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1.
RNA Biol ; 21(1): 17-31, 2024 Jan.
Article in English | MEDLINE | ID: mdl-39107918

ABSTRACT

Extracellular vesicles and nanoparticles (EVPs) are now recognized as a novel form of cell-cell communication. All cells release a wide array of heterogeneous EVPs with distinct protein, lipid, and RNA content, dependent on the pathophysiological state of the donor cell. The overall cargo content in EVPs is not equivalent to cellular levels, implying a regulated pathway for selection and export. In cancer, release and uptake of EVPs within the tumour microenvironment can influence growth, proliferation, invasiveness, and immune evasion. Secreted EVPs can also have distant, systemic effects that can promote metastasis. Here, we review current knowledge of EVP biogenesis and cargo selection with a focus on the role that extracellular RNA plays in oncogenesis and metastasis. Almost all subtypes of RNA have been identified in EVPs, with miRNAs being the best characterized. We review the roles of specific miRNAs that have been detected in EVPs and that play a role in oncogenesis and metastasis.


Subject(s)
Carcinogenesis , Drug Resistance, Neoplasm , Extracellular Vesicles , MicroRNAs , Neoplasm Metastasis , Neoplasms , Tumor Microenvironment , Humans , Extracellular Vesicles/metabolism , Drug Resistance, Neoplasm/genetics , Neoplasms/metabolism , Neoplasms/pathology , Neoplasms/genetics , MicroRNAs/genetics , MicroRNAs/metabolism , Carcinogenesis/genetics , Animals , Gene Expression Regulation, Neoplastic , Cell Communication
2.
bioRxiv ; 2024 Jun 10.
Article in English | MEDLINE | ID: mdl-38915549

ABSTRACT

Short-interfering RNA (siRNA) has gained significant interest for treatment of neurological diseases by providing the capacity to achieve sustained inhibition of nearly any gene target. Yet, achieving efficacious drug delivery throughout deep brain structures of the CNS remains a considerable hurdle. We herein describe a lipid-siRNA conjugate that, following delivery into the cerebrospinal fluid (CSF), is transported effectively through perivascular spaces, enabling broad dispersion within CSF compartments and through the CNS parenchyma. We provide a detailed examination of the temporal kinetics of gene silencing, highlighting potent knockdown for up to five months from a single injection without detectable toxicity. Single-cell RNA sequencing further demonstrates gene silencing activity across diverse cell populations in the parenchyma and at brain borders, which may provide new avenues for neurological disease-modifying therapies.

3.
bioRxiv ; 2024 May 22.
Article in English | MEDLINE | ID: mdl-38826470

ABSTRACT

Extracellular communication via the transfer of vesicles and nanoparticles is now recognized to play an important role in tumor microenvironment interactions. Cancer cells upregulate and secrete abundant levels of miR-100 and miR-125b that can alter gene expression by both cell- and non-cell-autonomous mechanisms. We previously showed that these miRNAs activate Wnt signaling in colorectal cancer (CRC) through noncanonical pairing with 5 negative regulators of Wnt signaling. To identify additional targets of miR-100 and miR-125b , we used bioinformatic approaches comparing multiple CRC cell lines, including knockout lines lacking one or both of these miRNAs. From an initial list of 96 potential mRNA targets, we tested 15 targets with 8 showing significant downregulation in the presence of miR-100 and miR-125b . Among these, Cingulin (CGN) and Protein tyrosine phosphatase receptor type-R (PTPRR) are downregulated in multiple cancers, consistent with regulation by increased levels of miR-100 and miR-125b. We also show that increased cellular levels of miR-100 and miR-125b enhance 3D growth and invasiveness in CRC and glioblastoma cell lines. Lastly, we demonstrate that extracellular transfer of miR-100 and miR-125b can silence both reporter and endogenous mRNA targets in recipient cells and also increase the invasiveness of recipient spheroid colonies when grown under 3D conditions in type I collagen.

