Journal: Communications Biology

Article Title: Topical application of calcitonin gene-related peptide as a regenerative, antifibrotic, and immunomodulatory therapy for corneal injury

doi: 10.1038/s42003-024-05934-y

Figure Lengend Snippet: a Schematic figure showing the method of mechanical injury. The epithelium and superficial stroma were removed using Algerbrush-II. b Assessment of CGRP protein levels using ELISA showed significantly reduced expression following corneal injury on days 1, 3, and 7. c The gene expression levels of the CGRP receptors, calcitonin receptor-like receptor (CLR), receptor activity-modifying protein (RAMP) 1, and RAMP2. RT-PCR showed upregulation of the RAMP1 on day 3 post-injury and returned to normal levels by day 7. ( n = 3–5 per group). The data were presented as mean ± standard error of mean (SEM) and comparison is determined by a one-way ANOVA test with pairwise comparison. * p < 0.05, ** p < 0.01. (Legend: Circle = naive, Inverted Triangle = Day 1, Square = Day 3, Upright Triangle = Day 7). Figure 1a was created with BioRender.com .

Article Snippet: We prepared topical eye drops from CGRP lyophilized powder (Bachem, Bubendorf, Switzerland, Cat. No. 4025897).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Gene Expression, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Comparison

Journal: Communications Biology

Article Title: Topical application of calcitonin gene-related peptide as a regenerative, antifibrotic, and immunomodulatory therapy for corneal injury

doi: 10.1038/s42003-024-05934-y

Figure Lengend Snippet: a Schematic figure showing the timeline of treatment and clinical examination following injury. b Corneal fluorescein staining was performed to compare the size of epithelial defects in CGRP- and PBS-treated mice at different time points post-injury. c The assessment of the wound area shows that CGRP treatment resulted in a significantly smaller wound area compared to PBS-treated controls across all time points from 24 h to 6 days ( n = 9 per group). d Representative slit lamp photographs of PBS and CGRP-treated eyes up to 14 days after injury. The corneas of the PBS-treated controls showed progressive stromal opacification, whereas CGRP treatment showed significantly lower corneal opacity. e The scoring of corneal opacity was performed in a blinded fashion and showed a significantly lower score in CGRP-treated mice ( n = 12 per group). The data were represented as mean ± SEM. The statistical significance was determined by unpaired t -test, * p < 0.05, ** p < 0.01. (Legend: Pink Square = PBS, Blue Triangle = CGRP). Figure 2a was created with BioRender.com .

Article Snippet: We prepared topical eye drops from CGRP lyophilized powder (Bachem, Bubendorf, Switzerland, Cat. No. 4025897).

Techniques: Staining

Journal: Communications Biology

Article Title: Topical application of calcitonin gene-related peptide as a regenerative, antifibrotic, and immunomodulatory therapy for corneal injury

doi: 10.1038/s42003-024-05934-y

Figure Lengend Snippet: a Representative AS-OCT images showed a significant increase in central corneal thickness (CCT) and stromal hyperreflectivity in PBS-treated mice, whereas they were comparable to naïve mice in the treated group on days 7 and 14 post-injury. b CGRP treatment resulted in significantly lower CCT compared to PBS-treated controls on days 7 and 14 ( n = 10 per group). c Representative IVCM images of the corneal epithelium, stroma, and endothelium. The analysis of IVCM images showed that CGRP treatment resulted in decreased stromal hyperreflectivity ( d ), scar depth ( e ), endothelial cell loss ( f ), and endothelial coefficient of variation ( g ) compared to PBS-treated mice at day 14 post-injury. h The histological analysis by hematoxylin and eosin staining showed reduced corneal thickness and inflammatory cell infiltration following CGRP compared to the PBS treatment (scale bar = 200 µm). d n = 5, e – g n = 6 per group). The data were represented as mean ± SEM, and the statistical significance was determined by one-way ANOVA ( b , d , f , g ) and unpaired t -test ( e ), * p < 0.05, ** p < 0.01, *** P < 0.001, **** P < 0.0001. (Legend: Black Circle = Naïve, Pink Square = PBS, Blue Triangle = CGRP).

Article Snippet: We prepared topical eye drops from CGRP lyophilized powder (Bachem, Bubendorf, Switzerland, Cat. No. 4025897).

