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

Calcitonin gene-related peptide (CGRP) is a multifunctional neuropeptide abundantly expressed by corneal nerves. Using a murine model of corneal mechanical injury, we found CGRP levels in the cornea significantly reduced after injury. Topical application of CGRP as an eye drop accelerates corneal epithelial wound closure, reduces corneal opacification, and prevents corneal edema after injury in vivo. CGRP promotes corneal epithelial cell migration, proliferation, and the secretion of laminin. It reduces TGF-ß1 signaling and prevents TGF-ß1-mediated stromal fibroblast activation and tissue fibrosis. CGRP preserves corneal endothelial cell density, morphology, and pump function, thus reducing corneal edema. Lastly, CGRP reduces neutrophil infiltration, macrophage maturation, and the production of inflammatory cytokines in the cornea. Taken together, our results show that corneal nerve-derived CGRP plays a cytoprotective, pro-regenerative, anti-fibrotic, and anti-inflammatory role in corneal wound healing. In addition, our results highlight the critical role of sensory nerves in ocular surface homeostasis and injury repair.

Novel Indolyl-Benzimidazole Compounds Promote in vitro Wound Healing and Osteogenic Differentiation of Pluripotent Cells.

BACKGROUND: Increasing or restoring Bone Morphogenetic Protein- (BMP-) signaling through administration of recombinant BMPs (rBMPs) has demonstrated therapeutic efficacy for treating bone fractures or to enhance repair following spinal surgeries. However, direct use of rBMPs has come up against significant obstacles like high cost and incidence of adverse effects. Recently, we reported our findings on the novel indolyl-benzimidazoles, SY-LB-35 and SY-LB-57, that fully activated BMP receptor signaling demonstrating activity profiles that mirrored rBMPs. Here, we explored the potential of these compounds to substitute for rBMPs in processes like wound healing and osteogenesis. METHODS: Cell-based assays including cell viability, short- and long-term phosphorylation, protein expression, wound healing and bone differentiation assays were carried out in the pluripotent myoblast C2C12 cell line with select assays performed in multiple cell lines. Several assays included conditions in the presence of a selective inhibitor of type I BMP receptor, Activin-like kinase 2 (ALK2), or inhibitors of BMP-stimulated downstream signaling. All assays were repeated at least 3 times with replicates per condition where indicated. Statistical tests were carried out using Student's two-tailed, t-test. RESULTS: Sustained activation of non-canonical BMP signaling pathways was observed after 24-hour exposure to SY-LB-35 and SY-LB-57. Moreover, this treatment increased the expression of targets of BMP-mediated transcription such as the Id1 transcription factor. SY-LB-35 and SY-LB-57 promoted substantial increases in cell viability in three distinct cell types and increased the rate of wound closure in scrape-wounded C2C12 cell cultures. Cell viability and wound closure induced by SY-LB compounds required ALK2-, PI3K- and p38-dependent pathways. In contrast, responses to SY-LB compounds were not affected by ERK inhibition. Expression of bone differentiation markers beginning at 4 hours and evidence of calcium deposition detected after 21 days in C2C12 cell cultures exposed to SY-LB-35 and SY-LB-57 demonstrated the osteogenic potential of these compounds. CONCLUSIONS: The functional similarities between these novel compounds and rBMPs indicates that SY-LB-35 or SY-LB-57, acting as potent activators of BMP receptor signaling and inducers of osteogenic processes, could potentially replace rBMPs for treating BMP-related pathologies such as bone fracture repair or other wound healing processes.

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

Calcitonin gene-related peptide (CGRP) is a multifunctional neuropeptide abundantly expressed by corneal nerves. Using a murine model of corneal mechanical injury, we found CGRP levels in the cornea to be significantly reduced after injury. Topical application of CGRP as an eye drop three times daily accelerates corneal epithelial wound closure, reduces corneal opacification, and prevents corneal edema after injury in vivo. We then used a series of in vitro and in vivo techniques to investigate the mechanisms underlying CGRP's functions. CGRP promotes corneal epithelial cell migration, proliferation, and the secretion of laminin. It reduces TGF-ß1 signaling and prevents TGF-ß1-mediated stromal fibroblast activation and tissue fibrosis. CGRP reduces corneal endothelial cell apoptosis and death, preserves cell density and morphology, and promotes their pump function, thus reducing edema. Lastly, CGRP reduces neutrophil infiltration, macrophage maturation, and the production of inflammatory cytokines in the cornea. Taken together, our results show that corneal nerve-derived CGRP plays a cyto-protective, pro-regenerative, anti-fibrotic, and anti-inflammatory role in corneal wound healing. Given that current treatment options for corneal injury and opacity are scarce, CGRP has significant therapeutic potential in this area of unmet medical needs. In addition, our results highlight the critical role of sensory nerves in ocular surface homeostasis and injury repair.

