Browsing by Author "Feinberg, Konstantin"
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Item 07. The Mechanism of NGF Signaling In Innervation-dependent Corneal Epithelial Renewal: New Topical Treatment For Neurotrophic Keratopathy(Wolters Kluwer, 2024-04-19) Hussain, Arif; Mirmoeini, Kaveh; Mulenga, Chilando; Crabtree, Jordan; Tajdaran, Kiana; Henrique, Mario; Blum, Noam; Shalom-Feuerstein, Ruby; Borschel, Gregory; Feinberg, Konstantin; Surgery, School of MedicineBackground: Corneal clarity is essential for vision. Limbal stem cells (LSCs), the source of transparent corneal epithelial cells, are located in the basal epithelium of the limbus at the corneal-conjunctival interface, where they interact with corneal sensory nerves. Besides protection, corneal nerves may stimulate LSC activity. Pathological corneal denervation can lead to ulcers, scarring, and opacification due to impaired healing from repetitive wounds. This condition, termed Neurotrophic Keratopathy (NK), a major cause of corneal blindness, and lacks a definitive cure. Corneal nerves release various trophic factors that regulate epithelial renewal. Nerve growth factor (NGF) has shown positive effects on corneal healing and maintenance in vivo. Topical recombinant human NGF is the only FDA-approved treatment for NK. However, NGF is not efficacious in 30% of cases, requires very frequent dosing, and costs $100k per course. Moreover, NGF’s ability to heal corneal ulcers is limited. Prior studies showed NGF stimulates proliferation and maintenance of cultured human LSCs, which express TrkA and p75NTR receptors, but didn’t establish a link between NGF signaling and corneal sensory innervation-mediated trophic regulation of epithelial renewal. Furthermore, the role and molecular mechanism of NGF signaling in LSC activity remains unidentified. We hypothesize that NGF, locally expressed in its mature and premature (proNGF) forms, regulates LSC activity-dependent homeostatic and wound-induced corneal epithelial renewal via differential activation of its receptors TrkA and p75NTR, a process dependent on corneal sensory innervation. Methods: A) We conducted in vivo experiments in wild-type and mutant mice and rats to elucidate the NGF signaling mechanism in corneal innervation-dependent epithelial renewal. We examined the effect of combinations of TrkA and p75NTR agonists and antagonists on the healing of experimentally wounded corneas, both intact and surgically denervated. B) To understand the role and mechanism of NGF signaling in LSC activity and its relevance to humans, we assessed the clonogenicity of cultured human LSCs (hLSCs) by pharmacologically modulating NGF receptors, as described in (A). Results: A) While inactivation of TrkA completely prevents healing of normally innervated cornea, inactivation of p75NTR combined with a single daily dose of NGF induces complete and rapid healing of denervated de-epithelialized corneas. B) NGF or specific p75NTR inhibitor THX-B supported colonies’ formation by hLSCs that were further augmented by combination of the two compounds. Conclusions: Corneal sensory nerve-associated expression of NGF in its both forms (NGF and proNGF) regulates the proliferation and differentiation of LSCs by differentially stimulating the activity of the NGF receptors. Combined pharmacological activation and inhibition of TrkA and p75NTR, respectively, will be applied in the development of a superior NGF-based treatment of NK.Item 72. The Role of TrkA And P75NTR NGF Receptors in Corneal Wound Healing(Wolters Kluwer, 2022) Tajdaran, Kiana; Feinberg, Konstantin; Mirmoeini, Seyed K.; Zhang, Jennifer; Gordon, Tessa; Ali, Asim; Borschel, Gregory; Surgery, School of MedicinePurpose: The cornea is the window through which we see the world and is one of the most densely innervated structures in the body. Besides providing protective sensory input, corneal nerves may also stimulate limbal stem cells (LSCs), governing corneal epithelial maintenance and recovery. Loss of corneal innervation, through injury, diabetes, tumors, infections, and even improper contact lens use, leads to neurotrophic keratopathy (NK), a degenerative corneal disease that is characterized by corneal epithelial breakdown, scarring, and permanent vision loss1. The only non-invasive treatment option for NK is human recombinant nerve growth factor (rhNGF), but the short half-life of exogenous neurotrophins-based therapies limits their effecacy2. Development of small molecule ligands for neurotrophin receptors that have more favorable pharmacokinetics and plasma stability showed promising results in the treatment of several neurodegenerative conditions in recent years3. In this study, we investigated the molecular mechanism of NK and the role of the NGF receptors, TrkA and p75NTR, in corneal healing. We hypothesized that TrkA inhibition would delay corneal wound healing and p75NTR inhibition would accelerate corneal healing. Establishing the roles of these receptors may enable novel topical therapeutics for NK. Methods: We used commercially available Ntrk1 mutant mice, whose TrkA receptors are inhibited by a mammalian kinase inhibitor (1-NM-PP1)4. Ntrk1 mice (n=20) were