Techniques: Staining

Journal: Communications Biology

Article Title: Topical application of calcitonin gene-related peptide as a regenerative, antifibrotic, and immunomodulatory therapy for corneal injury

doi: 10.1038/s42003-024-05934-y

Figure Lengend Snippet: a Human CEC were cultured and CGRP (1 μM for 24 h) led to an increased frequency of proliferating Ki67 + cells (arrowheads, green) (scale bar = 20 µm). b CGRP resulted in an increase in the Ki67 + cells in a dose-dependent manner ( n = 4 per group). c CEC were cultured to a monolayer and a linear scratch was created. d CGRP promoted CEC migration in a dose-dependent manner ( n = 3 per group). e RT-PCR data showed significantly increased laminin 332 expression in CEC by CGRP ( n = 3 per group). f CGRP (1 μM for 1 h) increased the phosphorylation of ERK (p-ERK antibody, red, (scale = 20 µm). g Western blot analysis confirmed the increased p-ERK levels with CGRP treatment. h Mouse corneas obtained (on day 4) from the CGRP-treated mice showed more Ki67 (green) staining in the epithelium compared to PBS-treated controls, (scale bar = 100 µm). i A higher level of laminin immunostaining (red) was also observed in the cornea derived from CGRP-treated mice compared to the PBS-treated controls (scale bar = 20 µm). The data were presented as mean ± SEM, and the statistical significance was determined by one-way ANOVA with pairwise comparison, * p < 0.05, ** p < 0.01, **** P < 0.0001.

Article Snippet: We prepared topical eye drops from CGRP lyophilized powder (Bachem, Bubendorf, Switzerland, Cat. No. 4025897).

Techniques: Cell Culture, Migration, Reverse Transcription Polymerase Chain Reaction, Expressing, Phospho-proteomics, Western Blot, Staining, Immunostaining, Derivative Assay, Comparison

Journal: Communications Biology

Article Title: Topical application of calcitonin gene-related peptide as a regenerative, antifibrotic, and immunomodulatory therapy for corneal injury

doi: 10.1038/s42003-024-05934-y

Figure Lengend Snippet: Murine corneal fibroblasts were cultured in a medium supplemented with 10 ng/ml TGF-β1 for 24 h, in the presence or absence of 1 µM CGRP. CGRP significantly decreased the TGF-β1-mediated expression of α-SMA assessed via RT-PCR ( a ), western blot ( b ), and immunostaining ( c ) in vitro ( n = 3 per group). Corneas derived from CGRP-treated mice showed significantly lower expression of TGF-β1 compared to PBS-treated controls in vivo in RT-PCR ( d , n = 4 per group), western blot ( e ), and immunostaining ( f , scale = 50 μm). Similarly, α-SMA levels in RT-PCR ( g , n = 4 per group), western blot ( h ), and immunostaining ( f ) were significantly elevated post-injury and decreased by CGRP treatment in vivo. The data were presented as mean ± SEM, and the statistical significance was determined by one-way ANOVA with pairwise comparison. * p < 0.05, ** p < 0.01, *** P < 0.001, **** P < 0.0001. (Legend in d , g : Black Circle = Naïve, Pink Square = PBS, Blue Triangle = CGRP).

Article Snippet: We prepared topical eye drops from CGRP lyophilized powder (Bachem, Bubendorf, Switzerland, Cat. No. 4025897).

Techniques: Cell Culture, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunostaining, In Vitro, Derivative Assay, In Vivo, Comparison

Journal: Communications Biology

Article Title: Topical application of calcitonin gene-related peptide as a regenerative, antifibrotic, and immunomodulatory therapy for corneal injury

doi: 10.1038/s42003-024-05934-y

Figure Lengend Snippet: a Mouse corneas were collected on Day 14 post-injury and CGRP treatment led to preserved zonula occludens-1 (ZO-1) and Na + /K + ATPase staining, compared to the PBS-treated controls (scale = 20 μm). Analysis of immunohistochemical images showed higher endothelial cell density ( b ) and lower coefficient of variation ( c ) in vivo ( n = 6 per group). d RT-PCR evaluation of mouse corneas showed higher gene expression of α1 and α3 isoforms of Na + /K + ATPase in CGRP-treated mice compared to controls ( n = 3 per group). The data were presented as mean ± SEM, and the statistical significance was determined by one-way ANOVA with pairwise comparison ( b – d ), * p < 0.05, ** p < 0.01, *** P < 0.001. (Legend: Black Circle = Naïve, Pink Square = PBS, Blue Triangle = CGRP).

