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The vesicant sulfur mustard (SM) is a chemical warfare agent that causes acute and chronic injury to the cornea and proximal anterior segment structures. Despite clinical evidence of SM-exposure causing unexplained retinal deficits, there have been no animal studies conducted to examine the retinal toxicity of this vesciant. The cardinal hallmark of retinal response to stressors or injury is the activation of reactive gliosis, a cellular process largely governed by Müller glia. Previously we showed that corneal exposure to sodium hydroxide elicits rapid induction of reactive gliosis and results in retinal degeneration in a dose-related manner. Based on this evidence, we hypothesized that the vesicant nitrogen mustard (NM), an analog of SM, may also elicit reactive gliosis. To test this idea, we developed a mouse model of NM ocular injury and investigated corneal and retinal effects focusing on citrullination, a posttranslational modification (PTM) of proteins. This PTM was recently linked to alkali injury and has also been shown to occur in retinal degenerative conditions. Here, we demonstrate that corneal exposure to 1% NM causes a synchronous activation of citrullination in both the cornea and retina with hypercitrullination becoming apparent temporally and manifesting with altered cellular expression characteristics. A key finding is that ocular citrullination occurs acutely as early as 1-h post-injury in both the cornea and retina, which underscores a need for expeditious interception of this acute corneal and retinal response. Moreover, exploiting dose response and temporal studies, we uncoupled NM-induced retinal citrullination from its induction of retinal gliosis. Our findings demonstrate that hypercitrullination is a common corneo-retinal mechanism that sensitizes the eye to NM injury and suggests that counteracting hypercitrullination may provide a suitable countermeasure to vesicant injury., Competing Interests: Declaration of competing interest None., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Retinal scarring with vision loss continues to be an enigma in individuals with advanced age-related macular degeneration (AMD). Müller glial cells are believed to initiate and perpetuate scarring in retinal degeneration as these glial cells participate in reactive gliosis and undergo hypertrophy. We previously showed in the murine laser-induced model of choroidal neovascularization that models wet-AMD that glial fibrillary acidic protein (GFAP) expression, an early marker of reactive gliosis, increases along with its posttranslational modification citrullination. This was related to increased co-expression of the citrullination enzyme peptidyl arginine deiminase-4 (PAD4), which also colocalizes to GFAP filaments. However, whether such hypercitrullination in Müller glial drives fibrotic pathology has remained understudied. Here, using male and female C57Bl6 mice subjected to laser injury, we investigated in a temporal study how citrullination impacts GFAP and PAD4 dynamics. We found that high molecular weight citrullinated species that accumulate in Müller glia corresponded with dynamic changes in GFAP and PAD4 showing their temporal redistribution from polymeric cytoskeletal to soluble protein fractions using immunostaining and western blot analysis. In conditional glial-specific PAD4 knockout (PAD4cKO) mice subjected to laser injury, there was a stark reduction of citrullination and of polymerized GFAP filaments. These injured PAD4cKO retinas showed improved lesion healing, as well as reduced fibronectin deposition in the subretinal space at 30 days. Taken together, these findings reveal that pathologically overexpressed PAD4 in reactive Müller glia governs GFAP filament dynamics and alters their stability, suggesting chronic PAD4-driven hypercitrullination may be a target for retinal fibrosis., (© 2022 Wiley Periodicals LLC.)

