Exogenous Calcitonin Gene-related Peptide Research Articles

Sensory nerves drive migration of dental pulp stem cells via the CGRP-Ramp1 axis in pulp repair

Dental pulp stem cells (DPSCs) are responsible for maintaining pulp structure and function after pulp injury. DPSCs migrate directionally to the injury site before differentiating into odontoblast-like cells, which is a prerequisite and a determinant in pulp repair. Increasing evidence suggests that sensory neuron-stem cell crosstalk is critical for maintaining normal physiological functions, and sensory nerves influence stem cells mainly by neuropeptides. However, the role of sensory nerves on DPSC behaviors after pulp injury is largely unexplored. Here, we find that sensory nerves released significant amounts of calcitonin gene-related peptide (CGRP) near the injury site, acting directly on DPSCs via receptor activity modifying protein 1 (RAMP1) to promote collective migration of DPSCs to the injury site, and ultimately promoting pulp repair. Specifically, sensory denervation leads to poor pulp repair and ectopic mineralization, in parallel with that DPSCs failed to be recruited to the injury site. Furthermore, in vitro evidence shows that sensory nerve-deficient microenvironment suppressed DPSC migration prominently among all related behaviors. Mechanistically, the CGRP-Ramp1 axis between sensory neurons and DPSCs was screened by single-cell RNA-seq analysis and immunohistochemical studies confirmed that the expression of CGRP rather than Ramp1 increases substantially near the damaged site. We further demonstrated that CGRP released by sensory nerves binds the receptor Ramp1 on DPSCs to facilitate cell collective migration by an indirect co-culture system using conditioned medium from trigeminal neurons, CGRP recombinant protein and antagonists BIBN4096. The treatment with exogenous CGRP promoted the recruitment of DPSCs, and ultimately enhanced the quality of pulp repair. Targeting the sensory nerve could therefore provide a new strategy for stem cell-based pulp repair and regeneration.

Calcitonin gene‑related peptide alleviates hyperoxia‑induced human alveolar cell injury via the CGRPR/TRPV1/Ca2+ axis.

Although exogenous calcitonin gene‑related peptide (CGRP) protects against hyperoxia‑induced lung injury (HILI), the underlying mechanisms remain unclear. The present study attempted to elucidate the molecular mechanism by which CGRP protects against hyperoxia‑induced alveolar cell injury. Human alveolar A549 cells were treated with 95% hyperoxia to establish a hyperoxic cell injury model. ELISA was performed to detect the CGRP secretion. Immunofluorescence, quantitative (q)PCR, and western blotting were used to detect the expression and localization of CGRP receptor (CGRPR) and transient receptor potential vanilloid 1 (TRPV1). Cell counting kit‑8 and flow cytometry were used to examine the proliferation and apoptosis of treated cells. Digital calcium imaging and patch clamp were used to analyze the changes in intracellular Ca2+ signaling and membrane currents induced by CGRP in A549 cells. The mRNA and protein expression levels of Cyclin D1, proliferating cell nuclear antigen (PCNA), Bcl‑2 and Bax were detected by qPCR and western blotting. The expression levels of CGRPR and TRPV1 in A549 cells were significantly downregulated by hyperoxic treatment, but there was no significant difference in CGRP release between cells cultured under normal air and hyperoxic conditions. CGRP promoted cell proliferation and inhibited apoptosis in hyperoxia, but selective inhibitors of CGRPR and TRPV1 channels could effectively attenuate these effects; TRPV1 knockdown also attenuated this effect. CGRP induced Ca2+ entry via the TRPV1 channels and enhanced the membrane non‑selective currents through TRPV1 channels. The CGRP‑induced increase in intracellular Ca2+ was reduced by inhibiting the phospholipase C (PLC)/protein kinase C (PKC) pathway. Moreover, PLC and PKC inhibitors attenuated the effects of CGRP in promoting cell proliferation and inhibiting apoptosis. In conclusion, exogenous CGRP acted by inversely regulating the function of TRPV1 channels in alveolar cells. Importantly, CGRP protected alveolar cells from hyperoxia‑induced injury via the CGRPR/TRPV1/Ca2+ axis, which may be a potential target for the prevention and treatment of the HILI.

