Compartmentalized citrullination in Muller glial endfeet during retinal degeneration.

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.

ER-stress response in retinal Müller glia occurs significantly earlier than amyloid pathology in the Alzheimer's mouse brain and retina.

Alzheimer's Disease (AD) pathogenesis is thought to begin up to 20 years before cognitive symptoms appear, suggesting the need for more sensitive diagnostic biomarkers of AD. In this report, we demonstrated pathological changes in retinal Müller glia significantly earlier than amyloid pathology in AD mouse models. By utilizing the knock-in NLGF mouse model, we surprisingly discovered an increase in reticulon 3 (RTN3) protein levels in the NLGF retina as early as postnatal day 30 (P30). Despite RTN3 being a canonically neuronal protein, this increase was noted in the retinal Müller glia, confirmed by immunohistochemical characterization. Further unbiased transcriptomic assays of the P30 NLGF retina revealed that retinal Müller glia were the most sensitive responding cells in this mouse retina, compared with other cell types including photoreceptor cells and ganglion neurons. Pathway analyses of differentially expressed genes in glia cells showed activation of ER stress response via the upregulation of unfolded protein response (UPR) proteins such as ATF4 and CHOP. Early elevation of RTN3 in response to challenges by toxic Aß likely facilitated UPR. Altogether, these findings suggest that Müller glia act as a sentinel for AD pathology in the retina and should aid for both intervention and diagnosis.

Peptidyl arginine deiminase 4 deficiency protects against subretinal fibrosis by inhibiting Müller glial hypercitrullination.

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.

A corneo-retinal hypercitrullination axis underlies ocular injury to nitrogen mustard.

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.

Corneal nonmyelinating Schwann cells illuminated by single-cell transcriptomics and visualized by protein biomarkers.

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.

Desmin deficiency is not sufficient to prevent corneal fibrosis.

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.

Expression of peptidylarginine deiminase 4 in an alkali injury model of retinal gliosis.

Citrullination is an important posttranslational modification that occurs during retinal gliosis. We examined the expression of peptidyl arginine deiminases (PADs) to identify the PADs that mediate citrullination in a model of alkali-induced retinal gliosis. Mouse corneas were exposed to 1.0 N NaOH and posterior eye tissue from injured and control uninjured eyes was evaluated for transcript levels of various PADs by reverse-transcription polymerase chain reaction (RT-PCR), and quantitative RT-PCR (qPCR). Retinas were also subjected to immunohistochemistry (IHC) for glial fibrillary acidic protein (GFAP), citrullinated species, PAD2, and PAD4 and tissue levels of GFAP, citrullinated species, and PAD4 were measured by western blots. In other experiments, the PAD4 inhibitor streptonigrin was injected intravitreally into injured eyes ex vivo to test inhibitory activity in an organ culture system. We found that uninjured retina and choroid expressed Pad2 and Pad4 transcripts. Pad4 transcript levels increased by day 7 post-injury (p < 0.05), whereas Pad2 levels did not change significantly (p > 0.05) by qPCR. By IHC, PAD2 was expressed in uninjured eyes along ganglion cell astrocytes, but in injured retina PAD2 was downregulated at 7 days. On the other hand, PAD4 showed increased staining in the retina upon injury revealing a pattern that overlapped with filamentous GFAP staining in Müller glial processes by 7 days. Injury-induced citrullination and soluble GFAP protein levels were reduced by PAD4 inhibition in western blot experiments of organ cultures. Together, our findings for the first time identify PAD4 as a novel injury-inducible druggable target for retinal gliosis.

Sustained activation of ERK1/2 MAPK in Schwann cells causes corneal neurofibroma.

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.

Citrullination of glial intermediate filaments is an early response in retinal injury.

