Browsing by Author "Sishtla, Kamakshi"
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Item An improved method for murine laser-induced choroidal neovascularization lesion quantification from optical coherence tomography images(Elsevier, 2022-08-02) Jensen, Nathan R.; Lambert-Cheatham, Nathan; Hartman, Gabriella D.; Muniyandi, Anbukkarasi; Park, Bomina; Sishtla, Kamakshi; Corson, Timothy W.; Ophthalmology, School of MedicineLaser-induced choroidal neovascularization (L-CNV) in murine models is a standard method for assessing therapies, genetics, and mechanisms relevant to the blinding eye disease neovascular or "wet" age-related macular degeneration. The ex vivo evaluation of these lesions involves confocal microscopy analysis. In vivo evaluation via optical coherence tomography (OCT) has previously been established and allows longitudinal assessment of lesion development. However, to produce robust data, evaluation of many lesions may be required, which can be a slow, arduous process. A prior, manual method for quantifying these lesions as ellipsoids from orthogonal OCT images was effective but time consuming. We therefore developed an OCT lesion quantification that is simplified, streamlined, and less time-consuming.Item Antiangiogenic Activity and Cytotoxicity of Triterpenoids and Homoisoflavonoids from Massonia pustulata and Massonia bifolia(Thieme, 2018-07) Schwikkard, Sianne L.; Whitmore, Hannah; Corson, Timothy W.; Sishtla, Kamakshi; Langat, Moses K.; Carew, Mark; Mulholland, Dulcie A.; Ophthalmology, School of MedicineThe Hyacinthaceae family (sensu APGII), with approximately 900 species in around 70 genera, plays a significant role in traditional medicine in Africa as well as across Europe and the Middle and Far East. The dichloromethane extract of the bulbs of Massonia pustulata (Hyacinthaceae sensu APGII) yielded two known homoisoflavonoids, (R)-5-hydroxy-3-(4-hydroxybenzyl)-7-methoxy-4-chromanone 1 and 5-hydroxy-3-(4-hydroxybenzyl)-7-methoxy-4-chromone 2 and four spirocyclic nortriterpenoids, eucosterol 3, 28-hydroxyeucosterol 4 and two previously unreported triterpenoid derivatives, (17S,23S)-17α,23-epoxy-3β,22β,29-trihydroxylanost-8-en-27,23-olide 5, and (17S, 23S)-17α,23-epoxy-28,29-dihydroxylanost-8-en-3-on-27,23-olide 6. Compounds 1, 2, 3, and 5 were assessed for cytotoxicity against CaCo-2 cells using a neutral red uptake assay. Compounds 1, 2, and 5 reduced cell viability by 70% at concentrations of 30, 100, and 100 µM, respectively. Massonia bifolia yielded three known homoisoflavonoids, (R)-(4′-hydroxy)-5-hydroxy-7-methoxy-4-chromanone 1, (R)-(4′-hydroxy)-5,7-dihydroxy-4-chromanone 7 and (R)-(3′-hydroxy-4′-methoxy)-5,7-dihydroxy-4-chromanone 9, two previously unreported homoisoflavonoids, (E)-3-benzylidene-(3′,4′-dihydroxy)-5-hydroxy-7-methoxy-4-chromanone 8 and (R)-(3′,4′-dihydroxy)-5-hydroxy-7-methoxy-4-chromanone 10, and a spirocyclic nortriterpenoid, 15-deoxoeucosterol 11. Compounds 1, 1Ac, 7, 8, 9, and 10 were screened for antiangiogenic activity against human retinal microvascular endothelial cells. Some compounds showed dose-dependent antiproliferative activity and blocked endothelial tube formation, suggestive of antiangiogenic activity.Item The Antiangiogenic Activity of Naturally-occurring and synthetic Homoisoflavonoids from the Hyacinthaceae (sensu APGII)(American Chemical Society, 2019-04-05) Schwikkard, Sianne; Whitmore, Hannah; Sishtla, Kamakshi; Sulaiman, Rania S.; Shetty, Trupti; Basavarajappa, Halesha D.; Waller, Catherine; Alqahtani, Alaa; Frankemoelle, Lennart; Chapman, Andy; Crouch, Neil; Wetschnig, Wolfgang; Knirsch, Walter; Andriantiana, Jacky; Mas-Claret, Eduard; Langat, Moses K.; Mulholland, Dulcie; Corson, Timothy W.; Ophthalmology, School of MedicineExcessive blood vessel formation in the eye is implicated in wet age-related macular degeneration, proliferative diabetic retinopathy, neovascular glaucoma, and retinopathy of prematurity, which are major causes of blindness. Small