Royce Mohan

Withaferin A is a potent inhibitor of angiogenesis.

The medicinal plant Withania somnifera is widely researched for its anti-inflammatory, cardioactive and central nervous system effects. In Ayurveda, the major Traditional Indian medicine system, extracts from W. somnifera are distinctively employed for the treatment of arthritis and menstrual disorders. Because these conditions involve angiogenic processes we hypothesized that the W. somnifera extracts might contain angiogenesis inhibitors. We employed an endothelial cell-sprouting assay to monitor the purification of substances from W. somnifera root extracts and isolated as the active principle the previously known natural product withaferin A. We show that withaferin A inhibits human umbilical vein endothelial cell (HUVEC) sprouting in three-dimensional collagen-I matrix at doses which are relevant to NF-kappa B-inhibitory activity. Withaferin A inhibits cell proliferation in HUVECs (IC50=12 nM) at doses that are significantly lower than those required for tumor cell lines through a process associated with inhibition of cyclin D1 expression. We propose that the inhibition of NF-kappa B by withaferin A in HUVECs occurs by interference with the ubiquitin-mediated proteasome pathway as suggested by the increased levels of poly-ubiquitinated proteins. Finally, withaferin A is shown to exert potent anti-angiogenic activity in vivo at doses that are 500-fold lower than those previously reported to exert anti-tumor activity in vivo. In conclusion, our findings identify a novel mode of action of withaferin A, which highlights the potential use of this natural product for cancer treatment or prevention.

Proteolytic mechanisms in corneal ulceration and repair

Abstract Corneal stromal ulceration is a devastating disorder that can cause blindness. Stromal ulceration was once thought to be a physical dissolution process, which even now is described as “melting.” However, a major paradigm change occurred about 25 years ago with the demonstration of extracellular matrix-degrading activity associated with tissues isolated from ulcerating corneas. Recent studies have identified the enzymes involved as specific members of the matrix metalloproteinase (MMP) family. These studies have further provided evidence that MMPs participate at all stages of the ulcerative process, from formation of the initiating epithelial defect to ulcer resolution and repair. Roles for MMPs in these processes are discussed in this review. Studies on corneal ulceration provide basic information about failure to heal, which is useful for understanding mechanisms common to other organ systems besides the cornea.

Gelatinase B/lacZ Transgenic Mice, a Model for Mapping Gelatinase B Expression during Developmental and Injury-related Tissue Remodeling

Matrix metalloproteinases (MMPs) drive normal tissue remodeling and are implicated in a wide range of pathologies. Although MMP activity is controlled at multiple levels, the primary regulation of MMP activity is transcriptional. The transcriptional promoter elements required for MMP gene expression in cultured cells have been defined, but this has not been extended to the in vivo situation. In this paper, we show that the DNA sequences between −522 and +19 of the rabbit gelatinase B gene (MMP-9) (as characterized in the transgenic mouse line 3445) constitute a minimal promoter that drives appropriate developmental and injury-induced reporter gene expression in transgenic mice. We further show that the expression and activity of three transcription factors (NF-κB, AP-2, and Sp1) that control the activity of the gelatinase B promoter are selectively induced in the epithelium migrating to heal a wound. Although promoter activity parallels expression of the endogenous gene in cell cultures, we show by several criteria that cell cultures cannot model many aspects of promoter regulation in vivo. This study reveals that the transgenic mouse line 3445 might be a useful model for investigating the regulation of gelatinase B expressionin vivo and for identifying and characterizing new drugs that can control gelatinase B gene transcription.

Developmental and tissue-specific expression of a tomato anionic peroxidase (tap1) gene by a minimal promoter, with wound and pathogen induction by an additional 5'-flanking region.

