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Hepatocyte growth factor mimetic protects lateral line hair cells from aminoglycoside exposure.

Uribe PM, Kawas LH, Harding JW, Coffin AB - Front Cell Neurosci (2015)

Bottom Line: We found that a Dihexa concentration of 1 μM confers optimal protection from acute treatment with either ototoxin.Pretreatment with Dihexa does not affect the amount of fluorescently tagged gentamicin that enters hair cells, indicating that Dihexa's protection is likely mediated by intracellular events and not by inhibiting aminoglycoside entry.Our data suggest that Dihexa confers protection of hair cells through an HGF-mediated mechanism and that Dihexa holds clinical potential for mitigating chemical ototoxicity.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrative Physiology and Neuroscience, Washington State University Pullman, WA, USA.

ABSTRACT
Loss of sensory hair cells from exposure to certain licit drugs (e.g., aminoglycoside antibiotics, platinum-based chemotherapy agents) can result in permanent hearing loss. Here we ask if allosteric activation of the hepatocyte growth factor (HGF) cascade via Dihexa, a small molecule drug candidate, can protect hair cells from aminoglycoside toxicity. Unlike native HGF, Dihexa is chemically stable and blood-brain barrier permeable. As a synthetic HGF mimetic, it forms a functional ligand by dimerizing with endogenous HGF to activate the HGF receptor and downstream signaling cascades. To evaluate Dihexa as a potential hair cell protectant, we used the larval zebrafish lateral line, which possesses hair cells that are homologous to mammalian inner ear hair cells and show similar responses to toxins. A dose-response relationship for Dihexa protection was established using two ototoxins, neomycin and gentamicin. We found that a Dihexa concentration of 1 μM confers optimal protection from acute treatment with either ototoxin. Pretreatment with Dihexa does not affect the amount of fluorescently tagged gentamicin that enters hair cells, indicating that Dihexa's protection is likely mediated by intracellular events and not by inhibiting aminoglycoside entry. Dihexa-mediated protection is attenuated by co-treatment with the HGF antagonist 6-AH, further evidence that HGF activation is a component of the observed protection. Additionally, Dihexa's robust protection is partially attenuated by co-treatment with inhibitors of the downstream HGF targets Akt, TOR and MEK. Addition of an amino group to the N-terminal of Dihexa also attenuates the protective response, suggesting that even small substitutions greatly alter the specificity of Dihexa for its target. Our data suggest that Dihexa confers protection of hair cells through an HGF-mediated mechanism and that Dihexa holds clinical potential for mitigating chemical ototoxicity.

No MeSH data available.


Related in: MedlinePlus

Dihexa protects hair cells from acute aminoglycoside treatment. (A) Dihexa confers dose-dependent protection from 200 μM neomycin with two peaks of protection at 10−6 M (1 μM) and 10−13 M (100 fM), with six concentrations of Dihexa providing significant protection (One-way ANOVA; F(9,70) = 20.53 p < 0.001). Control fish exposed to embryo medium (EM) only (inset top) and 1 μM Dihexa plus 200 μM neomycin (inset bottom) display bright DASPEI fluorescence while fish treated with 200 μM neomycin alone (inset middle) display dim to absent DASPEI fluorescence. Scale bar represents 250 μm and applies to all three images in (A). (B) 1 μM Dihexa provides robust protection from aminoglycoside treatment across multiple concentrations of neomycin (Two-way ANOVA; Dihexa: F(1,63) = 155.8 p < 0.001). (C) 1 μM Dihexa confers significant protection from acute gentamicin exposure across all concentrations of gentamicin tested (Two-way ANOVA; Dihexa: F(1,71) = 58.42 p < 0.001). (D) 1 μM Dihexa does not provide protection against chronic gentamicin exposure (Two-way ANOVA; Dihexa: F(1,81) = 1.458 p > 0.05). Asterisks indicate significant difference from aminoglycoside only control (*p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001). N = 7–11 animals per treatment, error bars represent ± s.e.m.
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Figure 2: Dihexa protects hair cells from acute aminoglycoside treatment. (A) Dihexa confers dose-dependent protection from 200 μM neomycin with two peaks of protection at 10−6 M (1 μM) and 10−13 M (100 fM), with six concentrations of Dihexa providing significant protection (One-way ANOVA; F(9,70) = 20.53 p < 0.001). Control fish exposed to embryo medium (EM) only (inset top) and 1 μM Dihexa plus 200 μM neomycin (inset bottom) display bright DASPEI fluorescence while fish treated with 200 μM neomycin alone (inset middle) display dim to absent DASPEI fluorescence. Scale bar represents 250 μm and applies to all three images in (A). (B) 1 μM Dihexa provides robust protection from aminoglycoside treatment across multiple concentrations of neomycin (Two-way ANOVA; Dihexa: F(1,63) = 155.8 p < 0.001). (C) 1 μM Dihexa confers significant protection from acute gentamicin exposure across all concentrations of gentamicin tested (Two-way ANOVA; Dihexa: F(1,71) = 58.42 p < 0.001). (D) 1 μM Dihexa does not provide protection against chronic gentamicin exposure (Two-way ANOVA; Dihexa: F(1,81) = 1.458 p > 0.05). Asterisks indicate significant difference from aminoglycoside only control (*p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001). N = 7–11 animals per treatment, error bars represent ± s.e.m.

