IU Indianapolis ScholarWorks :: Browsing by Author "Shetty, Trupti"

Browsing by Author "Shetty, Trupti"

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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 Medicine
Excessive 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).
Ferrochelatase is a therapeutic target for ocular neovascularization
(Wiley, 2017) Basavarajappa, Halesha D.; Sulaiman, Rania S.; Qi, Xiaoping; Shetty, Trupti; Babu, Sardar Sheik Pran; Sishtla, Kamakshi L.; Lee, Bit; Quigley, Judith; Alkhairy, Sameerah; Briggs, Christian M.; Gupta, Kamna; Tang, Buyun; Shadmand, Mehdi; Grant, Maria B.; Boulton, Michael E.; Seo, Seung-Yong; Corson, Timothy W.; Department of Ophthalmology, IU School of Medicine
Ocular neovascularization underlies major blinding eye diseases such as “wet” age-related macular degeneration (AMD). Despite the successes of treatments targeting the vascular endothelial growth factor (VEGF) pathway, resistant and refractory patient populations necessitate discovery of new therapeutic targets. Using a forward chemical genetic approach, we identified the heme synthesis enzyme ferrochelatase (FECH) as necessary for angiogenesis in vitro and in vivo. FECH is overexpressed in wet AMD eyes and murine choroidal neovascularization; siRNA knockdown of Fech or partial loss of enzymatic function in the Fechm1Pas mouse model reduces choroidal neovascularization. FECH depletion modulates endothelial nitric oxide synthase function and VEGF receptor 2 levels. FECH is inhibited by the oral antifungal drug griseofulvin, and this compound ameliorates choroidal neovascularization in mice when delivered intravitreally or orally. Thus, FECH inhibition could be used therapeutically to block ocular neovascularization.
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 Medicine
The 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.
Measurement of mitochondrial respiration in the murine retina using a Seahorse extracellular flux analyzer
(Elsevier, 2021-06) Shetty, Trupti; Park, Bomina; Corson, Timothy W.; Ophthalmology, School of Medicine
Mitochondrial metabolism is a critical mechanism that is deregulated in numerous retinal diseases. Here, we elaborate a protocol to quantify oxygen consumption rate as a measure of mitochondrial respiration directly from mouse retinal tissue pieces. Our procedure combines the use of Seahorse extracellular flux technology and ex vivo retinal tissue isolation and is robustly reproducible under different treatment conditions. This protocol allows direct assessment of mitochondrial function in response to drug treatments or genetic manipulation in mouse models.
Mitochondrial Heme Synthesis Enzymes as Therapeutic Targets in Vascular Diseases
(Frontiers, 2020-07-15) Shetty, Trupti; Corson, Timothy W.; Ophthalmology, School of Medicine
NF-κB Signaling Is Regulated by Fucosylation in Metastatic Breast Cancer Cells
(MDPI, 2020-12-12) Doud, Emma H.; Shetty, Trupti; Abt, Melissa; Mosley, Amber L.; Corson, Timothy W.; Mehta, Anand; Yeh, Elizabeth S.; Biochemistry and Molecular Biology, School of Medicine
A growing body of evidence indicates that the levels of fucosylation correlate with breast cancer progression and contribute to metastatic disease. However, very little is known about the signaling and functional outcomes that are driven by fucosylation. We performed a global proteomic analysis of 4T1 metastatic mammary tumor cells in the presence and absence of a fucosylation inhibitor, 2-fluorofucose (2FF). Of significant interest, pathway analysis based on our results revealed a reduction in the NF-κB and TNF signaling pathways, which regulate the inflammatory response. NF-κB is a transcription factor that is pro-tumorigenic and a prime target in human cancer. We validated our results, confirming that treatment of 4T1 cells with 2FF led to a decrease in NF-κB activity through increased IκBα. Based on these observations, we conclude that fucosylation is an important post-translational modification that governs breast cancer cell signaling.