divided into three groups, which received saline injection as a control. In one experimental group animals received TrkA inhibitor and the other group received both TrkA and p75 inhibitor for 5 days. On day six we removed the corneal epithelium with a 0.5 mm rotating brush. To measure epithelial healing, we performed digital imaging of fluorescein staining daily for four days after injury. We then harvested the corneas for immunofluorescent and biochemical analyses. Results: We observed a significant delay in corneal epithelial healing following TrkA inhibition. Further, we observed that topical p75NTR inhibition accelerated corneal wound healing. Conclusion: A selective TrkA agonist or p75NTR inhibition could represent new topical therapeutics for NK.Item A functional tacrolimus-releasing nerve wrap for enhancing nerve regeneration following surgical nerve repair(Wolters Kluwer, 2024) Daeschler, Simeon C.; So, Katelyn J. W.; Feinberg, Konstantin; Manoraj, Marina; Cheung, Jenny; Zhang, Jennifer; Mirmoeini, Kaveh; Santerre, J. Paul; Gordon, Tessa; Borschel, Gregory H.; Surgery, School of MedicineAxonal regeneration following surgical nerve repair is slow and often incomplete, resulting in poor functional recovery which sometimes contributes to lifelong disability. Currently, there are no FDA-approved therapies available to promote nerve regeneration. Tacrolimus accelerates axonal regeneration, but systemic side effects presently outweigh its potential benefits for peripheral nerve surgery. The authors describe herein a biodegradable polyurethane-based drug delivery system for the sustained local release of tacrolimus at the nerve repair site, with suitable properties for scalable production and clinical application, aiming to promote nerve regeneration and functional recovery with minimal systemic drug exposure. Tacrolimus is encapsulated into co-axially electrospun polycarbonate-urethane nanofibers to generate an implantable nerve wrap that releases therapeutic doses of bioactive tacrolimus over 31 days. Size and drug loading are adjustable for applications in small and large caliber nerves, and the wrap degrades within 120 days into biocompatible byproducts. Tacrolimus released from the nerve wrap promotes axon elongation in vitro and accelerates nerve regeneration and functional recovery in preclinical nerve repair models while off-target systemic drug exposure is reduced by 80% compared with systemic delivery. Given its surgical suitability and preclinical efficacy and safety, this system may provide a readily translatable approach to support axonal regeneration and recovery in patients undergoing nerve surgery.Item Advancing Nerve Regeneration: Translational Perspectives of Tacrolimus (FK506)(MDPI, 2023-08-14) Daeschler, Simeon C.; Feinberg, Konstantin; Harhaus, Leila; Kneser, Ulrich; Gordon, Tessa; Borschel, Gregory H.; Ophthalmology, School of MedicinePeripheral nerve injuries have far-reaching implications for individuals and society, leading to functional impairments, prolonged rehabilitation, and substantial socioeconomic burdens. Tacrolimus, a potent immunosuppressive drug known for its neuroregenerative properties, has emerged in experimental studies as a promising candidate to accelerate nerve fiber regeneration. This review investigates the therapeutic potential of tacrolimus by exploring the postulated mechanisms of action in relation to biological barriers to nerve injury recovery. By mapping both the preclinical and clinical evidence, the benefits and drawbacks of systemic tacrolimus administration and novel delivery systems for localized tacrolimus delivery after nerve injury are elucidated. Through synthesizing the current evidence, identifying practical barriers for clinical translation, and discussing potential strategies to overcome the translational gap, this review provides insights into the translational perspectives of tacrolimus as an adjunct therapy for nerve regeneration.Item Biohacking Nerve Repair: Novel Biomaterials, Local Drug Delivery, Electrical Stimulation, and Allografts to Aid Surgical Repair(MDPI, 2024-07-31) Crabtree, Jordan R.; Mulenga, Chilando M.; Tran, Khoa; Feinberg, Konstantin; Santerre, J. Paul; Borschel, Gregory H.; Surgery, School of MedicineThe regenerative capacity of the peripheral nervous system is limited, and peripheral nerve injuries often result in incomplete healing and poor outcomes even after repair. Transection injuries that induce a nerve gap necessitate microsurgical intervention; however, even the current gold standard of repair, autologous nerve graft, frequently results in poor functional recovery. Several interventions have been developed to augment the surgical repair of peripheral nerves, and the application of functional biomaterials, local delivery of bioactive substances, electrical stimulation, and allografts are among the most promising approaches to enhance innate healing across a nerve gap. Biocompatible polymers with optimized degradation rates, topographic features, and other functions provided by their composition have been incorporated into novel nerve conduits (NCs). Many of these allow for the delivery of drugs, neurotrophic