Article Snippet: We prepared topical eye drops from CGRP lyophilized powder (Bachem, Bubendorf, Switzerland, Cat. No. 4025897).

Techniques: Staining, Immunohistochemical staining, In Vivo, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Comparison

Journal: Communications Biology

Article Title: Topical application of calcitonin gene-related peptide as a regenerative, antifibrotic, and immunomodulatory therapy for corneal injury

doi: 10.1038/s42003-024-05934-y

Figure Lengend Snippet: a Lower frequencies of CD45 + cells in corneas derived from CGRP-treated mice compared to controls on day 1 and day 3 post-injury, ( n = 5 per group). b Representative flow cytometry plots show that CGRP treatment suppressed the infiltration of CD45 + into the cornea on day 3 post-injury compared to PBS-treated controls. The RT-PCR analysis showed that the CGRP treatment resulted in significantly lower expression of CXCL1 ( c ), IL-1β, TNF, and MMP-9 ( d ) on day 3 post-injury ( n = 3 per group). e Representative flow cytometry plot shows the frequencies of neutrophils (CD11b + Ly6G + ) and macrophage (CD11b + Ly6G - ) in the mice cornea. f The frequencies of neutrophils were significantly lower in corneas derived from CGRP-treated mice compared to controls, and the frequencies of macrophages were comparable in the two groups ( n = 3 per naive group, n = 4 per PBS and CGRP group). g CGRP treatment resulted in significant suppression of MCH-II, CCR2, and iNOS expression in CGRP-treated mice compared to the controls ( n = 3 per group). The data were presented as mean ± SEM, and the statistical significance was determined by one-way ANOVA with pairwise comparison ( a – f ) and unpaired t -test ( g ), * p < 0.05, ** p < 0.01, *** P < 0.00, **** P < 0.0001. (Legend: Black Circle = Naïve, Pink Square = PBS, Blue Triangle = CGRP).

Article Snippet: We prepared topical eye drops from CGRP lyophilized powder (Bachem, Bubendorf, Switzerland, Cat. No. 4025897).

Techniques: Derivative Assay, Flow Cytometry, Reverse Transcription Polymerase Chain Reaction, Expressing, Comparison

Journal: Communications Biology

Article Title: Topical application of calcitonin gene-related peptide as a regenerative, antifibrotic, and immunomodulatory therapy for corneal injury

doi: 10.1038/s42003-024-05934-y

Figure Lengend Snippet: Injury leads to nerve damage and a decrease in CGRP level in the cornea. Topical application of CGRP promotes corneal epithelial cell regeneration and restores the epithelial basement membrane, thus reducing the release of pro-inflammatory and pro-fibrotic mediators including TNF-α, TGF-β, IL-1, and CXCL1 into the stroma. This leads to reduced keratocyte activation and stromal fibrosis. In addition, CGRP reduces neutrophil infiltration, macrophage maturation, and the production of inflammatory cytokines. It reduces corneal endothelial cell loss and maintains its pump function. Clinically, topical application of CGRP as an eye drop accelerates epithelial closure, preserves transparency, and prevents scar formation and edema after corneal injury. The figure was created with BioRender.com.

Article Snippet: We prepared topical eye drops from CGRP lyophilized powder (Bachem, Bubendorf, Switzerland, Cat. No. 4025897).

Techniques: Membrane, Activation Assay

Journal: Investigative Ophthalmology & Visual Science

Article Title: CGRP/TSP1 Signaling Dampens Corneal Inflammation and Fibrosis by Targeting A2M During Corneal Stromal Wound Healing