4.
Nat Commun ; 15(1): 1581, 2024 Feb 21.
Article in English | MEDLINE | ID: mdl-38383524

ABSTRACT

The high potential of siRNAs to silence oncogenic drivers remains largely untapped due to the challenges of tumor cell delivery. Here, divalent lipid-conjugated siRNAs are optimized for in situ binding to albumin to improve pharmacokinetics and tumor delivery. Systematic variation of the siRNA conjugate structure reveals that the location of the linker branching site dictates tendency toward albumin association versus self-assembly, while the lipid hydrophobicity and reversibility of albumin binding also contribute to siRNA intracellular delivery. The lead structure increases tumor siRNA accumulation 12-fold in orthotopic triple negative breast cancer (TNBC) tumors over the parent siRNA. This structure achieves approximately 80% silencing of the anti-apoptotic oncogene MCL1 and yields better survival outcomes in three TNBC models than an MCL-1 small molecule inhibitor. These studies provide new structure-function insights on siRNA-lipid conjugate structures that are intravenously injected, associate in situ with serum albumin, and improve pharmacokinetics and tumor treatment efficacy.


Subject(s)
Antineoplastic Agents , Triple Negative Breast Neoplasms , Humans , RNA, Small Interfering , Myeloid Cell Leukemia Sequence 1 Protein/genetics , Triple Negative Breast Neoplasms/drug therapy , Triple Negative Breast Neoplasms/genetics , Cell Line, Tumor , Gene Silencing , Lipids/chemistry , Albumins/genetics
5.
bioRxiv ; 2024 Jan 16.
Article in English | MEDLINE | ID: mdl-38293013

ABSTRACT

5-fluorouracil (5-FU) has been used for chemotherapy for colorectal and other cancers for over 50 years. The prevailing view of its mechanism of action is inhibition of thymidine synthase leading to defects in DNA replication and repair. However, 5-FU is also incorporated into RNA causing toxicity due to defects in RNA metabolism, inhibition of pseudouridine modification, and altered ribosome function. Here, we examine the impact of 5-FU on the expression and export of small RNAs (sRNAs) into small extracellular vesicles (sEVs). Moreover, we assess the role of 5-FU in regulation of post-transcriptional sRNA modifications (PTxM) using mass spectrometry approaches. EVs are secreted by all cells and contain a variety of proteins and RNAs that can function in cell-cell communication. PTxMs on cellular and extracellular sRNAs provide yet another layer of gene regulation. We found that treatment of the colorectal cancer (CRC) cell line DLD-1 with 5-FU led to surprising differential export of miRNA snRNA, and snoRNA transcripts. Strikingly, 5-FU treatment significantly decreased the levels of pseudouridine on both cellular and secreted EV sRNAs. In contrast, 5-FU exposure led to increased levels of cellular sRNAs containing a variety of methyl-modified bases. Our results suggest that 5-FU exposure leads to altered expression, base modifications, and mislocalization of EV base-modified sRNAs.

6.
Am J Respir Crit Care Med ; 209(2): 153-163, 2024 Jan 15.
Article in English | MEDLINE | ID: mdl-37931077

ABSTRACT

Rationale: Multiciliated cell (MCC) loss and/or dysfunction is common in the small airways of patients with chronic obstructive pulmonary disease (COPD), but it is unclear if this contributes to COPD lung pathology. Objectives: To determine if loss of p73 causes a COPD-like phenotype in mice and explore whether smoking or COPD impact p73 expression. Methods: p73floxE7-E9 mice were crossed with Shh-Cre mice to generate mice lacking MCCs in the airway epithelium. The resulting p73Δairway mice were analyzed using electron microscopy, flow cytometry, morphometry, forced oscillation technique, and single-cell RNA sequencing. Furthermore, the effects of cigarette smoke on p73 transcript and protein expression were examined using in vitro and in vivo models and in studies including airway epithelium from smokers and patients with COPD. Measurements and Main Results: Loss of functional p73 in the respiratory epithelium resulted in a near-complete absence of MCCs in p73Δairway mice. In adulthood, these mice spontaneously developed neutrophilic inflammation and emphysema-like lung remodeling and had progressive loss of secretory cells. Exposure of normal airway epithelium cells to cigarette smoke rapidly and durably suppressed p73 expression in vitro and in vivo. Furthermore, tumor protein 73 mRNA expression was reduced in the airways of current smokers (n = 82) compared with former smokers (n = 69), and p73-expressing MCCs were reduced in the small airways of patients with COPD (n = 11) compared with control subjects without COPD (n = 12). Conclusions: Loss of functional p73 in murine airway epithelium results in the absence of MCCs and promotes COPD-like lung pathology. In smokers and patients with COPD, loss of p73 may contribute to MCC loss or dysfunction.