Discovery of a novel class of benzimidazoles as highly effective agonists of bone morphogenetic protein (BMP) receptor signaling.

Increasing or restoring Bone Morphogenetic Protein receptor signaling is an effective therapy for conditions such as bone fracture and pulmonary arterial hypertension. However, direct use of recombinant BMPs has encountered significant obstacles. Moreover, synthetic, full agonists of BMP receptor signaling have yet to be identified. Here, we report the discovery of a novel class of indolyl-benzimidazoles, synthesized using a one-pot synthetic methodology, which appear to mimic the biochemical and functional activity of BMPs. The first-in-series compounds, SY-LB-35 and SY-LB-57, stimulated significant increases in cell number and cell viability in the C2C12 myoblast cell line. Cell cycle analysis revealed that these compounds induced a shift toward proliferative phases. SY-LB-35 and SY-LB-57 stimulated canonical Smad and non-canonical PI3K/Akt, ERK, p38 and JNK intracellular signaling pathways, similar to BMP2-stimulated responses. Importantly, increases in Smad phosphorylation and cell viability were dependent on type I BMP receptor activity. Thus, these compounds robustly activate intracellular signaling in a BMP receptor-dependent manner and may signify the first known, full agonists of BMP receptor signaling. Moreover, discovery of small molecule activators of BMP pathways, which can be efficiently formulated and targeted to diseased or damaged areas, could potentially substitute recombinant BMPs for treatment of BMP-related pathologies.

Assessment of BMP responses in an in vitro model of acute ethanol toxicity.

The assay presented here was designed to assess the immediate effects of ethanol (EtOH) exposure on intracellular signaling activated by BMPs (Bone Morphogenetic Proteins). Previous reports of the relationship between EtOH exposure and BMP-dependent signaling have primarily assessed the expression of individual BMPs, changes in BMP target genes or effects on the phosphorylation level of key downstream mediators after days or weeks of in vivo EtOH exposure. What happens to BMP-stimulated signaling immediately following exposure to EtOH remains largely unexplored. Here, the early events of BMP-evoked intracellular signaling were examined in an in vitro model of acute EtOH toxicity. The BMP/Ethanol Stimulation Assay involved first stimulating cultured cells with recombinant BMPs. BMP-evoked intracellular signaling was then allowed to develop for 30 minutes. Next, the cells were exposed to a range of EtOH concentrations for an additional 30 minutes. Finally, the cultures were processed for Western blot analysis or immunofluorescent labeling. This short-term assay: • Permits investigation of EtOH exposure during the initial signaling events downstream of BMP receptor activation • Enables assessment of how the presence of BMPs might protect against cellular injury caused by toxic EtOH levels.

Non-Smad, BMP-dependent signaling protects against the effects of acute ethanol toxicity.

This study explores the effect of acute Ethanol (EtOH) exposure on Bone Morphogenetic Protein (BMP)-evoked intracellular signaling, and the concomitant morphological changes induced by EtOH in C2C12 cells and DRG (Dorsal root ganglion) neurons in an in vitro model related to Fetal Alcohol Syndrome Disorder (FASD). All assays were performed within 30 min of BMP stimulation to specifically investigate the earliest events occurring in BMP-evoked intracellular signaling pathways. We show that Smad phosphorylation and nuclear translocation stimulated by BMPs was not altered following acute exposure to EtOH. In contrast, acute EtOH exposure alone caused a striking concentration-dependent decrease in Akt phosphorylation, as well as a loss of adhesion in C2C12 cells. The addition of BMPs before exposure to EtOH was associated with maintenance of Akt phosphorylation, greater cell adhesion in C2C12 cells, and preservation of growth cone complexity in DRG neurons. Thus, for both C2C12 cells and DRG neurons, BMPs, acting through non-canonical BMP signaling pathways, appear to impart some protection against the profound effects of acute EtOH exposure on cellular adhesion and structure.