doi: 10.1167/iovs.67.1.10

Figure Lengend Snippet: CGRP–CALCRL/RAMP1 signaling acts upstream of TSP1 and A2M in vitro. ( A , B ) HTKs were treated with rhTSP1 (0, 1, and 10 µg/mL) for 24 hours, followed by quantitative analysis of A2M expression levels using both quantitative PCR ( A ) and western blot ( B ). ( C ) Representative image of CGRP receptors in HTKs. Scale bars : 25 µm. ( D ) Schematic diagram illustrating that BIBN4096 is a specific antagonist of the CALCRL/RAMP1 receptor complex. ( E ) HTKs were treated with CGRP (200 nM) in the presence or absence of BIBN4096 (1 µM) for 12 hours or 24 hours. Quantitative PCR analysis of THBS1 and A2M mRNA levels in HTKs at the indicated time points ( n = 6). ( F , G ) Western blot analysis of TSP1 and A2M protein levels in HTKs at the indicated time points ( n > 3). Human β-actin served as the internal loading control. h, hours. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: CGRP lyophilized powder (015-09; Phoenix Pharmaceuticals, Inc., Burlingame, CA, USA) was reconstituted in phosphate-buffered saline (PBS) to a final concentration of 40μg/ml.

Techniques: In Vitro, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: CGRP/TSP1 Signaling Dampens Corneal Inflammation and Fibrosis by Targeting A2M During Corneal Stromal Wound Healing

doi: 10.1167/iovs.67.1.10

Figure Lengend Snippet: Blockage of CGRP–CALCRL/RAMP1 signaling worsens corneal inflammation and fibrosis by downregulating TSP1 and A2M expression. ( A ) The expression levels of Ramp1 and Calcrl in the corneal stroma of WT mice. ( B ) Representative images of RAMP1 and CALCRL in the corneal stroma of WT mice. ( C ) Hypothetical schematic diagram of CGRP regulating TSP1 and A2M in the process of corneal stromal repair. ( D ) Schematic diagram of subconjunctival injection of BIBN4096 in WT mice. ( E , F ) Corneal opacity and central thickness were assessed by slit lamp and AS-OCT at indicated time points post-injury ( n = 8). ( G ) Representative images of CD45 staining at day 3 post-injury. ( H ) Quantitative PCR analysis of inflammation-related genes ( n = 6). ( I , J ) BIBN4096 treatment increased α-SMA protein and mRNA levels at day 7 post-injury ( n = 6). ( K – M ) The expression of TSP1 and A2M was analyzed by immunofluorescence staining, quantitative PCR, and western blot at day 7 after injury ( n = 6). ( N ) Following topical administration of PBS or CGRP in Thbs1 −/− mice with stromal injury, slit-lamp images were obtained at the indicated time points, and the corneal opacity was evaluated. ( O ) AS-OCT images were captured at day 7 post-injury, and the central corneal thickness was analyzed ( n = 6). The internal reference control was m -β-actin . Scale bars : 50 µm. UW, unwounded. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant.

Article Snippet: CGRP lyophilized powder (015-09; Phoenix Pharmaceuticals, Inc., Burlingame, CA, USA) was reconstituted in phosphate-buffered saline (PBS) to a final concentration of 40μg/ml.

Techniques: Expressing, Injection, Staining, Real-time Polymerase Chain Reaction, Immunofluorescence, Western Blot, Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: CGRP/TSP1 Signaling Dampens Corneal Inflammation and Fibrosis by Targeting A2M During Corneal Stromal Wound Healing

doi: 10.1167/iovs.67.1.10

Figure Lengend Snippet: Schematic of the CGRP/TSP1/A2M signaling axis in suppressing corneal inflammation and fibrosis following stromal injury. Upon corneal stromal injury (e.g., mechanical injury, chemical burn, fungal infection), CGRP is rapidly released from damaged nerve terminals and binds to the CALCRL–RAMP1 receptor complex expressed on keratocytes or fibroblasts. This binding induces the expression of TSP1, which in turn upregulates A2M. Through this mechanism, the CGRP/TSP1/A2M signaling axis suppresses immune cell infiltration, reduces corneal inflammation, and ameliorates stromal fibrosis. In contrast, disruption of this pathway via the CGRP receptor antagonist BIBN4096, Thbs1 deletion, or A2m knockdown exacerbated corneal lesions, leading to increased corneal opacity, heightened inflammation, and enhanced fibrosis.

Article Snippet: CGRP lyophilized powder (015-09; Phoenix Pharmaceuticals, Inc., Burlingame, CA, USA) was reconstituted in phosphate-buffered saline (PBS) to a final concentration of 40μg/ml.

Techniques: Infection, Binding Assay, Expressing, Disruption, Knockdown