molecule antiangiogenic drugs are strongly needed to supplement existing biologics. Homoisoflavonoids have been previously shown to have potent antiproliferative activities in endothelial cells over other cell types. Moreover, they demonstrated a strong antiangiogenic potential in vitro and in vivo in animal models of ocular neovascularization. Here, we tested the antiangiogenic activity of a group of naturally occurring homoisoflavonoids isolated from the family Hyacinthaceae and related synthetic compounds, chosen for synthesis based on structure-activity relationship observations. Several compounds showed interesting antiproliferative and antiangiogenic activities in vitro on retinal microvascular endothelial cells, a disease-relevant cell type, with the synthetic chromane, 46, showing the best activity (GI50 of 2.3 × 10-4 μM).Item Bufadienolides and anti-angiogenic homoisoflavonoids from Rhodocodon cryptopodus, Rhodocodon rotundus and Rhodocodon cyathiformis(Elsevier, 2020) Whitmore, Hannah; Sishtla, Kamakshi; Knirsch, Walter; Andriantiana, Jacky L.; Schwikkard, Sianne; Mas-Claret, Eduard; Nassief, Sarah M.; Isyaka, Sani M.; Corson, Timothy W.; Mulholland, Dulcie A.; Ophthalmology, School of MedicineBackground Homoisoflavonoids have been shown to have potent anti-proliferative activities in endothelial cells over other cell types and have demonstrated a strong antiangiogenic potential in vitro and in vivo in animal models of ocular neovascularization. Three species of Rhodocodon (Scilloideaea subfamily of the Asparagaceae family), endemic to Madagascar, R. cryptopodus, R. rotundus and R. cyathiformis, were investigated. Purpose To isolate and test homoisoflavonoids for their antiangiogenic activity against human retinal microvascular endothelial cells (HRECs), as well as specificity against other ocular cell lines. Methods Plant material was extracted at room temperature with EtOH. Compounds were isolated using flash column chromatography and were identified using NMR and CD spectroscopy and HRESIMS. Compounds were tested for antiproliferative effects on primary human microvascular retinal endothelial cells (HRECs), ARPE19 retinal pigment epithelial cells, 92–1 uveal melanoma cells, and Y79 retinoblastoma cells. HRECs exposed to compounds were also tested for migration and tube formation ability. Results Two homoisoflavonoids, 3S-5,7-dihydroxy-(3′-hydroxy-4′-methoxybenzyl)-4-chromanone (1) and 3S-5,7-dihydroxy-(4′-hydroxy-3′-methoxybenzyl)-4-chromanone (2), were isolated along with four bufadienolides. Compound 1 was found to be non-specifically antiproliferative, with GI50 values ranging from 0.21–0.85 μM across the four cell types, while compound 2 showed at least 100-fold specificity for HRECs over the other tested cell lines. Compound 1, with a 3S configuration, was 700 times more potent that the corresponding 3R enantiomer recently isolated from a Massonia species. Conclusion Select homoisoflavonoids have promise as antiangiogenic agents that are not generally cytotoxic.Item Design, synthesis and biological evaluation of photoaffinity probes of antiangiogenic homoisoflavonoids(Elsevier, 2016-09) Lee, Bit; Sun, Wei; Lee, Hyungjun; Basavarajappa, Halesha; Sulaiman, Rania S.; Sishtla, Kamakshi; Fei, Xiang; Corson, Timothy W.; Seo, Seung-Yong; Department of Ophthalmology, IU School of MedicineA naturally occurring homoisoflavonoid, cremastranone (1) inhibited angiogenesis in vitro and in vivo. We developed an analogue SH-11037 (2) which is more potent than cremastranone in human retinal microvascular endothelial cells (HRECs) and blocks neovascularization in animal models. Despite their efficacy, the mechanism of these compounds is not yet fully known. In the course of building on a strong foundation of SAR and creating a novel chemical tool for target identification of homoisoflavonoid-binding proteins, various types of photoaffinity probes were designed and synthesized in which benzophenone and biotin were