Muller glia (MG) play a central role in reactive gliosis, a stress response associated with rare and common retinal degenerative diseases, including age-related macular degeneration (AMD). The posttranslational modification citrullination​ targeting glial fibrillary acidic protein (GFAP) in MG was initially discovered in a panocular chemical injury model. Here, we report in the paradigms of retinal laser injury, a genetic model of spontaneous retinal degeneration (JR5558 mice) and human wet-AMD tissues that MG citrullination is broadly conserved. After laser injury, GFAP polymers that accumulate in reactive MG are citrullinated in MG endfeet and glial cell processes. The enzyme responsible for citrullination, peptidyl arginine deiminase-4 (PAD4), localizes to endfeet and associates with GFAP polymers. Glial cell-specific PAD4 deficiency attenuates retinal hypercitrullination in injured retinas, indicating PAD4 requirement for MG citrullination. In retinas of 1-mo-old JR5558 mice, hypercitrullinated GFAP and PAD4 accumulate in MG endfeet/cell processes in a lesion-specific manner. Finally, we show that human donor maculae from patients with wet-AMD also feature the canonical endfeet localization of hypercitrullinated GFAP. Thus, we propose that endfeet are a "citrullination bunker" that initiates and sustains citrullination in retinal degeneration., Competing Interests: Competing interest statement: J.A. is a cofounder of iVeena Holdings, iVeena Delivery Systems, and Inflammasome Therapeutics and has been a consultant for Allergan, Boehringer-Ingelheim, Immunovant, Olix Pharmaceuticals, Retinal Solutions, and Saksin LifeSciences unrelated to this work. J.A. and B.D.G. are cofounders of DiceRx., (Copyright © 2022 the Author(s). Published by PNAS.)

The cornea is the most innervated tissue in the human body. Myelinated axons upon inserting into the peripheral corneal stroma lose their myelin sheaths and continue into the central cornea wrapped by only nonmyelinating corneal Schwann cells (nm-cSCs). This anatomical organization is believed to be important for central vision. Here we employed single-cell RNA sequencing (scRNA-seq), microscopy, and transgenics to characterize these nm-cSCs of the central cornea. Using principal component analysis, uniform manifold approximation and projection, and unsupervised hierarchal cell clustering of scRNA-seq data derived from central corneal cells of male rabbits, we successfully identified several clusters representing different corneal cell types, including a unique cell cluster representing nm-cSCs. To confirm protein expression of cSC genes, we performed cross-species validation, employing corneal whole-mount immunostaining with confocal microscopy in mouse corneas. The expression of several representative proteins of nm-cSCs were validated. As the proteolipid protein 1 (PLP1) gene was also expressed in nm-cSCs, we explored the Plp1-eGFP transgenic reporter mouse line to visualize cSCs. Specific and efficient eGFP expression was observed in cSCs in adult mice of different ages. Of several putative cornea-specific SC genes identified, Dickkopf-related protein 1 was shown to be present in nm-cSCs. Taken together, our findings, for the first time, identify important insights and tools toward the study nm-cSCs in isolated tissue and adult animals. We expect that our results will advance the future study of nm-cSCs in applications of nerve repair, and provide a resource for the study of corneal sensory function., (© 2020 The Authors. Journal of Neuroscience Research published by Wiley Periodicals LLC.)

The type III intermediate filament (IF) proteins vimentin and desmin are sequentially overexpressed in stromal myofibroblasts over the period when fibrosis sets in after corneal injury. Prior findings have revealed vimentin-deficient mice are significantly protected from corneal fibrosis after alkali injury, which has implicated this IF protein as an important regulator of corneal fibrosis. It has remained as yet unproven whether desmin contributes in any significant manner to corneal fibrosis. Here we have employed desmin-deficient (Des KO) mice in the corneal alkali injury model and show that injured Des KO mice develop fibrosis and show similar levels of corneal opacity at 14 days post-injury as wild type (WT) mice and retain this phenotype even at 30d post injury. Des KO corneas from injured mice show upregulation of vimentin and alpha-smooth muscle actin expression to equivalent levels as WT corneas, illuminating that desmin deficiency does not interfere with myofibrobast differentiation. Employing the small molecule withaferin A (WFA), an inhibitor of vimentin, we show that WFA treatment causes the decrease in steady state levels of vimentin and serine 38 phosphorylated vimentin, the latter a biomarker associated with corneal fibrosis, and improved corneal clarity through blockade of myofibroblast differentiation. To investigate further the mechanism of fibrosis in desmin deficiency, we examined keratin 8 expression in the epithelium, and found reduced levels of this cytokeratin in injured Des KO corneas compared to WT corneas. This finding also corroborates the decrease of cell proliferation in injured Des KO corneas compared to that in WT corneas. The fibrotic phenotype of Des KO corneas also features abundant vascularization, further exemplifying the magnitude of corneal pathology. Together, these findings illuminate that desmin does not contribute significantly to corneal fibrosis in this injury model., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Recent studies have shown that constitutive activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) in Schwann cells (SCs) increases myelin thickness in transgenic mice. In this secondary analysis, we report that these transgenic mice develop a postnatal corneal neurofibroma with the loss of corneal transparency by age six months. We show that expansion of non-myelinating SCs, under the control of activated ERK1/2, also drive myofibroblast differentiation that derives from both SC precursors and resident corneal keratocytes. Further, these mice also harbor activated mast cells in the central cornea, which contributes to pathological corneal neovascularization and fibrosis. This breach of corneal avascularity and immune status is associated with the growth of the tumor pannus, resulting in a corneal stroma that is nearly four times its normal size. In corneas with advanced disease, some axons became ectopically myelinated, and the disruption of Remak bundles is evident. To determine whether myofibroblast differentiation was linked to vimentin, we examined the levels and phosphorylation status of this fibrotic biomarker. Concomitant with the early upregulation of vimentin, a serine 38-phosphorylated isoform of vimentin (pSer38vim) increased in SCs, which was attributed primarily to the soluble fraction of protein-not the cytoskeletal portion. However, the overexpressed pSer38vim became predominantly cytoskeletal with the growth of the corneal tumor. Our findings demonstrate an unrecognized function of ERK1/2 in the maintenance of corneal homeostasis, wherein its over-activation in SCs promotes corneal neurofibromas. This study is also the first report of a genetically engineered mouse that spontaneously develops a corneal tumor., (© 2017 Wiley Periodicals, Inc.)

