Papers by kailash gulshan
Pyroptosis executor Gasdermin (GsdmD) promotes atherosclerosis in mice and humans. Disulfiram (DS... more Pyroptosis executor Gasdermin (GsdmD) promotes atherosclerosis in mice and humans. Disulfiram (DSF) was recently shown to potently inhibit GsdmD, but the in-vivo efficacy and mechanism of DSF’s anti-atherosclerotic activity is yet to be explored. We used human/mouse macrophages and a hyperlipidemic mouse model of atherosclerosis to determine DSF anti-atherosclerotic efficacy and mechanism. DSF-fed hyperlipidemic apoE-/-mice showed significantly reduced IL-1β release upon in-vivo Nlrp3 inflammasome assembly and showed smaller atherosclerotic lesions (∼27% and 29% reduction in males and females, respectively). The necrotic core area was also smaller (∼50% and 46% reduction in DSF-fed males and females, respectively). DSF induced autophagy in macrophages, hepatocytes/liver, and in atherosclerotic plaques. DSF modulated other atheroprotective pathways such as efferocytosis, phagocytosis, and gut microbiota. DSF-treated macrophages showed enhanced phagocytosis/efferocytosis, with a mecha...
Biophysical journal, Feb 1, 2024
Scientific Reports, Jul 31, 2019
Miltefosine is an fDA approved oral drug for treating cutaneous and visceral leishmaniasis. Leish... more Miltefosine is an fDA approved oral drug for treating cutaneous and visceral leishmaniasis. Leishmania is a flagellated protozoa, which infects and differentiates in macrophages. Here, we studied the effects of Miltefosine on macrophage's lipid homeostasis, autophagy, and NLRP3 inflammasome assembly/ activity. Miltefosine treatment conferred multiple effects on macrophage lipid homeostasis leading to increased cholesterol release from cells, increased lipid-raft disruption, decreased phosphatidylserine (PS) flip from the cell-surface, and redistribution of phosphatidylinositol 4,5-bisphosphate (PIP2) from the plasma membrane to actin rich regions in the cells. Enhanced basal autophagy, lipophagy and mitophagy was observed in cells treated with Miltefosine vs. control. Miltefosine treated cells showed marked increased in phosphorylation of kinases involved in autophagy induction such as; Adenosine monophosphate-activated protein kinase (AMPK) and Unc-51 like autophagy activating kinase (ULK1). The Toll like receptor (TLR) signaling pathway was blunted by Miltefosine treatment, resulting in decreased TLR4 recruitment to cell-surface and ~75% reduction in LPS induced pro-IL-1β mRnA levels. Miltefosine reduced endotoxin-mediated mitochondrial reactive oxygen species and protected the mitochondrial membrane potential. Miltefosine treatment induced mitophagy and dampened NLRP3 inflammasome assembly. Collectively, our data shows that Miltefosine induced ABCA1 mediated cholesterol release, induced AMPK phosphorylation and mitophagy, while dampening NLRP3 inflammasome assembly and IL-1β release. Atherosclerosis, a sterile inflammatory disease, is the major cause of coronary artery disease (CAD). LDL cholesterol drives atherosclerosis by depositing LDL into the arterial intima, where it can be modified to induce endothelial cell activation and the recruitment of leukocytes; and, the uptake of modified LDL into macrophages leads to foam cell formation and a further amplification of inflammation 1-4. Accumulation of oxidized lipids and cholesterol crystals in plaques activate toll-like receptor (TLR) pathways and the assembly of the NLRP3 inflammasome 2,5. The NLRP3 inflammasome plays a key role in processing procaspase 1 resulting in subsequent caspase 1 mediated processing of pro IL-1β to generate active interleukin-1β (IL-1β). The role of IL-1β in promoting human CAD was highlighted by the recently concluded CANTOS trial, showing that anti-IL-1β therapy met the primary endpoint, a reduction in a composite of heart attack, stroke and cardiovascular death 6. In contrast to atherogenic pathways, the atheroprotective pathways such as autophagy and cholesterol efflux become increasingly dysfunctional in aging, advanced atherosclerotic plaques, and in animal models of atherosclerosis and diabetes 5,7-9. Thus, the simultaneous induction of atheroprotective pathways, along with dampening of atherogenic pathways may serve as a potent therapeutic treatment for CAD patients. Here, we report that Miltefosine, an FDA approved drug for treating visceral and cutaneous leishmaniasis, promoted cholesterol release, disrupted lipid-rafts and TLR4 signaling, increased cell-surface phosphatidylserine (PS) exposure, induced phosphatidylinositol 4,5-bisphosphate (PIP2) trafficking from plasma membrane (PM) to the cell interior. Miltefosine treated cells exhibited increased phosphorylation of autophagy inducing kinases AMPK1 and ULK1, leading to increased basal autophagy, lipophagy and mitophagy. The lipopolysaccharide (LPS) induced
PLOS ONE, Jan 16, 2020
The crystal structure of a C-terminal deletion of apolipoprotein A-I (apoA1) shows a large helica... more The crystal structure of a C-terminal deletion of apolipoprotein A-I (apoA1) shows a large helical bundle structure in the amino half of the protein, from residues 8 to 115. Using site directed mutagenesis, guanidine or thermal denaturation, cell free liposome clearance, and cellular ABCA1-mediated cholesterol efflux assays, we demonstrate that apoA1 lipidation can occur when the thermodynamic barrier to this bundle unfolding is lowered. The absence of the C-terminus renders the bundle harder to unfold resulting in loss of apoA1 lipidation that can be reversed by point mutations, such as Trp8Ala, and by truncations as short as 8 residues in the amino terminus, both of which facilitate helical bundle unfolding. Locking the bundle via a disulfide bond leads to loss of apoA1 lipidation. We propose a model in which the C-terminus acts on the N-terminus to destabilize this helical bundle. Upon lipid binding to the C-terminus, Trp8 is displaced from its interaction with Phe57, Arg61, Leu64, Val67, Phe71, and Trp72 to destabilize the bundle. However, when the C-terminus is deleted, Trp8 cannot be displaced, the bundle cannot unfold, and apoA1 cannot be lipidated.