Mentions: Treatment with Dihexa confers protection from neomycin in a dose-dependent manner (Figure 2A). Untreated controls labeled with the vital dye DASPEI display bright neuromast fluorescence (Figure 2A inset). In contrast, animals treated with 200 μM neomycin for thirty minutes exhibit dim or completely absent fluorescence. The greatest protection was found at 10−6 M (1 μM), where only a minor decrease from control hair cell survival scores was observed. An additional peak of protection was seen at 10−13 M (100 fM) but was inconsistent across experiments, so all additional experiments were run with 10−6 M (1 μM) Dihexa. Figure 2B shows that 10−6 M (1 μM) Dihexa significantly protects hair cells from a range of neomycin concentrations. Additionally, there was no observed toxicity of Dihexa treatment alone. Dihexa also protects hair cells from variable concentrations of acute gentamicin (Figure 2C).


Hepatocyte growth factor mimetic protects lateral line hair cells from aminoglycoside exposure.

Uribe PM, Kawas LH, Harding JW, Coffin AB - Front Cell Neurosci (2015)

Dihexa protects hair cells from acute aminoglycoside treatment. (A) Dihexa confers dose-dependent protection from 200 μM neomycin with two peaks of protection at 10−6 M (1 μM) and 10−13 M (100 fM), with six concentrations of Dihexa providing significant protection (One-way ANOVA; F(9,70) = 20.53 p < 0.001). Control fish exposed to embryo medium (EM) only (inset top) and 1 μM Dihexa plus 200 μM neomycin (inset bottom) display bright DASPEI fluorescence while fish treated with 200 μM neomycin alone (inset middle) display dim to absent DASPEI fluorescence. Scale bar represents 250 μm and applies to all three images in (A). (B) 1 μM Dihexa provides robust protection from aminoglycoside treatment across multiple concentrations of neomycin (Two-way ANOVA; Dihexa: F(1,63) = 155.8 p < 0.001). (C) 1 μM Dihexa confers significant protection from acute gentamicin exposure across all concentrations of gentamicin tested (Two-way ANOVA; Dihexa: F(1,71) = 58.42 p < 0.001). (D) 1 μM Dihexa does not provide protection against chronic gentamicin exposure (Two-way ANOVA; Dihexa: F(1,81) = 1.458 p > 0.05). Asterisks indicate significant difference from aminoglycoside only control (*p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001). N = 7–11 animals per treatment, error bars represent ± s.e.m.
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Figure 2: Dihexa protects hair cells from acute aminoglycoside treatment. (A) Dihexa confers dose-dependent protection from 200 μM neomycin with two peaks of protection at 10−6 M (1 μM) and 10−13 M (100 fM), with six concentrations of Dihexa providing significant protection (One-way ANOVA; F(9,70) = 20.53 p < 0.001). Control fish exposed to embryo medium (EM) only (inset top) and 1 μM Dihexa plus 200 μM neomycin (inset bottom) display bright DASPEI fluorescence while fish treated with 200 μM neomycin alone (inset middle) display dim to absent DASPEI fluorescence. Scale bar represents 250 μm and applies to all three images in (A). (B) 1 μM Dihexa provides robust protection from aminoglycoside treatment across multiple concentrations of neomycin (Two-way ANOVA; Dihexa: F(1,63) = 155.8 p < 0.001). (C) 1 μM Dihexa confers significant protection from acute gentamicin exposure across all concentrations of gentamicin tested (Two-way ANOVA; Dihexa: F(1,71) = 58.42 p < 0.001). (D) 1 μM Dihexa does not provide protection against chronic gentamicin exposure (Two-way ANOVA; Dihexa: F(1,81) = 1.458 p > 0.05). Asterisks indicate significant difference from aminoglycoside only control (*p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001). N = 7–11 animals per treatment, error bars represent ± s.e.m.
Mentions: Treatment with Dihexa confers protection from neomycin in a dose-dependent manner (Figure 2A). Untreated controls labeled with the vital dye DASPEI display bright neuromast fluorescence (Figure 2A inset). In contrast, animals treated with 200 μM neomycin for thirty minutes exhibit dim or completely absent fluorescence. The greatest protection was found at 10−6 M (1 μM), where only a minor decrease from control hair cell survival scores was observed. An additional peak of protection was seen at 10−13 M (100 fM) but was inconsistent across experiments, so all additional experiments were run with 10−6 M (1 μM) Dihexa. Figure 2B shows that 10−6 M (1 μM) Dihexa significantly protects hair cells from a range of neomycin concentrations. Additionally, there was no observed toxicity of Dihexa treatment alone. Dihexa also protects hair cells from variable concentrations of acute gentamicin (Figure 2C).