Withaferin A (WFA), initially identified as a compound that inhibits experimental angiogenesis, has been shown to bind to soluble vimentin (sVim) and other type III intermediate filament (IF) proteins. We review WFA's dose-related activities (Section 1), examining nanomolar concentrations effects on sVim in cell proliferation and submicromolar effects on lamellipodia and focal adhesion formation. WFA effects on polymeric IFs are especially interesting to the study of cell migration and invasion that depend on IF mechanical contractile properties. WFA interferes with NF-κB signaling, though this anti-inflammatory mechanism may occur via perturbation of sVim-protein complexes, and possibly also via targeting IκB kinase β directly. However, micromolar concentrations that induce vimentin cleavage to promote apoptosis may increasingly show off-target effects via targeting other IFs (neurofilaments and keratin) and non-IFs (tubulin, heat-shock proteins, proteasome). Thus, in Section 2, we describe our studies combining cell cultures with animal models of injury to validate relevant type III IF-targeting mechanisms of WFA. In Section 3, we illuminate from investigating myofibroblast differentiation how sVim phosphorylation may govern cell type-selective sensitivity to WFA, offering impetus for exploring vimentin phosphorylation isoforms as targets and biomarkers of fibrosis. These different WFA targets and activities are listed in a summary table., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Vimentin is a newly recognized target for corneal fibrosis. Using primary rabbit corneal fibroblasts and myofibroblasts we show that myofibroblasts, unlike fibroblasts, display impaired cell spreading and cell polarization, which is associated with increased levels of soluble serine-38 phosphorylated vimentin (pSer38Vim). This pSer38Vim isoform is inefficiently incorporated into growing vimentin intermediate filaments (IFs) of myofibroblasts during cell spreading, and as a result, myofibroblasts maintain higher soluble pSer38Vim levels compared to fibroblasts. Moreover, the soluble vimentin-targeting small molecule and fibrotic inhibitor withaferin A (WFA) causes a potent blockade of cell spreading selectively in myofibroblasts by targeting soluble pSer38Vim for hyperphosphorylation. WFA treatment does not induce vimentin hyperphosphorylation in fibroblasts. This hyperphosphorylated pSer38Vim species in WFA-treated myofibroblasts becomes complexed with adaptor protein filamin A (FlnA), and these complexes appear as short squiggles when displaced from focal adhesions. The extracellular-signal regulated kinase (ERK) is also phosphorylated (pERK) in response to WFA, but surprisingly, pERK does not enter the nucleus but remains bound to pSer38Vim in cytoplasmic complexes. Using a model of corneal alkali injury, we show that fibrotic corneas of wild type mice possess high levels of pERK, whereas injured corneas of vimentin-deficient (Vim KO) mice that heal with reduced fibrosis have highly reduced pERK expression. Finally, WFA treatment causes a decrease in pERK and pSer38Vim expression in healing corneas of wild type mice. Taken together, these findings identify a hereto-unappreciated role for pSer38Vim as an important determinant of myofibroblast sensitivity to WFA.