Calcitonin gene-related peptide inhibits neuronal apoptosis in heatstroke rats via PKA/p-CREB pathway

PurposeThe incidence of heatstroke (HS) is not particularly high; however, once it occurs, the consequences are serious. It is reported that calcitonin gene-related peptide (CGRP) is protective against brain injury in HS rats, but detailed molecular mechanisms need to be further investigated. In this study, we further explored whether CGRP inhibited neuronal apoptosis in HS rats via protein kinase A (PKA)/p-cAMP response element-binding protein (p-CREB) pathway. MethodsWe established a HS rat model in a pre-warmed artificial climate chamber with a temperature of (35.5 ± 0.5) °C and a relative humidity of 60% ± 5%. Heatstress was stopped once core body temperature reaches above 41 °C. A total of 25 rats were randomly divided into 5 groups with 5 animals each: control group, HS group, HS+CGRP group, HS+CGRP antagonist (CGRP8-37) group, and HS+CGRP+PKA/p-CREB pathway blocker (H89) group. A bolus injection of CGRP was administered to each rat in HS+CGRP group, CGRP8-37 (antagonist of CGRP) in HS+CGRP8-37 group, and CGRP with H89 in HS+CGRP+H89 group. Electroencephalograms were recorded and the serum concentration of S100B, neuron-specific enolase (NSE), neuron apoptosis, activated caspase-3 and CGRP expression, as well as pathological morphology of brain tissue were detected at 2 h, 6 h, and 24 h after HS in vivo. The expression of PKA, p-CREB, and Bcl-2 in rat neurons were also detected at 2 h after HS in vitro. Exogenous CGRP, CGRP8-37, or H89 were used to determine whether CGRP plays a protective role in brain injury via PKA/p-CREB pathway. The unpaired t-test was used between the 2 samples, and the mean ± SD was used for multiple samples. Double-tailed p < 0.05 was considered statistically significant. ResultsElectroencephalogram showed significant alteration of θ (54.50 ± 11.51 vs. 31.30 ± 8.71, F = 6.790, p = 0.005) and α wave (16.60 ± 3.21 vs. 35.40 ± 11.28, F = 4.549, p = 0.020) in HS group compared to the control group 2 h after HS. The results of triphosphate gap terminal labeling (TUNEL) showed that the neuronal apoptosis of HS rats was increased in the cortex (9.67 ± 3.16 vs. 1.80 ± 1.10, F = 11.002, p = 0.001) and hippocampus (15.73 ± 8.92 vs. 2.00 ± 1.00, F = 4.089, p = 0.028), the expression of activated caspase-3 was increased in the cortex (61.76 ± 25.13 vs. 19.57 ± 17.88, F = 5.695, p = 0.009) and hippocampus (58.60 ± 23.30 vs. 17.80 ± 17.62, F = 4.628, p = 0.019); meanwhile the expression of serum NSE (5.77 ± 1.78 vs. 2.35 ± 0.56, F = 5.174, p = 0.013) and S100B (2.86 ± 0.69 vs. 1.35 ± 0.34, F = 10.982, p = 0.001) were increased significantly under HS. Exogenous CGRP decreased the concentrations of NSE and S100B, and activated the expression of caspase-3 (0.41 ± 0.09 vs. 0.23 ± 0.04, F = 32.387, p < 0.001) under HS; while CGRP8-37 increased NSE (3.99 ± 0.47 vs. 2.40 ± 0.50, F = 11.991, p = 0.000) and S100B (2.19 ± 0.43 vs. 1.42 ± 0.30, F = 4.078, p = 0.025), and activated the expression caspase-3 (0.79 ± 0.10 vs. 0.23 ± 0.04, F = 32.387, p < 0.001). For the cell experiment, CGRP increased Bcl-2 (2.01 ± 0.73 vs. 2.15 ± 0.74, F = 8.993, p < 0.001), PKA (0.88 ± 0.08 vs. 0.37 ± 0.14, F = 20.370, p < 0.001), and p-CREB (0.87 ± 0.13 vs. 0.29 ± 0.10, F = 16.759, p < 0.001) levels; while H89, a blocker of the PKA/p-CREB pathway reversed the expression. ConclusionsCGRP can protect against HS-induced neuron apoptosis via PKA/p-CREB pathway and reduce activation of caspase-3 by regulating Bcl-2. Thus CGRP may be a new target for the treatment of brain injury in HS.

Elucidating the Ability of CGRP to Modulate Microvascular Events in Mouse Skin.