Frontiers in Cardiovascular Medicine
Editorial on the Research Topic Decoding complexity: new insights into the cellular and molecular... more Editorial on the Research Topic Decoding complexity: new insights into the cellular and molecular mechanisms of cardiovascular and metabolic diseases
Advances in Experimental Medicine and Biology
Journal of Biological Chemistry, 2020
High-density lipoprotein (HDL) metabolism is facilitated in part by scavenger receptor class B, t... more High-density lipoprotein (HDL) metabolism is facilitated in part by scavenger receptor class B, type 1 (SR-B1) that mediates HDL uptake into cells. Higher levels of HDL have been associated with protection in other diseases, however, its role in prostate cancer is not definitive. SR-B1 is up-regulated in prostate cancer tissue, suggesting a possible role of this receptor in tumor progression. Here, we report that knockout (KO) of SR-B1 in both human and mouse prostate cancer cell lines through CRISPR/Cas9-mediated genome editing reduces HDL uptake into the prostate cancer cells and reduces their proliferation in response to HDL. In vivo studies using syngeneic SR-B1 WT (SR-B1+/+) and SR-B1 KO (SR-B1−/−) prostate cancer cells in WT and apolipoprotein-AI KO (apoA1-KO) C57BL/6J mice revealed that WT hosts, containing higher levels of total and HDL-cholesterol, grew larger tumors than apoA1-KO hosts with lower levels of total and HDL-cholesterol. Furthermore, SR-B1−/− prostate cancer ce...
Genetics, 2006
The transcriptional mediator complex has emerged as an important component of transcriptional reg... more The transcriptional mediator complex has emerged as an important component of transcriptional regulation, yet it is largely unknown whether its subunits have differential functions in development. We demonstrate that the zebrafish mutation m885 disrupts a subunit of the mediator complex, Crsp34/Med27. To explore the role of the mediator in the control of retinal differentiation, we employed two additional mutations disrupting the mediator subunits Trap100/Med24 and Crsp150/Med14. Our analysis shows that loss of Crsp34/Med27 decreases amacrine cell number, but increases the number of rod photoreceptor cells. In contrast, loss of Trap100/Med24 decreases rod photoreceptor cells. Loss of Crsp150/Med14, on the other hand, only slightly reduces dopaminergic amacrine cells, which are absent from both crsp34m885 and trap100lessen mutant embryos. Our data provide evidence for differential requirements for Crsp34/Med27 in developmental processes. In addition, our data point to divergent funct...
The activities of NLRP3 and AIM2 inflammasomes and Gasdermin D (GsdmD), the final executor of Inf... more The activities of NLRP3 and AIM2 inflammasomes and Gasdermin D (GsdmD), the final executor of Inflammasome activity, are implicated in lung cancer pathophysiology but it's not clear if their contributions promote or retard lung cancer progression. GsdmD plays a role in release of interleukin-1beta (IL-1b), and the CANTOS trial and recent studies have highlighted a crucial role of IL-1b in promoting lung cancer. Expression of GsdmD was shown to be upregulated in human non-small cell lung cancer (NSCLC) tissue, but its contribution to in vivo lung cancer metastasis is not known. Using a metastatic Lewis Lung Carcinoma (LLC) cell model, we show that GsdmD knockout (GsdmD-/-) mice form significantly fewer cancer foci in lung, and exhibit markedly decreased lung cancer metastasis. Furthermore, GsdmD-/- mice show a significant ~ 50% increase in median survival rate vs. isogenic WT C57BL6J mice. The cleaved forms of GsdmD and IL-1b were detected in lung tumor tissue, indicating inflamm...