Withaferin A (WFA) is a natural product that binds to soluble forms of the type III intermediate filament (IF) vimentin. Currently, it is unknown under what pathophysiological contexts vimentin is druggable, as cytoskeltal vimentin-IFs are abundantly expressed. To investigate druggability of vimentin, we exploited rabbit Tenon's capsule fibroblast (RbTCF) cell cultures and the rabbit glaucoma filtration surgical (GFS) model of fibrosis. WFA potently caused G₀/G₁ cell cycle inhibition (IC₅₀ 25 nM) in RbTCFs, downregulating ubiquitin E3 ligase skp2 and inducing p27(Kip1) expression. Transforming growth factor (TGF)-ß-induced myofibroblast transformation caused development of cell spheroids with numerous elongated invadopodia, which WFA blocked potently by downregulating soluble vimentin and α-smooth muscle actin (SMA) expression. In the pilot proof-of-concept study using the GFS model, subconjunctival injections of a low WFA dose reduced skp2 expression in Tenon's capsule and increased p27(Kip1) expression without significant alteration to vimentin-IFs. This treatment maintains significant nanomolar WFA concentrations in anterior segment tissues that correspond to WFA's cell cycle targeting activity. A ten-fold higher WFA dose caused potent downregulation of soluble vimentin and skp2 expression, but as found in cell cultures, no further increase in p27(Kip1) expression was observed. Instead, this high WFA dose potently induced vimentin-IF disruption and downregulated α-SMA expression that mimicked WFA activity in TGF-ß-treated RbTCFs that blocked cell contractile activity at submicromolar concentrations. These findings illuminate that localized WFA injection to ocular tissues exerts pharmacological control over the skp2-p27(Kip1) pathway by targeting of soluble vimentin in a model of surgical fibrosis.

The type III intermediate filaments (IFs) are essential cytoskeletal elements of mechanosignal transduction and serve critical roles in tissue repair. Mice genetically deficient for the IF protein vimentin (Vim(-/-)) have impaired wound healing from deficits in myofibroblast development. We report a surprising finding made in Vim(-/-) mice that corneas are protected from fibrosis and instead promote regenerative healing after traumatic alkali injury. This reparative phenotype in Vim(-/-) corneas is strikingly recapitulated by the pharmacological agent withaferin A (WFA), a small molecule that binds to vimentin and down-regulates its injury-induced expression. Attenuation of corneal fibrosis by WFA is mediated by down-regulation of ubiquitin-conjugating E3 ligase Skp2 and up-regulation of cyclin-dependent kinase inhibitors p27(Kip1) and p21(Cip1). In cell culture models, WFA exerts G(2)/M cell cycle arrest in a p27(Kip1)- and Skp2-dependent manner. Finally, by developing a highly sensitive imaging method to measure corneal opacity, we identify a novel role for desmin overexpression in corneal haze. We demonstrate that desmin down-regulation by WFA via targeting the conserved WFA-ligand binding site shared among type III IFs promotes further improvement of corneal transparency without affecting cyclin-dependent kinase inhibitor levels in Vim(-/-) mice. This dissociates a direct role for desmin in corneal cell proliferation. Taken together, our findings illuminate a previously unappreciated pathogenic role for type III IF overexpression in corneal fibrotic conditions and also validate WFA as a powerful drug lead toward anti-fibrosis therapeutic development.