Oedema formation and polymorphonuclear leukocyte (neutrophil) accumulation are involved in both acute and chronic inflammation. Calcitonin gene-related peptide (CGRP) is a sensory neuropeptide that is released from stimulated sensory nerves. CGRP is a potent vasodilator neuropeptide, especially when administered to the cutaneous microvasculature, with a long duration of action. Here, we have investigated the ability of vasodilator amounts of CGRP to modulate oedema formation and neutrophil accumulation induced in the cutaneous microvasculature of the mouse. To learn more about the mechanism of action of endogenous CGRP, we have investigated the response to the inflammatory stimulants tumour necrosis factor alpha (TNFα) and carrageenan in three different murine models: a model where sensory nerves were depleted by resiniferatoxin (RTX); a pharmacological method to investigate the effect of a selective CGRP receptor antagonist; and a genetic approach using wildtype (WT) and αCGRP knockout (KO) mice. Our results show that exogenous CGRP potentiates oedema formation induced by substance P (SP) and TNFα. This is further supported by our findings from sensory nerve-depleted mice (in the absence of all neuropeptides), which indicated that sensory nerves are involved in mediating the oedema formation and neutrophil accumulation induced by TNFα, and also carrageenan in cutaneous microvasculature. Furthermore, endogenous CGRP was shown to contribute to this inflammatory response as carrageenan-induced oedema formation is attenuated in WT mice treated with the CGRP receptor antagonist, and in αCGRPKO mice. It is therefore concluded that CGRP can contribute to inflammation by promoting oedema formation in skin, but this response is dependent on the pro-inflammatory stimulus and circumstance.

Calcitonin gene-related peptide-induced hemodynamic changes in migraine with and without aura.

Exogenous calcitonin gene-related peptide (eCGRP) can induce CGRP-induced headaches (CGRP-IH) and aura in migraine with aura (MA). This implies a common pathophysiological mechanism of trigeminovascular sensitization (TVS) in migraine headaches and aura. The aim was to assess hemodynamic changes in cerebral circulation induced by eCGRP. We predicted that cerebral hemodynamic changes may differ between migraine without aura (MO) and MA. We included twenty participants with migraine, of whom 15 (75%) had MO, and 5 (25%) had MA. An intravenous infusion of eCGRP was administered. Polymodal recording of mean arterial velocity in MCA (vm MCA) and PCA (vm PCA), end-tidal carbon dioxide partial pressure (Et-CO2 ), mean arterial pressure (MAP), and heart rate (HR) was employed using transcranial Doppler sonography (TCD). The parameters were determined at different time points with single responses vm MCAtot , vm PCAtot , Et-CO2tot , MAPtot , and HRtot . The CGRP-IH appeared in five participants with MA (100%) and in 11 participants with MO (73.3%) (p=.530). The difference of changes in vm MCAtot (p=.014) and vm PCAtot (p=.004) was significant, whereas in Et-CO2tot (p=.658), MAPtot (p=.392), and HRtot (p=.686), it appeared to be non-significant. We found significant associations between vm MCAtot and MA (p=.023; OR=0.88; 95%C.I. 0.78-0.98), and vm PCAtot and MA (p=.018; OR=0.85; 95%C.I. 0.74-0.97). Cerebral hemodynamics differs between MO and MA, indicating a pronounced vasodilatation and TVS in MA, which could induce aura.

Calcitonin Gene-Related Peptide Influences Bone-Tendon Interface Healing Through Osteogenesis: Investigation in a Rabbit Partial Patellectomy Model.

Background:Calcitonin gene-related peptide (CGRP), which has been shown to play an important role in osteogenesis during fracture repair, is also widely distributed throughout the tendon and ligament. Few studies have focused on the role of CGRP in repair of the bone-tendon interface (BTI).Purpose:To explore the effect of CGRP expression on BTI healing in a rabbit partial patellectomy model.Study Design:Controlled laboratory study.Methods:A total of 60 mature rabbits were subjected to a partial patellectomy and then randomly assigned to CGRP, CGRP-antagonist, and control groups. In the CGRP-antagonist group, the CGRP receptor antagonist BIBN4096BS was administered to block CGRP receptors. The patella–patellar tendon complex was harvested at 8 and 16 weeks postoperatively and subjected to radiographic, microlaser Raman spectroscopy, histologic, and biomechanical evaluation.Results:Radiographic data showed that local CGRP expression improved the growth parameters of newly formed bone, including area and volumetric bone mineral density (P < .05 for both). Raman spectroscopy revealed that the relative bone mineral composition increased in the CGRP group compared with in the control group and the CGRP-antagonist group (P < .05 for both). Histologic testing revealed that the CGRP group demonstrated better integration, characterized by well-developed trabecular bone expansion from the residual patella and marrow cavity formation, at the 8- and 16-week time points. Mechanical testing demonstrated that the failure load, ultimate strength, and stiffness in the CGRP group were significantly higher than those in the control group (P < .05 for all), whereas these parameters in the CGRP-antagonist group were significantly lower compared with those in the control group at 16 weeks after surgery (P < .05 for all).Conclusion:Increasing the local concentration of CGRP in the early stages of BTI healing enhanced osteogenesis in a rabbit partial patellectomy model and promoted healing of the BTI injury, whereas treatment using a CGRP antagonist had the opposite effect. However, exogenous CGRP expression did not induce novel bone remolding.Clinical Relevance:CGRP may have potential as a new therapy for BTI injuries or may be added to postoperative regimens to facilitate healing.

