy to treat ischemic diseases by stimulating blood vessel growth to rescue local blood perfusion. Therefore, injured microvasculature may be repaired by stimulating resident endothelial cells or circulating endothelial colony forming cells (ECFCs) or by autologous cell-based therapy. Endothelial Ca2+ signals represent a crucial player in angiogenesis and vasculogenesis; indeed, several angiogenic stimuli induce neovessel formation through an increase in intracellular Ca2+ concentration. Several members of the Transient Receptor Potential (TRP) channel superfamily are expressed and mediate Ca2+-dependent functions in vascular endothelial cells and in ECFCs, the only known truly endothelial precursor. TRP Vanilloid 1 (TRPV1), a polymodal cation channel, is emerging as an important player in endothelial cell migration, proliferation, and tubulogenesis, through the integration of several chemical stimuli. Herein, we first summarize TRPV1 structure and gating mechanisms. Next, we illustrate the physiological roles of TRPV1 in vascular endothelium, focusing our attention on how endothelial TRPV1 promotes angiogenesis. In particular, we describe a recent strategy to stimulate TRPV1-mediated pro-angiogenic activity in ECFCs, in the presence of a photosensitive conjugated polymer. Taken together, these observations suggest that TRPV1 represents a useful target in the treatment of ischemic diseases.
... Even though the cognate VEGF receptor (VEGFR) and TRPV1 are coexpressed on corneal fibroblasts (53), it is unclear if the Ca 2+ transients mediating VEGF-induced stromal angiogenesis stem from VEGFR-mediated activation of TRPV1 (13,54,55). A clear indication that neovascularization and the increased expression of VEGF following corneal chemical injury may depend on TRPV1 expression, is that those tissue responses were markedly attenuated in TRPV1 knockout mice (53). ...
... Even though the cognate VEGF receptor (VEGFR) and TRPV1 are coexpressed on corneal fibroblasts (53), it is unclear if the Ca 2+ transients mediating VEGF-induced stromal angiogenesis stem from VEGFR-mediated activation of TRPV1 (13,54,55). A clear indication that neovascularization and the increased expression of VEGF following corneal chemical injury may depend on TRPV1 expression, is that those tissue responses were markedly attenuated in TRPV1 knockout mice (53). Besides neovascularization, VEGF upregulation contributes to increased endothelial cell proliferation and migration in a bovine wound healing model (56,57). ...
This study was undertaken to determine if crosstalk among the transient receptor potential (TRP) melastatin 8 (TRPM8), TRP vanilloid 1 (TRPV1), and vascular endothelial growth factor (VEGF) receptor triad modulates VEGF-induced Ca 2+ signaling in human corneal keratocytes. Using RT-PCR, qPCR and immunohistochemistry, we determined TRPV1 and TRPM8 gene and protein coexpression in a human corneal keratocyte cell line (HCK) and human corneal cross sections. Fluorescence Ca 2+ imaging using both a photomultiplier and a single cell digital imaging system as well as planar patch-clamping measured relative intracellular Ca 2+ levels and underlying whole-cell currents. The TRPV1 agonist capsaicin increased both intracellular Ca 2+ levels and whole-cell currents, while the antagonist capsazepine (CPZ) inhibited them. VEGF-induced Ca 2+ transients and rises in whole-cell currents were suppressed by CPZ, whereas a selective TRPM8 antagonist, AMTB, increased VEGF signaling. In contrast, an endogenous thyroid hormone-derived metabolite 3-Iodothyronamine (3-T 1 AM) suppressed increases in the VEGF-induced current. The TRPM8 agonist menthol increased the currents, while AMTB suppressed this response. The VEGF-induced increases in Ca 2+ influx and their underlying ionic currents stem from crosstalk between VEGFR and TRPV1, which can be impeded by 3-T 1 AM-induced TRPM8 activation. Such suppression in turn blocks VEGF-induced TRPV1 activation. Therefore, crosstalk between TRPM8 and TRPV1 inhibits VEGFR-induced activation of TRPV1.
... Expression of active form of TGFβ1 in an incision-injured cornea was evaluated by performing immunohistochemistry with a selective anti TGFβ1 antibody (Saika et al. 2005a;Okada et al. 2011;Flanders et al. 1991;Flanders et al. 1989;Thompson et al. 1989). Our previous study showed that this form of TGFβ1 is not present in a healthy mouse cornea (Tomoyose et al. 2015;Saika. 2004). ...
The present study attempts to elucidate the role of TRPV1 cation channel receptor on primary repair in an incision-wounded mouse cornea in vivo. Previous study revealed that blocking TRPV1 suppressed myofibroblast formation and expression of transforming growth factor β1 (TGFβ1) in cultured keratocytes or ocular fibroblasts. Male C57BL/6 (wild-type; WT) mice and male C57BL/6 Trpv1-null (KO) mice incurred a full-thickness incision injury (1.8 mm in length, limbus to limbus) in the central cornea of one eye with a surgical blade under general and topical anesthesia. The injury was not sutured. On days 0, 5, and 10, the eyes were enucleated, processed for histology, immunohistochemistry, and real-time RT-PCR gene expression analysis to evaluate the effects of the loss of TRPV1 on primary healing. Electron microscopy observation was also performed to know the effect of the loss of TRPV1 on ultrastructure of keratocytes. The results showed that the loss of Trpv1 gene delayed closure of corneal stromal incision with hindered myofibroblast transdifferentiation along with declines in the expression of collagen Ia1 and TGFβ1. Inflammatory cell infiltration was not affected by the loss of TRPV1. Ultrastructurally endoplasmic reticulum of TRPV1-null keratocytes was more extensively dilated as compared with WT keratocytes, suggesting an impairment of protein secretion by TRPV1-gene knockout. These results indicate that injury-related TRPV1 signal is involved in healing of stromal incision injury in a mouse cornea by selectively stimulating TGFβ-induced granulation tissue formation.
Corneal injury-associated inflammation could induce inward-growing neovascularization from the periphery of the tissue. Such neovascularization could cause stromal opacification and curvature disturbance, and both potentially impairs visual function. We determined the effects of the loss of transient receptor potential vanilloid 4 (TRPV4) expression on the development of neovascularization in the corneal stroma in mice by producing a cauterization injury in the central area of the cornea. New vessels are immunohistochemically labeled with anti-TRPV4 antibodies. TRPV4 gene knockout suppressed the growth of such CD31-labelled neovascularization in association with suppression of infiltration of macrophages and tissue mRNA expression of vascular endothelial cell growth factor-A level. Treatment of cultured vascular endothelial cells with supplementation of HC-067047 (0.1 μM, 1 μM, or 10 μM), a TRPV4 antagonist, attenuated the formation of a tube-like structure with sulforaphane (15 μM, for positive control) that modeled new vessel formation. In conclusion, the TRPV4 signal is involved in injury-induced macrophagic inflammation and neovascularization activity by vascular endothelial cells in a mouse corneal stroma. TRPV4 could be a therapeutic target to prevent unfavorable post-injury neovascularization in the cornea.