Excess cholesterol induces foam cell formation, NLRP3 inflammasome activation, and IL-1β release ... more Excess cholesterol induces foam cell formation, NLRP3 inflammasome activation, and IL-1β release in atherosclerotic plaques. We have shown previously that Miltefosine increased cholesterol release and dampened NLRP3 inflammasome assembly in macrophages. Here, we show that Miltefosine reduced LPS-induced choline uptake by macrophages and attenuated NLRP3 inflammasome assembly in mice. Miltefosine-fed mice showed reduced plasma IL-1β in a polymicrobial cecal slurry injection model of systemic inflammation. Miltefosine-fed mice showed increased reverse cholesterol transport from macrophages to plasma, liver, and feces. Hyperlipidemic apoE−/−mice fed with Miltefosine showed significantly reduced weight gain and markedly reduced atherosclerotic lesions vs. control mice. 16S rDNA sequencing and analysis showed alterations in the gut microbiota profile of Miltefosine-fed hyperlipidemic apoE−/−vs. control mice, with the most notable changes inRomboutsiaandBacteroidetes species. Taken togeth...
Activation of inflammasomes, such as Nlrp3 and Aim2, can exacerbate atherosclerosis in mice and h... more Activation of inflammasomes, such as Nlrp3 and Aim2, can exacerbate atherosclerosis in mice and humans. Gasdermin D (GsdmD) serves as a final executor of inflammasome activity, by generating membrane pores for the release of mature Interleukin-1beta (IL-1β). Inflammation dampens reverse cholesterol transport (RCT) and promotes atherogenesis, while anti-IL-1β antibodies were shown to reduce cardiovascular disease in humans. Though Nlrp3/AIM2 and IL-1β nexus is an emerging atherogenic pathway, the direct role of GsdmD in atherosclerosis is not yet fully clear. Here, we used in-vivo Nlrp3 inflammasome activation to show that the GsdmD-/- mice release ~80% less IL-1β vs WT mice. The GsdmD-/- macrophages were more resistant to Nlrp3 inflammasome mediated reduction in cholesterol efflux, showing ~26% decrease vs. ~60% reduction in WT macrophages. GsdmD expression in macrophages exacerbated foam cell formation in an IL-1β dependent fashion. The GsdmD-/- mice were resistant to Nlrp3 inflamm...
Arteriosclerosis, Thrombosis, and Vascular Biology, 2016
Introduction: apoAI-ABCA1 mediated nascent HDL assembly promotes cholesterol efflux and reverse c... more Introduction: apoAI-ABCA1 mediated nascent HDL assembly promotes cholesterol efflux and reverse cholesterol transport. Cell surface phosphatidylinositol 4,5- bisphosphate (PIP2) mediates this process, but whether PIP2 is effluxed via apoA1-ABCA1 pathway and is carried in plasma is unknown. Objective: To determine if PIP2 is effluxed during ABCA1-dependent nHDL biogenesis, whether PIP2 is found on plasma HDL, and if PIP2 can be delivered to target cells via SRB1. Methods and Results: We previously reported at this meeting that: 1) apoAI binds to PIP2 with a K d of ~100 nM; 2) ABCA1 expression translocates PIP2 from the inner to outer leaflet of the plasma membrane; 3) cell surface PIP2 is required for ABCA1-dependent cellular binding of apoA1 and cholesterol efflux. Here, we report that PIP2 incubation with lipid-free apoA1 promoted monomeric vs. dimeric apoA1. We found that ABCA1 expression increased efflux of PIP2 by ~20-fold in RAW264.7 cells labeled with [3H]myo-inositol, and cha...
Arteriosclerosis, Thrombosis, and Vascular Biology, 2014
Objectives: Insulin resistance-induced hyperlipidemia and hyperlipoproteinemia are risk factors o... more Objectives: Insulin resistance-induced hyperlipidemia and hyperlipoproteinemia are risk factors of atherosclerosis. Spingolipid ceramide is involved in insulin resistance and lipoprotein metabolism, and sphingomyelin, a ceramide metabolite, is an independent risk factor for CVD. Here we studied the role of spingolipids in insulin resistance associated atherosclerosis. Methods: Low density lipoprotein receptors knockout (LDLR -/- ) mice, a diet induced mouse model of insulin resistance and atherosclerosis, were fed a standard chow diet or a western diet (high fat with 0.2% cholesterol) for 12 weeks with or without myriocin, an inhibitor of de novo ceramide synthesis. A targeted lipidomics approach was used for hepatic and plasma profiling of ceramides. Metabolic 2 H 2 O-labeling technique was applied to quantify turnover rates of hepatic and plasma lipids and lipoproteins, including ApoB and ApoAI - the principle proteins of VLDL/LDL and HDL. Hepatic and intestinal expression of gene...
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Papers by kailash gulshan