Calcitonin gene-related peptide alleviates hypertrophic scar formation by inhibiting the inflammation.

The present study aims to explore the roles of calcitonin gene-related peptide (CGRP) in the hypertrophic scar and its underlying mechanism. The levels of CGRP were determined in human hypertrophic scar and mouse cutaneous scar using ELISA and Western blot. In in vivo studies, A cutaneous excision mouse model was established and treated with exogenous CGRP or CGRP antagonist. In in vitro studies, bone marrow-derived macrophages (BMDMs) were isolated and treated with exogenous CGRP in the presence of lipopolysaccharide (LPS). qRT-PCR and Western blot were applied to determine the mRNA and protein levels of scar formation and inflammation-related genes, respectively. Flow cytometry was operated to determine the populations of macrophages in the scar. Elevated levels of CGRP were observed in the hypertrophic scar. In the cutaneous excision mouse model, treatment of exogenous CGRP or CGRP antagonist-affected scar formation-related genes including Col1, Tgfb1, and α-SMA, inflammation-related genes including Il1b, Il6, Tnfa, and Ccl2, and CD45+F4/80+ macrophage. In LPS-induced BMDMs, treatment of exogenous CGRP also altered inflammation-related genes by regulating NF-κB and ERK signaling pathways. The ameliorated effects of CGRP on inflammation in hypertrophic scar formation are associated with its regulative effects on NF-κB and ERK signaling pathways.

Calcitonin Gene-Related Peptide Protects Against Cardiovascular Dysfunction Independently of Nitric Oxide In Vivo.

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Vasomotor action of androgens in the mesenteric artery of hypertensive rats. Role of perivascular innervation.

Androgens may exert cardiovascular protective actions by regulating the release and function of different vascular factors. In addition, testosterone (TES) and its 5-reduced metabolites, 5α- and 5β-dihydrotestosterone (5α- and 5β-DHT) induce vasorelaxant and hypotensive effects. Furthermore, hypertension has been reported to alter the release and function of the neurotransmitters nitric oxide (NO), calcitonin gene-related peptide (CGRP) and noradrenaline (NA). Since the mesenteric arteries possess a dense perivascular innervation and significantly regulate total peripheral vascular resistance, the objective of this study was to analyze the effect of TES, 5α- and 5β-DHT on the neurogenic release and vasomotor function of NO, CGRP and NA. For this purpose, the superior mesenteric artery from male spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto (WKY) rats was used to analyze: (i) the effect of androgens (10 nM, incubated for 30 min) on the neurogenic release of NO, CGRP and NA and (ii) the vasoconstrictor-response to NA and the vasodilator responses to the NO donor, sodium nitroprusside (SNP) and exogenous CGRP. The results showed that TES, 5α- or 5β-DHT did not modify the release of NO, CGRP or NA induced by electrical field stimulation (EFS) in the arteries of SHR; however, in the arteries of WKY rats androgens only caused an increase in EFS-induced NO release. Moreover, TES, and especially 5β-DHT, increased the vasodilator response induced by SNP and CGRP in the arteries of SHR. These findings could be contributing to the hypotensive/antihypertensive efficacy of 5β-DHT previously described in conscious SHR and WKY rats, pointing to 5β- DHT as a potential drug for the treatment of hypertension.