PURPOSE: A hallmark of retinal gliosis is the increased detection and modification of the type III intermediate filament (IF) proteins vimentin and glial fibrillary acidic protein (GFAP). Here, we investigated vimentin and GFAP in Müller glia in a mouse model of alkali injury, focusing on the posttranslational modification of citrullination. METHODS: Mice were injured by corneal exposure to 1.0 N NaOH, and eyes were enucleated at different time points following injury. The levels of soluble and cytoskeletal forms of IF proteins and citrullination were measured using western blot analysis. Citrullinated GFAP was identified by immunoprecipitation followed by two-dimensional (2D) isoelectric focusing-polyacrylamide gel electrophoresis (IEF-PAGE) western blotting using a specific antibody that recognizes citrullinated GFAP. Vimentin, GFAP, and citrullinated proteins were localized in the retina by immunohistochemistry (IHC). Drug treatments were investigated in retinal explant cultures of posterior eyecups obtained from mouse eyes that were injured in vivo. RESULTS: Detection of GFAP in injured retinas increased over a period of 1 to 7 days, showing increased levels in both soluble and cytoskeletal forms of this IF protein. The global level of citrullinated proteins was also induced over this period, with low-salt buffer extraction showing the most abundant early changes in citrullination. Using IHC, we found that GFAP filaments assembled at Müller glial end feet, growing in size with time through the inner layers of the retina at 1-3 h postinjury. Interestingly, over this early time period, levels of soluble citrullinated proteins also increased within the retina, as detected by western blotting, coincident with the localization of the citrullinated epitopes on growing GFAP filaments and existing vimentin filaments by 3 h after injury. Taking advantage of the in vivo injury model to promote a robust gliotic response, posterior eyecups from 7-day postinjured eyes were treated in explant cultures with the peptidyl arginine deiminase inhibitor Cl-amidine, which was found to reduce global citrullination. Surprisingly, the detection of injury-induced high-molecular-weight GFAP species containing citrullinated epitopes was also reduced by Cl-amidine treatment. Using a low dose of the potent type III IF drug withaferin A (WFA), we showed that Cl-amidine treatment in combination with WFA reduced global protein citrullination further, suggesting that GFAP may be a key component of pathological citrullinated targets. CONCLUSIONS: Our findings illuminate citrullination as a potential novel target for trauma-induced retinal gliosis. We also propose that strategies for combining drugs targeting type III IFs and citrullination may potentiate tissue repair, which is an idea that needs to be validated in vivo.

Early retinal inflammatory biomarkers in the middle cerebral artery occlusion model of ischemic stroke.

PURPOSE: The transient middle cerebral artery occlusion (MCAO) model of stroke is one of the most commonly used models to study focal cerebral ischemia. This procedure also results in the simultaneous occlusion of the ophthalmic artery that supplies the retina. Retinal cell death is seen days after reperfusion and leads to functional deficits; however, the mechanism responsible for this injury has not been investigated. Given that the eye may have a unique ocular immune response to an ischemic challenge, this study examined the inflammatory response to retinal ischemia in the MCAO model. METHODS: Young male C57B/6 mice were subjected to 90-min transient MCAO and were euthanized at several time points up to 7 days. Transcription of inflammatory cytokines was measured with quantitative real-time PCR, and immune cell activation (e.g., phagocytosis) and migration were assessed with ophthalmoscopy and flow cytometry. RESULTS: Observation of the affected eye revealed symptoms consistent with Horner's syndrome. Light ophthalmoscopy confirmed the reduced blood flow of the retinal arteries during occlusion. CX3CR1-GFP reporter mice were then employed to evaluate the extent of the ocular microglia and monocyte activation. A significant increase in green fluorescent protein (GFP)-positive macrophages was seen throughout the ischemic area compared to the sham and contralateral control eyes. RT-PCR revealed enhanced expression of the monocyte chemotactic molecule CCL2 early after reperfusion followed by a delayed increase in the proinflammatory cytokine TNF-α. Further analysis of peripheral leukocyte recruitment by flow cytometry determined that monocytes and neutrophils were the predominant immune cells to infiltrate at 72 h. A transient reduction in retinal microglia numbers was also observed, demonstrating the ischemic sensitivity of these cells. Blood-eye barrier permeability to small and large tracer molecules was increased by 72 h. Retinal microglia exhibited enhanced phagocytic activity following MCAO; however, infiltrating myeloid cells were significantly more efficient at phagocytizing material at all time points. Immune homeostasis in the affected eye was largely restored by 7 days. CONCLUSIONS: This work demonstrates that there is a robust inflammatory response in the eye following MCAO, which may contribute to a worsening of retinal injury and visual impairment. These results mirror what has been observed in the brain after MCAO, suggesting a conserved inflammatory signaling response to ischemia in the central nervous system. Imaging of the eye may therefore serve as a useful non-invasive prognostic indicator of brain injury after MCAO. Future studies are needed to determine whether this inflammatory response is a potential target for therapeutic manipulation in retinal ischemia.

Corneal antifibrotic switch identified in genetic and pharmacological deficiency of vimentin.

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.

Withaferin A targets intermediate filaments glial fibrillary acidic protein and vimentin in a model of retinal gliosis.

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.

A robust model for simultaneously inducing corneal neovascularization and retinal gliosis in the mouse eye.

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.

LMP2-specific inhibitors: chemical genetic tools for proteasome biology.

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.

The tumor inhibitor and antiangiogenic agent withaferin A targets the intermediate filament protein vimentin.

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.

Cell lines and transgenic mice expressing a matrix metalloproteinase-9 promoter-driven reporter gene: potential for assay of ultraviolet light effects and light-inhibiting compounds.

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.

Small molecule anti-angiogenic probes of the ubiquitin proteasome pathway: potential application to choroidal neovascularization.

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 Use of Withaferin A to Study Intermediate Filaments.

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 ß 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.

Vimentin Phosphorylation Underlies Myofibroblast Sensitivity to Withaferin A In Vitro and during Corneal Fibrosis.

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.