factors, and whole cells locally to nerve repair sites, mitigating adverse effects that limit their systemic use. The electrical stimulation of repaired nerves in the perioperative period has shown benefits to healing and recovery in human trials, and novel biomaterials to enhance these effects show promise in preclinical models. The use of acellular nerve allografts (ANAs) circumvents the morbidity of donor nerve harvest necessitated by the use of autografts, and improvements in tissue-processing techniques may allow for more readily available and cost-effective options. Each of these interventions aid in neural regeneration after repair when applied independently, and their differing forms, benefits, and methods of application present ample opportunity for synergistic effects when applied in combination.Item Congenital disorder of glycosylation – one size does not fit all: a parent’s perspective(Sage, 2022-08-22) Feinberg, Konstantin; Surgery, School of MedicineThis article is written by the parent of a child living with PMM2-congenital disorder of glycosylation (abbreviated to PMM2-CDG). It provides a parental perspective of the journey taken from diagnosis to present day and details the effect of off-label treatment with epalrestat.Item Foretinib mitigates cutaneous nerve fiber loss in experimental diabetic neuropathy(Springer Nature, 2022-05-19) Daeschler, Simeon C.; Zhang, Jennifer; Gordon, Tessa; Borschel, Gregory H.; Feinberg, Konstantin; Surgery, School of MedicineDiabetes is by far, the most common cause of neuropathy, inducing neurodegeneration of terminal sensory nerve fibers associated with loss of sensation, paresthesia, and persistent pain. Foretinib prevents die-back degeneration in cultured sensory and sympathetic neurons by rescuing mitochondrial activity and has been proven safe in prospective clinical trials. Here we aimed at investigating a potential neuroprotective effect of Foretinib in experimental diabetic neuropathy. A mouse model of streptozotocin induced diabetes was used that expresses yellow fluorescent protein (YFP) in peripheral nerve fibers under the thy-1 promoter. Streptozotocin-injected mice developed a stable diabetic state (blood glucose > 270 mg/dl), with a significant reduction of intraepidermal nerve fiber density by 25% at 5 weeks compared to the non-diabetic controls. When diabetic mice were treated with Foretinib, a significantly greater volume of the cutaneous nerve fibers (67.3%) in the plantar skin was preserved compared to vehicle treated (37.8%) and non-treated (44.9%) diabetic mice while proximal nerve fiber morphology was not affected. Our results indicate a neuroprotective effect of Foretinib on cutaneous nerve fibers in experimental diabetic neuropathy. As Foretinib treated mice showed greater weight loss compared to vehicle treated controls, future studies may define more sustainable treatment regimen and thereby may allow patients to take advantage of this neuroprotective drug in chronic neurodegenerative diseases like diabetic neuropathy.Item QS8: The Roles of the TrkA and p75NTR NGF Receptors in Corneal Wound Healing(Wolters Kluwer, 2021-07-26) Tajdaran, Kiana; Feinberg, Konstantin; Mirmoeini, Seyed Kaveh; Zhang, Jennifer; Gordon, Tessa; Borschel, Gregory; Surgery, School of MedicinePurpose: The cornea is the window through which we see the world and is one of the most densely innervated structures in the body. Besides providing protective sensory input, corneal nerves have been postulated to stimulate limbal stem cells (LSCs), hence governing corneal epithelial maintenance and recovery. Loss of corneal innervation, through injury, diabetes, tumors, infections, and even improper contact lens use, leads to neurotrophic keratopathy (NK), a degenerative corneal disease that is characterized by corneal epithelial breakdown, scarring, and permanent vision loss1. The only non-invasive treatment option for NK is the human recombinant nerve growth factor (rhNGF), but the short half-life of exogenous neurotrophins-based therapies make this therapeutic approach less effective2. Development of the small molecule ligands for neurotrophins receptors that have better pharmacokinetics and plasma stability showed promising results in the treatment of several neurodegenerative conditions in the recent years3. In this study, we were prompted to investigate the molecular mechanism of NK and the role of NGF receptors, TrkA and p75NTR, in corneal healing. We hypothesized that TrkA inhibition would delay corneal wound healing and p75NTR inhibition accelerates corneal healing. This knowledge will lay the basis for a new non-invasive approach for NK. Methods: For this experiment, we took advantage of commercially available Ntrk1 mutant mice, which allow for pharmacological inhibition of TrkA receptor with an inhibitor known as not mammalian kinase inhibitor (1-NM-PP1)4. Ntrk1 mice (n=20) were divided into three groups, which received saline injection as a control. In one experimental group animals were received TrkA inhibitor and the other group received both TrkA and p75 inhibitor for 5 days. On day six we removed the corneal epithelium with a 0.5 mm rotating brush. To measure