Dietary capsaicin normalizes CGRP peptidergic DRG neurons in experimental diabetic peripheral neuropathy

Diabetic sensory neuropathy leads to impairment of peripheral sensory nerves and downregulation of calcitonin gene-related peptide (CGRP) in a functionally specific subset of peripheral sensory neurons mediating pain. Whether CGRP plays a neuroprotective role in peripheral sensory nerve is unclear. We evaluated alterations in noxious thermal sensation and downregulation of CGRP in the 8 weeks after induction of diabetes in rats. We supplemented capsaicin in the diet of the animals to upregulate CGRP and reversed the downregulation of the neuropeptide in the dorsal root ganglion (DRG) neurons dissociated from the diabetic animals, via gene transfection and exogenous CGRP, to test disease-preventing and disease-limiting effects of CGRP. Significant preservation of the nociceptive sensation, CGRP in spinal cord and DRG neurons, and number of CGRP-expressing neurons was found in the diabetic animals given capsaicin. Improvement in the survival of the neurons and the outgrowth of neurites was achieved in the neurons transfected by LV-CGRP or by exogenous CGRP, paralleling the correction of abnormalities of intracellular reactive oxygen species and mitochondrial transmembrane potentials. The results suggest that downregulation of CGRP impairs viability, regeneration and function of peripheral sensory neurons while capsaicin normalizes the CGRP peptidergic DRG neurons and function of the sensory nerves.

Calcitonin Gene-Related Peptide Enhances Distraction Osteogenesis by Increasing Angiogenesis.

Distraction osteogenesis (DO) is a well-established surgical technique for treating bone defect and limb lengthening. The major drawback of DO is the long treatment period as the external fixator has to be kept in place until consolidation is completed. Calcitonin gene-related peptide (CGRP) has been reported to promote angiogenesis by affecting endothelial progenitor cells (EPCs) in limb ischemia and wound healing. Thus, the goal of this study was to evaluate the angiogenic effect of exogenous CGRP on bone regeneration in a rat DO model. Exogenous CGRP was directly injected into the bone defect after each cycle of distraction in vivo. Microcomputed tomography, biomechanical test, and histological analysis were performed to assess the new bone formation. Angiography and immunofluorescence were performed to assess the formation of blood vessels. CD31+CD144+ EPCs in the bone defect were quantified with flow cytometry. In in vitro study, bone marrow stem cells (BMSCs) were used to investigate the effect of CGRP on EPCs production during endothelial differentiation. Our results showed that CGRP significantly promoted bone regeneration and vessel formation after consolidation. CGRP significantly increased the fraction of CD31+CD144+EPCs and the capillary density in the bone defect at the end of distraction phase. CGRP increased EPC population in the endothelial differentiation of BMSCs in vitro by activating PI3K/AKT signaling pathway. Furthermore, differentiated EPCs rapidly assembled into tube-like structures and promoted osteogenic differentiation of BMSCs. In conclusion, CGRP increased EPC population and promoted blood vessel formation and bone regeneration at the defect region in a DO model. Impact statement Distraction osteogenesis (DO) is a well-established surgical technique for limb lengthening and bone defect. The disadvantage of this technique is that external fixator is needed to be kept in place for about 12 months. This may result in increased risk of infection, financial burden, and negative psychological impacts. In this study, we have injected calcitonin gene-related peptide (CGRP) into the defect region after distraction and found that CGRP enhanced vessel formation and bone regeneration in a rat DO model. This suggests that a controlled delivery system for CGRP could be developed and applied clinically for accelerating bone regeneration in patients with DO.

Exogenous CGRP Regulates Apoptosis and Autophagy to Alleviate Traumatic Brain Injury Through Akt/mTOR Signalling Pathway.

With millions of traumatic brain injury (TBI) patients every year, TBI is regarded as one of the leading causes of human death and disability. Calcitonin gene-related peptide (CGRP) has been domenstrated to be a potential therapeutic target for TBI. However, the detailed effect and underlying mechanism of CGRP on the injured brain after TBI has hardly been investigated. In this work, we established TBI models of mice and injected CGRP before and after modelling to study its effects on the brain lesion, neurological functions and behaviours, neuron apoptosis and autophagy after TBI. Impacts of introduced CGRP on the activation of Akt/mTOR signalling in the cortical tissues surrounding injured areas after TBI were also evaluated. It was found that CGRP was reduced after TBI, and gradually restored over time. CGRP administration significantly restored the brain lesion induced by TBI. The permeability of blood-brain barrier and brain edema was increased dramatically after TBI, which was ameliorated by exogenous CGRP. Moreover, several neurological behaviour tests were performed, showing that CGRP introduction also relieved the cognitive abilities of mice which were impaired after TBI. Enhancing apoptosis and autophagy of neurons in the cortical tissues of injury sites following TBI were also alleviated by CGRP administration. Besides, CGRP-treated brain cortical tissues showed increased activation of Akt/mTOR signalling after TBI. Therefore, the results suggest that exogenous CGRP plays a neuroprotective role in the injuryed brain after TBI, to relieve cell apoptosis and autophagy, at least partially through Akt/mTOR signalling pathway. This finding also provides more evidence for the treatment of TBI through introducing exogenous CGRP or its related drugs.