Purpose: To develop an animal model for simultaneously eliciting corneal angiogenesis and retinal gliosis that will enable the assessment of inhibitor efficacy on these two pathological processes in separate anatomic sites of the ocular globe., Methods: Four to six week-old mice in a C57BL/6J background were anesthetized and 0.15 N NaOH was applied to the cornea, followed by mechanical scraping of the epithelium from limbus and central cornea. After this injury, mice were treated with vehicle or with an inhibitor (withaferin A [WFA]), which were delivered by intraperitoneal injection, to assess the pharmacological effects on angiogenesis and/or gliosis. Mice were sacrificed after 14 days and tissues (corneas and retinas) were prepared for analysis of corneal neovascularization and retinal gliosis by immunohistochemistry and western blotting, respectively. This protocol was also suited for studying earlier disease end points, for assessment of drug dose efficacy or genetic influences and the entire procedure and this analysis was completed in 16-17 days., Results: Both corneal angiogenesis and retinal gliosis were maximally sustained at fourteen days following chemical and mechanical injury of the cornea. 1) Injured corneas showed abundant CD31+ staining, with new blood vessels branching out from the limbus to the central cornea. WFA treatment potently inhibited corneal neovascularization. 2) Retinal gliosis in injured mice was associated with upregulated expression of glial fibrillary acidic protein (GFAP) that appeared as polymeric filaments and soluble forms expressed in reactive Müller glial cells. WFA treatment potently downregulated the expression of soluble and filamentous GFAP; the latter protein was fragmented., Conclusions: We have developed a mouse model for investigating retinal gliosis and corneal neovascularization. We used this model to demonstrate the simultaneous inhibitory effects of WFA on both of these disease processes. Retinal gliosis occurs in several major degenerative conditions of the eye, including age-related macular degeneration, where angiogenesis is also a prevailing pathological feature. Thus, inhibitors of both gliosis and angiogensis used as combination therapy are currently being explored for treatment of such complex diseases. The model presented here affords a very simple preclinical assay for screening combination of drugs or polypharmacological agents and reduces the numbers of animals because of the different anatomic sites of these pathologies. Finally, given that endogenous mediators elicit angiogenesis and gliosis in this model, the combination of genetics and pharmacology can be exploited to study drug mechanisms and for target validation in vivo.

Gliosis is a biological process that occurs during injury repair in the central nervous system and is characterized by the overexpression of the intermediate filaments (IFs) glial fibrillary acidic protein (GFAP) and vimentin. A common thread in many retinal diseases is reactive Müller cell gliosis, an untreatable condition that leads to tissue scarring and even blindness. Here, we demonstrate that the vimentin-targeting small molecule withaferin A (WFA) is a novel chemical probe of GFAP. Using molecular modeling studies that build on the x-ray crystal structure of tetrameric vimentin rod 2B domain we reveal that the WFA binding site is conserved in the corresponding domain of tetrameric GFAP. Consequently, we demonstrate that WFA covalently binds soluble recombinant tetrameric human GFAP at cysteine 294. In cultured primary astrocytes, WFA binds to and down-regulates soluble vimentin and GFAP expression to cause cell cycle G(0)/G(1) arrest. Exploiting a chemical injury model that overexpresses vimentin and GFAP in retinal Müller glia, we demonstrate that systemic delivery of WFA down-regulates soluble vimentin and GFAP expression in mouse retinas. This pharmacological knockdown of soluble IFs results in the impairment of GFAP filament assembly and inhibition of cell proliferative response in Müller glia. We further show that a more severe GFAP filament assembly deficit manifests in vimentin-deficient mice, which is partly rescued by WFA. These findings illustrate WFA as a chemical probe of type III IFs and illuminate this class of withanolide as a potential treatment for diverse gliosis-dependent central nervous system traumatic injury conditions and diseases, and for orphan IF-dependent pathologies.

The natural product withaferin A (WFA) exhibits antitumor and antiangiogenesis activity in vivo, which results from this drug's potent growth inhibitory activities. Here, we show that WFA binds to the intermediate filament (IF) protein, vimentin, by covalently modifying its cysteine residue, which is present in the highly conserved alpha-helical coiled coil 2B domain. WFA induces vimentin filaments to aggregate in vitro, an activity manifested in vivo as punctate cytoplasmic aggregates that colocalize vimentin and F-actin. WFA's potent dominant-negative effect on F-actin requires vimentin expression and induces apoptosis. Finally, we show that WFA-induced inhibition of capillary growth in a mouse model of corneal neovascularization is compromised in vimentin-deficient mice. These findings identify WFA as a chemical genetic probe of IF functions, and illuminate a potential molecular target for withanolide-based therapeutics for treating angioproliferative and malignant diseases.