Methods for Studying TNFα-Induced Autophagy.

Autophagy is an evolutionarily conserved cellular mechanism in eukaryotes that plays an important role in the maintenance of cellular homeostasis. The autophagy process maintains protein homeostasis by recycling damaged organelles and degrading many long-lived proteins in conjunction with the ubiquitin-proteasome system. Cytokines are low-molecular-weight secreted proteins that regulate a broad range of biological activities. For instance, pro-inflammatory cytokines such as tumor necrosis factor-α (TNFα) induce inflammation, autophagy, and apoptotic cell death. In this chapter, we discuss experimental techniques such as immunoblotting and fluorescence microscopy that can be utilized to measure autophagy in response to TNFα treatment.

The Availability and Affordability of Cardiovascular Medicines for Secondary Prevention in Tehran Province (Iran).

Availability and affordability of medicines are crucial to achieving success in prevention programs, particularly in developing countries. The aim of this study was to determine the availability and affordability of cardiovascular medicines for secondary prevention in Tehran province of Iran. A cross-sectional survey was conducted in Tehran province in 2015, using the 2nd edition of the World Health Organization/Health Action International methodology. Data on the availability and affordability of 21 selected cardiovascular medicines were collected from the public and private healthcare sectors. A total of 120 facilities were included in the survey and the medicines in this survey were both original and lowest-price generic. Lowest-price generic equivalent medicines were highly available (> 80%) in almost all pharmacies of both public and private sectors, while the availability of original brand medicines was highly poor in public and private pharmacies. The median price ratios were 0.72 to 0.76 for generic medicines. The treatment of cardiovascular diseases with lowest-price generic equivalent medicines was generally affordable; moreover, less than a single day's wage was adequate to purchase a monthꞌs supply of the lowest priced generic of the surveyed medicines. The availability of the selected generic medicines for the secondary prevention of cardiovascular diseases is high in both public and private sectors and they were affordable for low-paid unskilled government workers in the province. The result of this study demonstrates that the supply policies pertaining to generic medicines have been implemented successfully.

Initial Results of a Prospective Study and Identification of New Strategies to Increase Traceability of Plasma-derived Medicines.

Plasma medicine is an innovative and emerging field used in a broad range of medical conditions. The present study focused on consumption and documentation pattern of plasma-derived medicines in a teaching hospital. A two-step study was conducted from October to December 2015. During the first phase, the patient records receiving plasma-derived medicines including Coagulation Factor VIII, IX, Prothrombin Complex Concentrate, Factor VIII/Von Wilberand Complex, Anti-Hepatitis B Immunoglobulin, Intravenous Immunoglobulin, Anti-Tetanus Immunoglobulin, and Albumin were checked to assess recording details of these medications at the time of administration. Adverse events reported with the mentioned products were examined from traceability viewpoint. The second step concentrated on practical strategies to improve documentation status of plasma-derived medicines in the hospital. We proposed national guideline as the first strategy and a new barcoding system to track and identify drug information of plasma medicines. Of the expected drug information, only generic name, dosage from, and strength were recorded after administration. Post-marketing safety surveillance of the plasma products was poor similarly. Unavailability of suitable instructions was the main reason for documentation deficiency. A guideline was designed and implemented to inform healthcare professionals about essentials of appropriate documentation for plasma-derived medicines. Updated results of the ongoing phase will be submitted soon. Our survey highlights the importance of documentation as a key component of plasma-derived medicines surveillance within the hospitals.

Interactive involvement of hippocampal cAMP/PKA and cyclooxygenase-2 signaling pathways in spatial learning in the Morris water maze.