The anti-inflammatory regulation of calcitonin gene-related peptide in mouse Aspergillus fumigatus keratitis.

To analyze the impact of calcitonin gene-related peptide (CGRP) in mouse keratitis after Aspergillus fumigatus (A. fumigatus) infection. C57BL/6 mice were treated subconjunctivally with different concentrations of exogenous CGRP, and BALB/c mice were treated with CGRP8-37 (a CGRP antagonist) before corneas were infected with A. fumigatus. The cornea was assessed under the slit-lamp and the clinical score was recorded. The mRNA levels of IL-1β, TNF-α, IL-6, and MIP-2 were detected by quantitative real-time polymerase chain reaction (PCR), while the protein level of IL-1β was determined by Western blotting. In vitro, RAW264.7 cells were used to investigate NLRP3 and IL-1β expression induced by A. fumigatus after the pretreatment of exogenous CGRP or CGRP8-37. Cytokines expression in RAW264.7 cells was evaluated by real-time PCR and Western blotting. Using exogenous CGRP resulted in down-regulated synthesis of IL-1β and MIP-2 stimulated by A. fumigatus in C57BL/6 mice keratitis, and the synthesis of IL-1β, MIP-2 and IL-6 was up-regulated in BALB/c mice corneas after the pretreatment with CGRP8-37. Pretreatment with exogenous CGRP and CGRP8-37 did not influence TNF-α mRNA levels either in BALB/c or C57BL/6 mice keratitis. The levels of NLRP3 and IL-1β were both reduced in A. fumigatus stimulated-macrophages after treatment with exogenous CGRP. And CGRP8-37 pretreatment would increase NLRP3 and IL-1β levels. CGRP may alleviate the inflammatory reaction in mice keratitis after infection with A. fumigatus. The anti-inflammatory effect may be related to the inhibition of NLRP3 expression by CGRP.

Lasmiditan inhibits calcitonin gene-related peptide release in the rodent trigeminovascular system.

Migraine headache pathophysiology involves trigeminovascular system activation, calcitonin gene-related peptide (CGRP) release, and dysfunctional nociceptive transmission. Triptans are 5-HT1B/1D/(1F) receptor agonists that prejunctionally inhibit trigeminal CGRP release, but their vasoconstrictor properties limit their use in migraine patients with cardiovascular disease. By contrast, lasmiditan is a novel antimigraine and selective 5-HT1F receptor agonist devoid of vasoconstrictor properties. On this basis, this study has investigated the modulation of trigeminal CGRP release by lasmiditan. For this purpose, we have comparatively analysed the inhibition of several components of the trigeminovascular system induced by lasmiditan and sumatriptan through: ex vivo KCl-induced CGRP release from isolated dura mater, trigeminal ganglion, and trigeminal nucleus caudalis of mice; and in vivo dural vasodilation in the rat closed-cranial window model induced by endogenous (electrical stimulation and capsaicin) and exogenous CGRP. The ex vivo release of CGRP was similarly inhibited by sumatriptan and lasmiditan in all trigeminovascular system components. In vivo, intravenous (i.v.) lasmiditan or higher doses of sumatriptan significantly attenuated the vasodilatory responses to endogenous CGRP release, but not exogenous CGRP effects. These data suggest that lasmiditan prejunctionally inhibits CGRP release in peripheral and central trigeminal nerve terminals. Because lasmiditan is a lipophilic drug that crosses the blood-brain barrier, additional central sites of action remain to be determined.

Expression and role of calcitonin gene-related peptide in mouse Aspergillus fumigatus keratitis.

To investigate the expression and role of calcitonin gene-related peptide (CGRP) in the mouse models induced by Aspergillus fumigatus (A. fumigatus). C57BL/6 mice were randomized into a control group and A. fumigatus keratitis group. The cornea photography was assessed under the slit lamp and the clinical score was recorded after infection. Western blot, real-time polymerase chain reaction (PCR) and immunohistofluorescence analysis were applied to detect CGRP expression in cornea of both groups. In vitro, tests were conducted with C57BL/6 mice macrophages to investigate CGRP expression after interaction with A. fumigatus. Cytokines expression induced by exogenous CGRP and the antagonist CGRP8-37 in A. fumigatus-exposed macrophages was evaluated by real-time PCR and ELISA. The cornea expression of CGRP was significantly elevated in C57BL/6 mice corneas and macrophages after A. fumigatus infection. After treatment with exogenous CGRP, the levels of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and IL-6 were reduced, and IL-10 level was increased in the A. fumigatus stimulated-macrophages. However, IL-1β, TNF-α and IL-6 levels were upregulated after pretreatment of CGRP8-37. But the mRNA levels of MIP-2, TGF-β and IL-10 were not changed. This study provides evidence that A. fumigatus increased CGRP expression. CGRP may play a protective role against inflammation in A. fumigatus keratitis.