The immunoproteasome, having been linked to neurodegenerative diseases and hematological cancers, has been shown to play an important role in MHC class I antigen presentation. However, its other pathophysiological functions are still not very well understood. This can be attributed mainly to a lack of appropriate molecular probes that can selectively modulate the immunoproteasome catalytic subunits. Herein, we report the development of molecular probes that selectively inhibit the major catalytic subunit, LMP2, of the immunoproteasome. We show that these compounds irreversibly modify the LMP2 subunit with high specificity. Importantly, LMP2-rich cancer cells compared to LMP2-deficient cancer cells are more sensitive to growth inhibition by the LMP2-specific inhibitor, implicating an important role of LMP2 in regulating cell growth of malignant tumors that highly express LMP2.

Acute and chronic exposure to ultraviolet (UV) wavelengths in sunlight can cause adverse reactions in exposed areas of the skin and corneas. UV exposure up-regulates the synthesis of Matrix Metalloproteinses (MMPs) and evidence suggests these enzymes mediate tissue damage. Therefore MMP gene activity can serve as a surrogate marker for bioassays. In this study, we tested the possible utility for this purpose of two stably transfected cell lines (from mouse keratinocytes and rabbit epithelial-like corneal cells) and a transgenic mouse line (line 3445), each harboring a DNA construct containing a bacterial beta-galactosidase (LacZ) reporter gene driven by the rabbit MMP-9 transcriptional promoter. We observed only a weak 2-fold maximal induction of LacZ reporter gene expression in the mouse epidermal cell line after exposure to UV-B irradiation (5, 10, 40 mJ/cm2) and no significant expression of the reporter gene in the rabbit epithelial-like cell line. Similarly negative results were obtained when primary corneal epithelial cells from human and rabbit were exposed to different doses of UV-B irradiation and endogenous MMP-9 gene expression was assayed by zymography and immunoprecipitation analysis. In contrast, when skin from 3-day-old transgenic mouse line 3445 was exposed to UV-B and UV-A, a clear dose-dependent induction of the LacZ reporter gene occurred and the location of gene expression was dependent on the wave-length of irradiation. These results suggest that line 3445 transgenic mice may serve as a useful tool to quantitatively and qualitatively assess the biological effects of UV light and the efficacy of therapeutic agents.

Purpose: To characterize the angiogenic and inflammatory responses of human choroidal endothelial cells (HCECs) to stimulators and inhibitors of the ubiquitin proteasome pathway (UPP)., Methods: The regulation of the UPP by the inhibitor withaferin A and its congener, withanolide D, two natural products derived from the medicinal plant Withania somnifera was assessed in the three-dimensional endothelial cell sprouting assay (3D-ECSA), by using HCEC- and human umbilical vein endothelial cell (HUVEC)-derived spheroids embedded in a collagen I matrix. Western blot analysis was used to investigate the effect of withanolides on IkappaB-alpha, polyubiquitination, and heme oxygenase (HO)-1 regulation in HCEC and HUVEC cultures., Results: HCECs, like HUVECs, responded to fibroblast growth factor-2, vascular endothelial growth factor, and tumor necrosis factor (TNF)-alpha stimulation and sprouted vessel-like structures in collagen I matrix. However, HCECs were slower to generate these sprouting vessels, when compared with HUVECs. The extent of inhibition of endothelial cell sprouting in 3D matrix, the blockade of TNF-alpha-induced IkappaB-alpha degradation, levels of global polyubiquitinated proteins, and induced production of HO-1 in response to treatment by the withanolides in cultured endothelial cells was similarly regulated between HCECs and HUVECs., Conclusions: HCECs share with HUVECs a similar response to UPP inhibitors, suggesting that this well-conserved pathway that regulates angioinflammatory mechanisms could be exploited for drug-targeting in the development of novel agents for CNV treatment.