Subject(s)
Emphysema , Pulmonary Disease, Chronic Obstructive , Pulmonary Emphysema , Animals , Humans , Mice , Epithelium/metabolism , Lung , Pulmonary Disease, Chronic Obstructive/pathology
7.
Arthritis Rheumatol ; 76(5): 684-695, 2024 May.
Article in English | MEDLINE | ID: mdl-38111131

ABSTRACT

OBJECTIVE: High-density lipoprotein (HDL) has well-characterized anti-atherogenic cholesterol efflux and antioxidant functions. Another function of HDL uncharacterized in rheumatoid arthritis (RA) is its ability to transport microRNAs (miRNAs) between cells and thus alter cellular function. The study's purpose was to determine if HDL-miRNA cargo is altered and affects inflammation in RA. METHODS: HDL-microRNAs were characterized in 30 RA and 30 control participants by next generation sequencing and quantitative polymerase chain reaction. The most abundant differentially expressed miRNA was evaluated further. The function of miR-1246 was assessed by miRNA mimics, antagomiRs, small interfering RNA knockdown, and luciferase assays. Monocyte-derived macrophages were treated with miR-1246-loaded HDL and unmodified HDL from RA and control participants to measure delivery of miR-1246 and its effect on interleukin-6 (IL-6). RESULTS: The most abundant miRNA on HDL was miR-1246; it was significantly enriched two-fold on HDL from RA versus control participants. HDL-mediated miR-1246 delivery to macrophages significantly increased IL6 expression 43-fold. miR-1246 delivery significantly decreased DUSP3 1.5-fold and DUSP3 small interfering RNA knockdown increased macrophage IL6 expression. Luciferase assay indicated DUSP3 is a direct target of miR-1246. Unmodified HDL from RA delivered 1.6-fold more miR-1246 versus control participant HDL. Unmodified HDL from both RA and control participants attenuated activated macrophage IL6 expression, but this effect was significantly blunted in RA so that IL6 expression was 3.4-fold higher after RA versus control HDL treatment. CONCLUSION: HDL-miR-1246 was increased in RA versus control participants and delivery of miR-1246 to macrophages increased IL-6 expression by targeting DUSP3. The altered HDL-miRNA cargo in RA blunted HDL's anti-inflammatory effect.


Subject(s)
Arthritis, Rheumatoid , Interleukin-6 , Lipoproteins, HDL , Macrophages , MicroRNAs , Humans , Arthritis, Rheumatoid/genetics , Arthritis, Rheumatoid/metabolism , MicroRNAs/metabolism , Lipoproteins, HDL/pharmacology , Lipoproteins, HDL/metabolism , Middle Aged , Male , Female , Interleukin-6/metabolism , Macrophages/metabolism , Case-Control Studies , Inflammation/metabolism , Adult , Aged
8.
J Extracell Vesicles ; 12(11): e12366, 2023 11.
Article in English | MEDLINE | ID: mdl-37885043