The influence of receptor activity-modifying protein-1 overexpression on angiogenesis in mouse brain capillary endothelial cells.

Receptor activity-modifying protein-1 (RAMP1) is highly expressed in the heart and vasculature, indicating that it might be related to the vascular system. However, the effects of RAMP1 on angiogenesis and the intrinsic mechanisms underlying this process remain unclear. Here, we verified that RAMP1 is a critical regulator of angiogenesis in a mouse brain capillary endothelial cell line (bEnd.3). We first constructed a RAMP1 overexpression lentiviral vector system and stably transfected bEnd.3 cells. We further showed that RAMP1 overexpression could lead to bEnd.3 migration and capillary tube formation in Matrigel without exogenous calcitonin gene-related peptide (CGRP) treatment. At the same time, RAMP1 overexpression had little effect on proliferation. More importantly, vascular endothelial growth factor (VEGF) and CGRP expression levels were not significantly higher in RAMP1-overexpressing cells than in control cells (P > 0.05), indicating that RAMP1 did not function through upregulating VEGF or CGRP expression in bEnd.3 cells. Strikingly, RAMP1 transfection increased adrenomedullin 2 (AM2) expression levels ( P < 0.05). Taken together, these data contribute to a better understanding of the molecular mechanisms of RAMP1 in angiogenesis.

Calcitonin gene-related peptide inhibits the cardiac fibroblasts senescence in cardiac fibrosis via up-regulating klotho expression

It has been documented cardiac fibroblasts as the predominant cell population undergoing senescence in heart. Calcitonin gene-related peptide (CGRP) exhibits a wide range of cardiovascular protective effects. Whether CGRP protects against cardiac fibroblasts senescence in cardiac fibrosis remains unknown. Here, we detected the down-regulation of CGRP concomitant with senescence in fibrotic myocardium, both hypertension- induced left ventricular fibrosis in SHR rats and hypoxia-induced right ventricular fibrosis in pulmonary artery hypertension rats. Exogenous CGRP inhibited the cardiac fibroblasts senescence and senescence-associated secretory phenotype (SASP) induced by TGF-β1, which was abolished by CGRP8–37, a selective CGRP receptor antagonist. Moreover, the expression of klotho, an anti-senescence protein, was down-regulated in fibrotic myocardium, and CGRP up-regulated the klotho expression in TGF-β1-treated cardiac fibroblasts. Klotho knockdown by siRNA reversed the inhibition of CGRP on senescence and SASP induced by TGF-β1 in cardiac fibroblasts. These results suggested that CGRP inhibited the cardiac fibroblasts senescence and SASP in cardiac fibrosis via up-regulating klotho expression.

Ryanodine- and CaMKII-dependent release of endogenous CGRP induces an increase in acetylcholine quantal size in neuromuscular junctions of mice.

ObjectiveThe aim of this study was to identify the mechanism responsible for an increase in miniature endplate potentials (MEPPs) amplitude, induced by ryanodine as an agonist of ryanodine receptors in mouse motor nerve terminals.MethodsUsing intracellular microelectrode recordings of MEPPs and evoked endplate potentials (EPPs), the changes in spontaneous and evoked acetylcholine release in motor synapses of mouse diaphragm neuromuscular preparations were studied.ResultsRyanodine (0.1 μM) increased both the amplitudes of MEPPs and EPPs to a similar extent (up to 130% compared to control). The ryanodine effect was prevented by blockage of receptors of calcitonin gene‐related peptide (CGRP) by a truncated peptide CGRP 8‐37. Endogenous CGRP is stored in large dense‐core vesicles in motor nerve terminals and may be released as a co‐transmitter. The ryanodine‐induced increase in MEPPs amplitude may be fully prevented by inhibition of vesicular acetylcholine transporter by vesamicol or by blocking the activity of protein kinase A with H‐89, suggesting that endogenous CGRP is released in response to the activation of ryanodine receptors. Activation of CGRP receptors can, in turn, upregulate the loading of acetylcholine into synaptic vesicles, which will increase the quantal size. This new feature of endogenous CGRP activity looks similar to recently described action of exogenous CGRP in motor synapses of mice. The ryanodine effect was prevented by inhibitors of Ca/Calmodulin‐dependent kinase II (CaMKII) KN‐62 or KN‐93. Inhibition of CaMKII did not prevent the increase in MEPPs amplitude, which was caused by exogenous CGRP.ConclusionsWe propose that the activity of presynaptic CaMKII is necessary for the ryanodine‐stimulated release of endogenous CGRP from motor nerve terminals, but CaMKII does not participate in signaling downstream the activation of CGRP‐receptors followed by quantal size increase.