Bottom Line: We found that a Dihexa concentration of 1 μM confers optimal protection from acute treatment with either ototoxin.Pretreatment with Dihexa does not affect the amount of fluorescently tagged gentamicin that enters hair cells, indicating that Dihexa's protection is likely mediated by intracellular events and not by inhibiting aminoglycoside entry.Our data suggest that Dihexa confers protection of hair cells through an HGF-mediated mechanism and that Dihexa holds clinical potential for mitigating chemical ototoxicity.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrative Physiology and Neuroscience, Washington State University Pullman, WA, USA.

ABSTRACT
Loss of sensory hair cells from exposure to certain licit drugs (e.g., aminoglycoside antibiotics, platinum-based chemotherapy agents) can result in permanent hearing loss. Here we ask if allosteric activation of the hepatocyte growth factor (HGF) cascade via Dihexa, a small molecule drug candidate, can protect hair cells from aminoglycoside toxicity. Unlike native HGF, Dihexa is chemically stable and blood-brain barrier permeable. As a synthetic HGF mimetic, it forms a functional ligand by dimerizing with endogenous HGF to activate the HGF receptor and downstream signaling cascades. To evaluate Dihexa as a potential hair cell protectant, we used the larval zebrafish lateral line, which possesses hair cells that are homologous to mammalian inner ear hair cells and show similar responses to toxins. A dose-response relationship for Dihexa protection was established using two ototoxins, neomycin and gentamicin. We found that a Dihexa concentration of 1 μM confers optimal protection from acute treatment with either ototoxin. Pretreatment with Dihexa does not affect the amount of fluorescently tagged gentamicin that enters hair cells, indicating that Dihexa's protection is likely mediated by intracellular events and not by inhibiting aminoglycoside entry. Dihexa-mediated protection is attenuated by co-treatment with the HGF antagonist 6-AH, further evidence that HGF activation is a component of the observed protection. Additionally, Dihexa's robust protection is partially attenuated by co-treatment with inhibitors of the downstream HGF targets Akt, TOR and MEK. Addition of an amino group to the N-terminal of Dihexa also attenuates the protective response, suggesting that even small substitutions greatly alter the specificity of Dihexa for its target. Our data suggest that Dihexa confers protection of hair cells through an HGF-mediated mechanism and that Dihexa holds clinical potential for mitigating chemical ototoxicity.

No MeSH data available.


Related in: MedlinePlus