epithelial healing, we performed digital imaging of fluorescein staining daily for four days after injury. We then harvested the corneas for immunofluorescent and biochemical analyses. Results: Our results show a significant delay in corneal epithelial healing following TrkA inhibition and acceleration in corneal healing after p75NTR inhibition. Conclusion: A selective TrkA agonist or p75NTR inhibitors could be a new therapeutic approach for NK.Item Schwann Cells Are Key Regulators of Corneal Epithelial Renewal(Association for Research in Vision and Ophthalmology (ARVO), 2023) Mirmoeini, Kaveh; Tajdaran, Kiana; Zhang, Jennifer; Gordon, Tessa; Ali, Asim; Kaplan, David R.; Feinberg, Konstantin; Borschel, Gregory H.; Surgery, School of MedicinePurpose: Corneal sensory nerves protect the cornea from injury. They are also thought to stimulate limbal stem cells (LSCs) to produce transparent epithelial cells constantly, enabling vision. In other organs, Schwann cells (SCs) associated with tissue-innervating axon terminals mediate tissue regeneration. This study defines the critical role of the corneal axon-ensheathing SCs in homeostatic and regenerative corneal epithelial cell renewal. Methods: SC localization in the cornea was determined by in situ hybridization and immunohistochemistry with SC markers. In vivo SC visualization and/or ablation were performed in mice with inducible corneal SC-specific expression of tdTomato and/or Diphtheria toxin, respectively. The relative locations of SCs and LSCs were observed with immunohistochemical analysis of harvested genetically SC-prelabeled mouse corneas with LSC-specific antibodies. The correlation between cornea-innervating axons and the appearance of SCs was ascertained using corneal denervation in rats. To determine the limbal niche cellular composition and gene expression changes associated with innervation-dependent epithelial renewal, single-cell RNA sequencing (scRNA-seq) of dissociated healthy, de-epithelized, and denervated cornea limbi was performed. Results: We observed limbal enrichment of corneal axon-associated myelinating and non-myelinating SCs. Induced local genetic ablation of SCs, although leaving corneal sensory innervation intact, markedly inhibited corneal epithelial renewal. scRNA-seq analysis (1) highlighted the transcriptional heterogenicity of cells populating the limbal niche, and (2) identified transcriptional changes associated with corneal innervation and during wound healing that model potential regulatory paracrine interactions between SCs and LSCs. Conclusions: Limbal SCs are required for innervation-dependent corneal epithelial renewal.Item Schwann Cells Are Required for Efficient Corneal Wound Healing(Wolters Kluwer, 2021) Mirmoeini, Seyed Kaveh; Feinberg, Konstantin; Tajdaran, Kiana; Zhang, Jennifer; Gordon, Tessa; Borschel, Gregory H.; Surgery, School of MedicineBACKGROUND: Corneal nerves play a crucial role in maintaining corneal health, which includes regulation of activity of limbal stem cell (LSC). Their loss leads to neurotrophic keratopathy (NK), with corneal ulceration, scarring, and ultimately, blindness. Having identified nerve-ensheathing Schwann cells (SC) in the corneal limbus, we hypothesize that SCs, via paracrine interaction with LSC, play a key role in corneal epithelial maintenance and healing. In this study we wanted to (1) Define the role of SCs in corneal healing; (2) Determine the paracrine interaction between the limbal SCs and LSC. METHODS: (1) Local corneal ablation of SCs was induced in a genetically modified mouse where the topical application of tamoxifen induced SCs apoptosis. The corneal epithelium was then removed with an Amoils brush under anesthesia and fluorescein was used to assess healing over 4 days. (2) We performed single-cell RNA expression analysis of 10,000 cells derived from dissociated rat limbus with droplet-based high throughput 10× Genomics to identify ~3000 genes. We used the data to predict possible ligand-receptor interactions between the limbal SCs and LSC. RESULTS: (1) Ablation of SCs impaired corneal wound healing in mouse cornea, suggesting the involvement of SC in innervation-dependent corneal epithelial recovery. (2) Genomic analysis suggested the presence of paracrine crosstalk between SCs and LSCs, and relevant downstream intracellular signaling events in LSCs. The latter included activation of Notch signaling and VEGF-mediated cell migration and inhibition of apoptosis. Further expression analysis comparing the limbal region of healthy and wounded corneas indicated significant changes in the expression of jag1, Pdgfa, Tgfb1, and Ptn genes by SCs. All of these genes could potentially play a role in corneal recovery. CONCLUSIONS: Our findings (i) describe the presence of a high volume of SCs at the limbus, located in close spatial vicinity to LSCs, (ii) demonstrate the importance of the limbal SCs for corneal wound healing, and (iii) suggest the presence of paracrine SC-LSC interaction that may be responsible for the limbal nerve-mediated activation of LSCs during homeostasis or the epithelial wound healing after injury. These findings suggest new therapeutic targets for treating NK.