attached to homoisoflavanonoids using PEG linkers on either the C-3′ or C-7 position. Notably, the photoaffinity probes linking on the phenol group of the C-3′ position retain excellent activity of inhibiting retinal endothelial cell proliferation with up to 72 nM of GI50.Item Enantioselective Synthesis of Homoisoflavanones by Asymmetric Transfer Hydrogenation and Their Biological Evaluation for Antiangiogenic Activity(ACS Publications, 2019-08-05) Heo, Myunghoe; Lee, Bit; Sishtla, Kamakshi; Fei, Xiang; Lee, Sanha; Park, Soojun; Yuan, Yue; Lee, Seul; Kwon, Sangil; Lee, Jungeun; Kim, Sanghee; Corson, Timothy W.; Seo, Seung-Yong; Ophthalmology, School of MedicineNeovascular eye diseases are a major cause of blindness. Excessive angiogenesis is a feature of several conditions, including wet age-related macular degeneration, proliferative diabetic retinopathy, and retinopathy of prematurity. Development of novel anti-angiogenic small molecules for the treatment of neovascular eye disease is essential to provide new therapeutic leads for these diseases. We have previously reported the therapeutic potential of anti-angiogenic homoisoflavanone derivatives with efficacy in retinal and choroidal neovascularization models, although these are racemic compounds due to the C3-stereogenic center in the molecules. This work presents asymmetric synthesis and structural determination of anti-angiogenic homoisoflavanones and pharmacological characterization of the stereoisomers. We describe an enantioselective synthesis of homoisoflavanones by virtue of ruthenium-catalyzed asymmetric transfer hydrogenation accompanying dynamic kinetic resolution, providing a basis for the further development of these compounds into novel experimental therapeutics for neovascular eye diseases.Item Heme Synthesis Inhibition Blocks Angiogenesis via Mitochondrial Dysfunction(Elsevier, 2020-07-19) Shetty, Trupti; Sishtla, Kamakshi; Park, Bomina; Repass, Matthew J.; Corson, Timothy W.; Ophthalmology, School of MedicineThe relationship between heme metabolism and angiogenesis is poorly understood. The final synthesis of heme occurs in mitochondria, where ferrochelatase (FECH) inserts Fe2+ into protoporphyrin IX to produce proto-heme IX. We previously showed that FECH inhibition is antiangiogenic in human retinal microvascular endothelial cells (HRECs) and in animal models of ocular neovascularization. In the present study, we sought to understand the mechanism of how FECH and thus heme is involved in endothelial cell function. Mitochondria in endothelial cells had several defects in function after heme inhibition. FECH loss changed the shape and mass of mitochondria and led to significant oxidative stress. Oxidative phosphorylation and mitochondrial Complex IV were decreased in HRECs and in murine retina ex vivo after heme depletion. Supplementation with heme partially rescued phenotypes of FECH blockade. These findings provide an unexpected link between mitochondrial heme metabolism and angiogenesis.Item Identification of Novel Pathways Regulated by APE1/Ref-1 in Human Retinal Endothelial Cells(MDPI, 2023-01) Mijit, Mahmut; Liu, Sheng; Sishtla, Kamakshi; Hartman, Gabriella D.; Wan, Jun; Corson, Timothy W.; Kelley, Mark R.; Ophthalmology, School of MedicineAPE1/Ref-1 (apurinic/apyrimidinic endonuclease 1, APE1 or APEX1; redox factor-1, Ref-1) is a dual-functional enzyme with crucial roles in DNA repair, reduction/oxidation (redox) signaling, and RNA processing and metabolism. The redox function of Ref-1 regulates several transcription factors, such as NF-κB, STAT3, HIF-1α, and others, which have been implicated in multiple human diseases, including ocular angiogenesis, inflammation, and multiple cancers. To better understand how APE1 influences these disease processes, we investigated the effects of APEX1 knockdown (KD) on gene expression in human retinal endothelial cells. This abolishes both DNA repair and redox signaling functions, as well as RNA interactions. Using