Patient preferences in retinal drug delivery
(Nature, 2021) Jacobs, Brandon; Palmer, Nicholas; Shetty, Trupti; Dimaras, Helen; Hajrasouliha, Amir; Jusufbegovic, Denis; Corson, Timothy W.; Ophthalmology, School of Medicine
Retinal vascular diseases (RVDs) are often treated with intravitreally (IVT) injected drugs, with relatively low patient compliance and potential risks. Ongoing research explores alternative RVD treatments, including eye drops and oral tablets. This study surveyed RVD patients treated with IVT injections to establish factors influencing low compliance rates while gauging treatment delivery method preferences. Demographics, perspectives, and treatment preferences were collected via IRB-approved, self-administered survey sent to Glick Eye Institute patients treated via IVT injections. Demographics, diagnoses, and treatments were ascertained from respondents’ medical records. Gender, age, and number of IVT injections received were used as stratifications. Five-level Likert-style scales and t-tests evaluated responses and stratification comparisons. The most common diagnoses in the respondent population (n = 54; response rate = 5%) were age-related macular degeneration, macular edema, and diabetic retinopathy. Respondents had varying levels of education, income, and age. Most (83%) admitted feeling anxious prior to their first IVT injection, but 80% reported willingness to receive IVT injections indefinitely, with a preference for ophthalmologist visits every 1–3 months. Eye drops would be preferred over IVT injections by 76% of respondents, while 65% preferred oral tablets, due to several perceived negative factors of IVT injections and positive factors for eye drops. Stratified groups did not differ in responses to survey questions. RVD patients will accept IVT injections for vision preservation, but alternative delivery methods like eye drops or oral tablets would be preferred. Thus, development of eye drop and oral therapeutics for RVD treatment is further emphasized by these findings.
Ref-1/APE1 Inhibition with Novel Small Molecules Blocks Ocular Neovascularization
(ASPET, 2018-10) Sardar Pasha, Sheik Pran Babu; Sishtla, Kamakshi; Sulaiman, Rania S.; Park, Bomina; Shetty, Trupti; Shah, Fenil; Fishel, Melissa L.; Wikel, James H.; Kelley, Mark R.; Corson, Timothy W.; Ophthalmology, School of Medicine
Ocular neovascular diseases like wet age-related macular degeneration are a major cause of blindness. Novel therapies are greatly needed for these diseases. One appealing antiangiogenic target is reduction-oxidation factor 1–apurinic/apyrimidinic endonuclease 1 (Ref-1/APE1). This protein can act as a redox-sensitive transcriptional activator for nuclear factor (NF)-κB and other proangiogenic transcription factors. An existing inhibitor of Ref-1’s function, APX3330, previously showed antiangiogenic effects. Here, we developed improved APX3330 derivatives and assessed their antiangiogenic activity. We synthesized APX2009 and APX2014 and demonstrated enhanced inhibition of Ref-1 function in a DNA-binding assay compared with APX3330. Both compounds were antiproliferative against human retinal microvascular endothelial cells (HRECs; GI50 APX2009: 1.1 μM, APX2014: 110 nM) and macaque choroidal endothelial cells (Rf/6a; GI50 APX2009: 26 μM, APX2014: 5.0 μM). Both compounds significantly reduced the ability of HRECs and Rf/6a cells to form tubes at mid-nanomolar concentrations compared with control, and both significantly inhibited HREC and Rf/6a cell migration in a scratch wound assay, reducing NF-κB activation and downstream targets. Ex vivo, APX2009 and APX2014 inhibited choroidal sprouting at low micromolar and high nanomolar concentrations, respectively. In the laser-induced choroidal neovascularization mouse model, intraperitoneal APX2009 treatment significantly decreased lesion volume by 4-fold compared with vehicle (P < 0.0001, ANOVA with Dunnett’s post-hoc tests), without obvious intraocular or systemic toxicity. Thus, Ref-1 inhibition with APX2009 and APX2014 blocks ocular angiogenesis in vitro and ex vivo, and APX2009 is an effective systemic therapy for choroidal neovascularization in vivo, establishing Ref-1 inhibition as a promising therapeutic approach for ocular neovascularization.