The Transient Receptor Potential Ankyrin Type 1 Plays a Critical Role in Cortical Spreading Depression

The transient receptor potential ankyrin type-1 (TRPA1) channels have been proposed as a potential target for migraine therapy. Yet the role of cortical TRPA1 channels in migraine mechanism has not been fully understood. Cortical spreading depression (CSD) is known as an underlying cause of migraine aura. The aim of this study is to investigate if cortical TRPA1 activity is required for CSD genesis and propagation. A mouse brain slice CSD model with intrinsic optical imaging was applied for TRPA1 signaling pharmacology. The results showed that the TRPA1 agonist, umbellulone, facilitated the propagation of submaximal CSD. Correspondingly, an anti-TRPA1 antibody and two selective TRPA1 antagonists, A967079 and HC-030031, prolonged the CSD latency and reduced magnitude, indicating a reduced cortical susceptibility to CSD under TRPA1 deactivation. Furthermore, the TRPA1 agonist, allyl-isothiocyanate (AITC), reversed the suppression of CSD by HC-030031, but not by A967079. Interestingly, the inhibitory action of A967079 on CSD was reversed by exogenous calcitonin-gene-related peptide (CGRP). Consistent to TRPA1 deactivation, the prolonged CSD latency was observed by an anti-CGRP antibody in the mouse brain slice, which was reversed by exogenous CGRP. We conclude that cortical TRPA1 is critical in regulating cortical susceptibility to CSD, which involves CGRP. The data strongly suggest that deactivation of TRPA1 channels and blockade of CGRP would have therapeutic benefits in preventing migraine with aura.

Vasorelaxation to the Nitroxyl Donor Isopropylamine NONOate in Resistance Arteries Does Not Require Perivascular Calcitonin Gene-Related Peptide.

Nitroxyl (HNO) donors offer considerable therapeutic potential for the treatment of hypertension-related cardiovascular disorders, particularly heart failure, as they combine an inotropic action with peripheral vasodilation. Angeli's salt is the only HNO donor whose mechanism has been studied in depth, and recently, Angeli's salt vasodilation was suggested to be indirect and caused by calcitonin gene-related peptide (CGRP) released from perivascular nerves after HNO activates TRPA1 (transient receptor potential cation channel subfamily A member 1) channels. We investigated resistance artery vasorelaxation to the HNO donor, isopropylamine NONOate (IPA/NO), one of the structures providing a template for therapeutic development. Wire myography in combination with measurements of smooth muscle membrane potential was used to characterize the effect of IPA/NO in mesenteric resistance arteries. Immunohistochemistry was assessed in pressurized arteries. IPA/NO concentration dependently hyperpolarized and relaxed arteries precontracted with the α1-adrenoreceptor agonist, phenylephrine. These effects were blocked by the soluble guanylyl cyclase inhibitor, ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) but not by the KATP channel inhibitor, glibenclamide. Vasorelaxation persisted in the presence of raised [K+]o, used to block hyperpolarization, capsaicin to deplete perivascular CGRP, or HC030031 (2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)-N-(4 isopropylphenyl) acetamide) to block TRPA1 receptors. Without preconstriction, hyperpolarization to IPA/NO was suppressed by glibenclamide, capsaicin, or HC030031. Hyperpolarization but not vasorelaxation to exogenous CGRP was inhibited with glibenclamide. Thus, vascular hyperpolarization is not necessary for vasorelaxation to the HNO donor IPA/NO, even though both effects are cGMP dependent. The reduced hyperpolarization after depletion of perivascular CGRP or block of TRPA1 receptors indicates some release of CGRP, but this does not contribute to HNO vasorelaxation. Therefore, HNO-TRPA1-CGRP signaling does not seem important for vasodilation to IPA/NO in resistance arteries.