INTRODUCTION: Accumulated evidence shows that the cAMP-PKA signaling pathway plays a key role in memory functions. Cyclooxygenase-2, a critical player in neuroinflammation, has been confirmed in the pathogenesis of neurodegenerative diseases. This study is aimed to assess the effect of the interaction of cAMP-PKA and cyclooxygenase pathways on spatial memory acquisition in animal models. MATERIAL AND METHODS: In the present study, the effects of the four-day bilateral intra-hippocampal infusions of H-89 as a protein kinase AII inhibitor (10 µM/side), celecoxib (0.1 M/side) as a selective cyclooxygenase-2 inhibitor, cele-coxib/H-89 and bucladesine (10 µM/side)/celecoxib/H-89 on spatial memory acquisition in the Morris water maze were investigated. Control animals received bilateral intra-hippocampal infusions of dimethyl sulfoxide. Rats were trained for 4 days; each day included one block of four trials. Post-training probe trial tests were performed on day five. RESULTS: A bilateral intra-hippocampal infusion of H-89 and celecoxib led to a significant impairment in spatial learning compared to the controls through a notable decrease in escape latency and traveled distance. But, combination treatment of animals with celecoxib/H-89 and bucladesine/celecoxib/H-89 could considerably reverse celecoxib and H-89-induced spatial memory acquisition impairments in the Morris water maze. CONCLUSIONS: These results indicate the probable regulatory effects of cAMP/PKA and cyclooxygenase-2 signaling pathways on spatial memory acquisition in the Morris water maze.

Efficacy of a telephone-based intervention among patients with type-2 diabetes; a randomized controlled trial in pharmacy practice.

Background Pharmacists' interventions to improve outcomes of diabetes management have been promising. However, evidence on using telephone-based interventions in pharmacy practice are limited, particularly in developing countries. Objective To evaluate the efficacy of a telephone-based intervention to improve care and clinical outcomes in type-2 diabetes. Setting A referral community pharmacy and drug information center. Method We conducted a two-armed randomized controlled trial on 100 patients with type-2 diabetes. The intervention consisted of 16 telephone calls in 3 month by a trained pharmacist working in an academic drug information center, while the control group received usual care. Before random allocation, patients attended a live education session delivered by pharmacists to learn the basics of diabetes care and to confirm the eligibility criteria. Assessments were performed at baseline, month-3 (after intervention), and month-9 (follow-up). Main outcome measure Hemoglobin A1c (HbA1c). Results Eighty four patient completed the trial. Baseline variables were comparable between the two groups and the baseline value of hemoglobin A1c was 8.00 ± 1.44 in the study population. HbA1c was significantly improved in both groups at month-3 (6.97 ± 1.41 vs. 7.09 ± 1.78) and remained steady at month-9 (6.96 ± 1.44 vs. 7.26 ± 1.85). Lipid profile showed small improvements in the intervention group but was not significant. The adherence score and self-care score improvement was significantly higher in the intervention group at month-3 and were maintained at month-9. Conclusion Medication adherence and self-care significantly improved in the telephone-based intervention group. However, the improvement of clinical outcomes might have been diluted due to the live diabetes education session.

A community pharmacy-based cardiovascular risk screening service implemented in Iran.

BACKGROUND: Cardiovascular disease is a major health concern around the world. OBJECTIVE: To assess the outcomes and feasibility of a pharmacy-based cardiovascular screening in an urban referral community pharmacy in Iran. METHODS: A cross sectional study was conducted in a referral community pharmacy. Subjects aged between 30-75 years without previous diagnose of cardiovascular disease or diabetes were screened. Measurement of all major cardiovascular risk factors, exercise habits, medical conditions, medications, and family history were investigated. Framingham risk score was calculated and high risk individuals were given a clinical summary sheet signed by a clinical pharmacist and were encouraged to follow up with their physician. Subjects were contacted one month after the recruitment period and their adherence to the follow up recommendation was recorded. RESULTS: Data from 287 participants were analyzed and 146 were referred due to at least one abnormal laboratory test. The results showed 26 patients with cardiovascular disease risk greater than 20%, 32 high systolic blood pressure, 22 high diastolic blood pressures, 50 high total cholesterol levels, 108 low HDL-C levels, and 22 abnormal blood glucose levels. Approximately half of the individuals who received a follow up recommendation had made an appointment with their physician. Overall, 15.9% of the individuals received medications and 15.9% received appropriate advice for risk factor modification. Moreover, 7.5% were under evaluation by a physician. CONCLUSION: A screening program in a community pharmacy has the potential to identify patients with elevated cardiovascular risk factor. A plan for increased patient adherence to follow up recommendations is required.

The role of nitric oxide in the PKA inhibitor induced spatial memory deficits in rat: involvement of choline acetyltransferase.