Retinal Phenotyping of Ferrochelatase Mutant Mice Reveals Protoporphyrin Accumulation and Reduced Neovascular Response
(ARVO, 2021-02-01) Sardar Pasha, S. P. B.; Shetty, Trupti; Lambert-Cheatham, Nathan A.; Sishtla, Kamakshi; Mathew, Deepa; Muniyandi, Anbukkarasi; Patwari, Neeta; Bhatwadekar, Ashay D.; Corson, Timothy W.; Ophthalmology, School of Medicine
Purpose: Heme depletion, through inhibition of ferrochelatase (FECH), blocks retinal and choroidal neovascularization. Both pharmacologic FECH inhibition and a partial loss-of-function Fech mutation (Fechm1Pas) are associated with decreased neovascularization. However, the ocular physiology of Fechm1Pas mice under basal conditions has not been characterized. Here, we aimed to characterize the retinal phenotype of Fechm1Pas mice. Methods: We monitored retinal vasculature at postnatal day 17, 2 months, and 6 months in Fechm1Pas homozygotes, heterozygotes, and their wild-type littermates. We characterized Fech substrate protoporphyrin (PPIX) fluorescence in the eye (excitation = 403 nm, emission = 628 nm), retinal function by electroretinogram, visual acuity by optomotor reflex, and retinal morphology by optical coherence tomography and histology. We stained vasculature using isolectin B4 and fluorescein angiography. We determined endothelial sprouting of retinal and choroidal tissue ex vivo and bioenergetics of retinal punches using a Seahorse flux analyzer. Results: Fundi, retinal vasculature, venous width, and arterial tortuosity showed no aberrations. However, VEGF-induced retinal and choroidal sprouting was decreased in Fechm1Pas mutants. Homozygous Fechm1Pas mice had pronounced buildup of PPIX in the posterior eye with no damage to visual function, bioenergetics, and integrity of retinal layers. Conclusions: Even with a buildup of PPIX in the retinal vessels in Fechm1Pas homozygotes, the vasculature remains normal. Notably, stimulus-induced ex vivo angiogenesis was decreased in Fechm1Pas mutants, consistent with reduced pathologic angiogenesis seen previously in neovascular animal models. Our findings indicate that Fechm1Pas mice are a useful model for studying the effects of heme deficiency on neovascularization due to Fech blockade.
The Role of Heme Synthesis in Endothelial Mitochondrial Function and Ocular Angiogenesis
(2020-08) Shetty, Trupti; Corson, Timothy W.; Bhatwadekar, Ashay D.; Hoffmann-Longtin, Krista J.; Jerde, Travis J.; Lu, Tao; Sullivan, William J., Jr.
Abnormal blood vessel growth from pre-existing blood vessels, termed pathological angiogenesis, is a common characteristic of vascular diseases like proliferative diabetic retinopathy (PDR) and wet age-related macular degeneration (wet AMD). Retinal detachment, hemorrhage, and loss of vision are only some of the debilitating consequences of advanced pathological angiogenesis. Current therapeutics targeting these blood vessels are ineffective in many patients. We previously identified a novel angiogenesis target, ferrochelatase (FECH), from the heme synthesis pathway, and found that FECH inhibition is antiangiogenic in cell and animal models. Heme synthesis occurs in mitochondria, where FECH inserts Fe2+ into protoporphyrin IX (PPIX) to produce heme. However, the relationship between heme metabolism and angiogenesis is poorly understood. I sought to understand the mechanism of how FECH and thus, heme is involved in endothelial cell function. First, I determined the energetic state of retinal and choroidal endothelial cells, previously uncharacterized. I found that mitochondria in endothelial cells had several functional defects after heme inhibition. FECH loss changed the shape of mitochondria and depleted expression of genes maintaining mitochondrial dynamics. FECH blockade elevated oxidative stress and depolarized mitochondrial membrane potential. Heme depletion had negative effects on cellular metabolism, affecting oxidative phosphorylation and glycolysis. Mitochondrial complex IV of the electron transport chain (cytochrome c oxidase) was decreased in cultured human retinal endothelial cells and in murine retina ex vivo after FECH inhibition. Supplementation with heme partially rescued phenotypes of FECH blockade. Additionally, I discovered that partial loss-of-function Fech mutation in mice caused PPIX accumulation with no change in normal vasculature, as assessed by fundoscopy. These findings provide an unexpected link between mitochondrial heme metabolism and angiogenesis. My studies identify a novel role of a heme synthesis enzyme in blood vessel formation and provide an opportunity to exploit these findings therapeutically for patients with PDR and wet AMD.

Browsing by Author "Shetty, Trupti"

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