The tomato anionic peroxidase genes (tap1 and tap2) are induced by wounding and pathogen attack. The 5′-flanking region of tap1 confers wound- and pathogen-inducible β-glucuronidase (GUS) expression in tobacco plants transformed with a tap1/GUS chimeric fusion gene construct. A series of nested 5′ promoter deletions in the tap1/GUS fusion gene construct was created, and introduced into tobacco protoplasts via polyethylene glycol-mediated DNA transfer. A −202 construct (where the transcriptional start site is denoted +1) and larger tap1 promoter constructs showed constitutive GUS expression. A 2-fold increase in GUS expression over the high constitutive levels was observed with −358 bp and larger tap1 constructs when protoplasts were incubated with elicitor preparations from Verticillium albo-atrum. In tobacco plants transformed with the tap1 promoter deletion/GUS fusion gene constructs, wounding caused induction of GUS expression by 20 h that increased 6- to 18-fold by 72 h. The region between −202 and −358 of the tap1 promoter conferred wound responsiveness. GUS was also found to be expressed in the epidermis and trichomes in the aerial parts of transgenic plants. High-level GUS expression was observed in the nodal region of stems that was associated with the leaf traces. GUS that was absent in very young flower buds was found in the subsequent developmental stages in the pistils, ovaries and anthers. The developmentally regulated tissue-specific expression of GUS was found with all constructs containing the −202 and larger promoters whereas wound and pathogen induction required −358 or larger promoter. These results suggest that the tap1 gene, which was heretofore thought to be expressed only upon wounding or pathogen attack, plays a role in normal developmental processes of the plant and this gene acquired additional 5′-flanking promoter for the purpose of responding to wounding and fungal attack.

Induction of a tomato anionic peroxidase gene (tap1) by wounding in transgenic tobacco and activation of tap1/GUS and tap2/GUS chimeric gene fusions in transgenic tobacco by wounding and pathogen attack.

The anionic peroxidase genes of tomato, tap1 and tap2, are induced by wounding in tomato fruits and by elicitor treatment in cell suspension cultures. These homologous genes code for anionic peroxidases that are postulated to cause polymerization of the phenolic residues into wall polymers in wound-healing and pathogen-infected tissues. An expression construct containing the entire TAP1 gene with its 5′ and 3′ flanking sequences was introduced into tobacco by Agrobacterium tumefaciens-mediated gene transfer. Also, constructs containing the 5′ upstream regions of tap1 and tap2 including sequences coding for their respective putative leader peptides fused translationally to the β-glucuronidase (GUS) reporter gene were made and introduced into tobacco. Northern blot analysis of transcripts from wound-healing leaf tissues of transformants containing tap1 showed that the introduced gene was being transcribed in the heterologous host. The induction of tap1 transcripts in the wound-healing transgenic tobacco tissues was observed by 48 h and increased over time period of 84 h. Wounding also led to expression of GUS in tap1/GUS and tap2/GUS transformants and GUS activity was localized to the wound site. Activation of the tap1 and tap2 promoters in wound-healing transgenic tobacco tissues showed a GUS expression profile that correlated with the postulated role for anionic peroxidases in phenolic polymerization in suberizing tissues. Inoculation of tap1/GUS and tap2/GUS transformant leaves with fungal conidia from Fusarium solani f. sp. pisi caused expression of GUS in locally inoculated regions, and GUS expression increased over a period of four days.

Small-molecule inhibitors of the cell cycle

The cell cycle remains an attractive target for the development of small-molecule inhibitors for use as both novel chemotherapeutics and research probes. Given the importance of cytoskeletal dynamics and cyclin-dependent kinases for cell-cycle progression, much interest has focused on the identification of anti-mitotic agents and kinase inhibitors. However recent advances in cell-based screening technologies and an increased interest in inhibitors with greater specificity are beginning to influence the search for novel cell-cycle inhibitors.

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 G0/G1 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.

Corneal Antifibrotic Switch Identified in Genetic and Pharmacological Deficiency of Vimentin

Background: Withaferin A (WFA) is a vimentin-targeting inhibitor that has potent anti-proliferative activity. Results: WFA protects against corneal fibrosis by down-regulating injury-induced vimentin to exert epithelial cell cycle arrest and inhibit myofibroblast expression, which is a mechanism closely mimicked in vimentin-deficient mice during injury healing. Conclusion: Vimentin is a novel fibrosis target. Significance: Ocular fibrotic conditions that overexpress vimentin could be treatable with WFA. 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 p27Kip1 and p21Cip1. In cell culture models, WFA exerts G2/M cell cycle arrest in a p27Kip1- 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 Effectively Targets Soluble Vimentin in the Glaucoma Filtration Surgical Model of Fibrosis

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 Tenons 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 p27Kip1 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 Tenons capsule and increased p27Kip1 expression without significant alteration to vimentin-IFs. This treatment maintains significant nanomolar WFA concentrations in anterior segment tissues that correspond to WFAs 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 p27Kip1 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-p27Kip1 pathway by targeting of soluble vimentin in a model of surgical fibrosis.

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.