The natural product withaferin A (WFA) is a potent angiogenesis inhibitor and it targets the ubiquitin-proteasome pathway in vascular endothelial cells. We generated a biotinylated affinity analog WFA-LC(2)B for use as a probe to study angiogenesis. WFA-LC(2)B inhibits angiogenic sprouting in vitro and it causes levels of ubiquitinated proteins to increase in tumor necrosis factor-alpha-treated human umbilical vein endothelial cells, confirming the retention of WFA's biological activity. We show that WFA-LC(2)B forms protein adducts in endothelial cells which are competed by free WFA in vivo. This WFA-LC(2)B analog will be useful to isolate the biological target of WFA.

Small molecules designed to specifically activate or inactivate protein functions have been useful to study biological processes. PROTACS are small molecule chimera which comprise a ligand and a peptide recognition motif for an E3 ligase. These novel reagents exploit the ubiquitin-mediated proteasome degradation pathway to target the ligand-bound protein for intracellular degradation. Here, we report that an estrogen receptor (ER)-targeting PROTACS that causes degradation of ER is able to potently inhibit endothelial cell differentiation in a three-dimensional angiogenic sprouting assay. These findings support the use of ER-targeting PROTACS as probes of angiogenesis.

Purpose: Expression of the genes for collagenase and interleukin-1alpha (IL-1alpha) are induced as stromal cells become activated to the repair fibroblast phenotype after injury to the cornea. This investigation examines the mechanisms whereby expression of these genes is inhibited by transforming growth factor-beta (TGF-beta), dexamethasone (DEX), or retinoic acid (RET A)., Methods: A model of freshly isolated cultures of corneal stromal cells and early passage cultures of corneal fibroblasts was used in these studies. This model reproduces the events of stromal cell activation in the corneal wound., Results: In early passage cultures of corneal fibroblasts, expression of collagenase is under obligatory control by autocrine IL-1alpha. IL-1alpha controls its own expression through an autocrine feedback loop that is dependent on transcription factor NF-kappaB. TGF-beta, DEX, and RET A were each effective inhibitors of collagenase gene expression in these cells. Furthermore, these agents have the capacity to inhibit expression of IL-1alpha and this was correlated with their ability to affect DNA-binding activity of NF-kappaB. However, TGF-beta, DEX, and RET A were also effective inhibitors of the low level of collagenase expressed by freshly isolated corneal stromal cells that cannot express IL-1alpha., Conclusions: In cells with an active IL-1alpha autocrine loop there are at least two distinct signaling pathways by which collagenase gene expression can be modulated. The results of this study demonstrate that TGF-beta, DEX, and RET A differentially inhibit collagenase and IL-1alpha gene expression. This information will be useful in the design of therapeutic modalities for fibrotic disease in the cornea and other parts of the eye.

Purpose: The matrix metalloproteinase gelatinase B is synthesized by cells at the leading edge of the corneal epithelium migrating to heal a wound. Recent data from the authors' laboratory suggest that excessive synthesis contributes to repair defects. The goal of the study reported here was to investigate mechanisms controlling gelatinase B production by corneal epithelial cells., Methods: Freshly isolated cultures of corneal epithelial cells and early passage stromal fibroblasts from rabbit were used for these studies., Results: In a previous study, it was found that the cytokine interleukin (IL)-1alpha is released into the culture medium of corneal epithelial cells more efficiently when they are plated at low density with limited cell-cell contact than when plated at high density. In this study, we show that production of gelatinase B by these cells is similarly affected by cell plating density. However, it is further demonstrated that these two events are not dependent on one another but occur in parallel: IL-1alpha does not regulate gelatinase B production (synthesis), nor was there evidence that any other secreted autocrine cytokine acts as mediator. Instead, our data suggest that gelatinase B production is downregulated directly by high cell density and indicate a connection to the level of protein kinase C activity. Nevertheless, the anticancer agent suramin, which blocks collagenase synthesis by interfering with autocrine cytokine-receptor interactions, still inhibits synthesis of gelatinase B., Conclusions: Unlike collagenase synthesis by corneal stromal fibroblasts, production (synthesis) of gelatinase B does not appear to be controlled by secreted autocrine cytokines but can still be inhibited by suramin. Suramin may make an effective therapeutic agent for controlling pathologic overproduction of gelatinase B in corneal ulcers.