RNA-seq analysis, we identified the crucial signaling pathways affected following APEX1 KD, with subsequent validation by qRT-PCR. Gene expression data revealed that multiple genes involved in DNA base excision repair, other DNA repair pathways, purine or pyrimidine metabolism signaling, and histidine/one carbon metabolism pathways were downregulated by APEX1 KD. This is in contrast with the alteration of pathways by APEX1 KD in human cancer lines, such as pancreatic ductal adenocarcinoma, lung, HeLa, and malignant peripheral nerve sheath tumors. These results highlight the unique role of APE1/Ref-1 and the clinical therapeutic potential of targeting APE1 and pathways regulated by APE1 in the eye. These findings provide novel avenues for ocular neovascularization treatment.Item Kif14 overexpression accelerates murine retinoblastoma development(Wiley, 2016-10) O'Hare, Michael; Shadmand, Mehdi; Sulaiman, Rania S.; Sishtla, Kamakshi; Sakisaka, Toshiaki; Corson, Timothy W.; Department of Ophthalmology, IU School of MedicineThe mitotic kinesin KIF14 has an essential role in the recruitment of proteins required for the final stages of cytokinesis. Genomic gain and/or overexpression of KIF14 has been documented in retinoblastoma and a number of other cancers, such as breast, lung and ovarian carcinomas, strongly suggesting its role as an oncogene. Despite evidence of oncogenic properties in vitro and in xenografts, Kif14's role in tumor progression has not previously been studied in a transgenic cancer model. Using a novel Kif14 overexpressing, simian virus 40 large T-antigen retinoblastoma (TAg-RB) double transgenic mouse model, we aimed to determine Kif14's role in promoting retinal tumor formation. Tumor initiation and development in double transgenics and control TAg-RB littermates were documented in vivo over a time course by optical coherence tomography, with subsequent ex vivo quantification of tumor burden. Kif14 overexpression led to an accelerated initiation of tumor formation in the TAg-RB model and a significantly decreased tumor doubling time (1.8 vs. 2.9 weeks). Moreover, overall percentage tumor burden was also increased by Kif14 overexpression. These data provide the first evidence that Kif14 can promote tumor formation in susceptible cells in vivo.Item Observations on spontaneous tumor formation in mice overexpressing mitotic kinesin Kif14(Nature, 2018) Sishtla, Kamakshi; Pitt, Natalie; Shadmand, Mehdi; O'Hare, Michael N.; Sulaiman, Rania S.; Sinn, Anthony L.; Condon, Keith; Pollok, Karen E.; Sandusky, George E.; Corson, Timothy W.; Ophthalmology, School of MedicineThe KIF14 locus is gained and overexpressed in various malignancies, with prognostic relevance. Its protein product, a mitotic kinesin, accelerates growth of normal mammary epithelial cells in vitro and retinoblastoma tumours in a mouse model, while KIF14 knockdown blocks growth of brain, liver, ovarian, breast, prostate, and other tumour cells and xenografts. However, the tumour-initiating effects of Kif14 overexpression have not been studied. We aged a cohort of Kif14-overexpressing transgenic mice and wild-type littermates and documented survival, cause of death, and tumour burden. The Kif14 transgene was expressed in all tissues examined, and was associated with increased proliferation marker expression. Neither mouse weights nor overall survival differed between genotypes. However, Kif14 transgenic mice showed a higher incidence of fatal lymphomas (73 vs. 50%, p = 0.03, Fisher’s exact test), primarily follicular and diffuse B-cell lymphomas. Non-tumour findings included a bilateral ballooning degeneration of lens in 12% of Kif14 transgenic mice but no wild-type mice (p = 0.02). Overall, this work reveals a novel association of Kif14 overexpression with lymphoma but suggests that Kif14 does not have as prominent a role in initiating cancer in other cell types as it does in accelerating tumour development in response to other oncogenic insults.