Several lines of evidence show that cAMP-PKA signaling pathway plays critical role in memory functions and suggest nitric oxide as an important modulator in learning and memory. In this study, we assessed the effects of intra-hippocampal infusion of H-89, a selective PKAII inhibitor, and 1400 W, a selective inducible nitric oxide synthase (iNOS) inhibitor, on spatial memory in rats. By using the Morris water maze, spatial memory retention parameters were examined 48 h after the infusions through measuring escape latency, traveled distance, and swimming speed. The rats receiving intra-hippocampal infusions of 1400 W (100 µM/side) showed a significant reduction (*P<0.05) in escape latency and traveled distance in comparison with the control saline group. In contrast, a significant increase (**P<0.01) in escape latency and traveled distance was observed after infusion of 10 µM H-89. Moreover, among combination groups, co-administration of 1400 W (400 µM/side) with 10 µM/side of H-89 caused a significant reduction (*P<0.05) in escape latency and traveled distance in comparison with the H-89 group. Also, we evaluated the molecular effects of 1400 W on the expression of choline acetyltransferase (ChAT), a cholinergic marker, in the CA1 region of the hippocampus and medial septal area (MSA). Immunohistochemical analysis of post-training bilateral intra-hippocampal infusion of 1400 W revealed a significant increase in ChAT immunoreactivity levels in both the CA1 and the MSA regions. Overall, the results suggest that 1400 W has protective effect against H89-induced spatial memory impairment. Moreover, the observed memory improvements caused by 1400 W infusions, might be due to interaction of iNOS with the cholinergic system.

Effects of Selective iNOS Inhibitor on Spatial Memory in Recovered and Non-Recovered Ketamine Induced Anesthesia.

Nitric oxide (NO) is thought to be involved in spatial learning and memory in several brain areas such as hippocampus. This study examined the effects of post-training intrahippocampal microinjections of 1400W as a selective iNOS inhibitor on spatial memory, in anesthetized and non-anesthetized situations in rats. In the present work, 4-day training trials of animals were conducted. Spatial memory was tested 48 hours after the drug infusions. For microinjection of 1400W into CA1 region of the hippocampus in conscious animals, guide cannula was implanted into the CA1 area and 1400W was infused after recovery from surgical anesthesia. In anesthetized animals, 1400W was microinjected directly into CA1 region by Hamilton syringe during anesthesia. After completion of training, 1400W (10, 50 and 100 µM/side) were microinjected bilaterally (1 µL/side) and testing trials were performed 48 h after drug infusions in both groups of cannulated and non-cannulated rats. Significant reduction was observed in escape latency and traveled distance in animals that received 1400W (100 µM/side, *p < 0.05) via cannula after recovery in comparison with control group. Also, microinjection of 1400W (100 µM/side) in post recovery phase caused a significant (***p < 0.001) reduction in time and distance of finding the hidden platform in comparison with anesthetized situation. These findings suggest that 1400W has a significant improvement on spatial memory and memory enhancement induced by iNOS inhibitor can be affected by anesthesia in a period of time.

Effects of Selective iNOS Inhibitor on Spatial Memory in Recovered and Non-recovered Ketamine Induced-anesthesia in Wistar Rats.

Nitric oxide (NO) is thought to be involved in spatial learning and memory in several brain areas such as hippocampus. This study examined the effects of post-training intrahippocampal microinjections of 1400W as a selective inducible nitric oxide synthase (iNOS) inhibitor on spatial memory, in both anesthetized and non-anesthetized situations in rats. In the present work, 4-day training trials of animals were conducted. Spatial memory was tested 48 h after the drug infusions. For microinjection of 1400W into CA1 region of the hippocampus in conscious animals, guide cannula was implanted into the CA1 area and 1400W was infused after recovery from surgical anesthesia. In anesthetized animals, 1400W was microinjected directly into CA1 region by Hamilton syringe during anesthesia. After completion of training, 1400W (10, 50 and 100 µM/side) were microinjected bilaterally (1 µL/side) and testing trials were performed 48 h after drug infusions in both groups of cannulated and non-cannulated rats. Significant reduction was observed in escape latency and traveled distance in animals that received 1400W (100 µM/side, * P < 0.05) via cannula after recovery in comparison with control group. Moreover, microinjection of 1400W (100 µM/side) in post recovery phase also caused a significant (*** P < 0.001) reduction in time and distance of finding the hidden platform in comparison with anesthetized situation. These results suggest that 1400W has a significant improvement on spatial memory, and memory enhancement induced by iNOS inhibitor can be affected by anesthesia in a period of time.