ABSTRACT

Extracellular vesicle (EV)-carried miRNAs can influence gene expression and functional phenotypes in recipient cells. Argonaute 2 (Ago2) is a key miRNA-binding protein that has been identified in EVs and could influence RNA silencing. However, Ago2 is in a non-vesicular form in serum and can be an EV contaminant. In addition, RNA-binding proteins (RBPs), including Ago2, and RNAs are often minor EV components whose sorting into EVs may be regulated by cell signaling state. To determine the conditions that influence detection of RBPs and RNAs in EVs, we evaluated the effect of growth factors, oncogene signaling, serum, and cell density on the vesicular and nonvesicular content of Ago2, other RBPs, and RNA in small EV (SEV) preparations. Media components affected both the intravesicular and extravesicular levels of RBPs and miRNAs in EVs, with serum contributing strongly to extravesicular miRNA contamination. Furthermore, isolation of EVs from hollow fiber bioreactors revealed complex preparations, with multiple EV-containing peaks and a large amount of extravesicular Ago2/RBPs. Finally, KRAS mutation impacts the detection of intra- and extra-vesicular Ago2. These data indicate that multiple cell culture conditions and cell states impact the presence of RBPs in EV preparations, some of which can be attributed to serum contamination.


Subject(s)
Argonaute Proteins , Extracellular Vesicles , MicroRNAs , Extracellular Vesicles/metabolism , MicroRNAs/metabolism , Argonaute Proteins/metabolism
9.
bioRxiv ; 2023 Feb 15.
Article in English | MEDLINE | ID: mdl-36824780

ABSTRACT

The high potential for therapeutic application of siRNAs to silence traditionally undruggable oncogenic drivers remains largely untapped due to the challenges of tumor cell delivery. Here, siRNAs were optimized for in situ binding to albumin through C18 lipid modifications to improve pharmacokinetics and tumor delivery. Systematic variation of siRNA conjugates revealed a lead structure with divalent C18 lipids each linked through three repeats of hexaethylene glycol connected by phosphorothioate bonds. Importantly, we discovered that locating the branch site of the divalent lipid structure proximally (adjacent to the RNA) rather than at a more distal site (after the linker segment) promotes association with albumin, while minimizing self-assembly and lipoprotein association. Comparison to higher albumin affinity (diacid) lipid variants and siRNA directly conjugated to albumin underscored the importance of conjugate hydrophobicity and reversibility of albumin binding for siRNA delivery and bioactivity in tumors. The lead conjugate increased tumor siRNA accumulation 12-fold in orthotopic mouse models of triple negative breast cancer over the parent siRNA. When applied for silencing of the anti-apoptotic oncogene MCL-1, this structure achieved approximately 80% MCL1 silencing in orthotopic breast tumors. Furthermore, application of the lead conjugate structure to target MCL1 yielded better survival outcomes in three independent, orthotopic, triple negative breast cancer models than an MCL1 small molecule inhibitor. These studies provide new structure-function insights on optimally leveraging siRNA-lipid conjugate structures that associate in situ with plasma albumin for molecular-targeted cancer therapy.

10.
J Lipid Res ; 64(2): 100328, 2023 02.
Article in English | MEDLINE | ID: mdl-36626966

ABSTRACT

HDL are dynamic transporters of diverse molecular cargo and play critical roles in lipid metabolism and inflammation. We have previously reported that HDL transport both host and nonhost small RNAs (sRNA) based on quantitative PCR and sRNA sequencing approaches; however, these methods require RNA isolation steps which have potential biases and may not isolate certain forms of RNA molecules from samples. HDL have also been reported to accept functional sRNAs from donor macrophages and deliver them to recipient endothelial cells; however, using PCR to trace HDL-sRNA intercellular communication has major limitations. The present study aims to overcome these technical barriers and further understand the pathways involved in HDL-mediated bidirectional flux of sRNAs between immune cells. To overcome these technical limitations, SYTO RNASelect, a lipid-penetrating RNA dye, was used to quantify a) overall HDL-sRNA content, b) bidirectional flux of sRNAs between HDL and immune cells, c) HDL-mediated intercellular communication between immune cells, and d) HDL-mediated RNA export changes in disease. Live cell imaging and loss-of-function assays indicate that the endo-lysosomal system plays a critical role in macrophage storage and export of HDL-sRNAs. These results identify HDL as a substantive mediator of intercellular communication between immune cells and demonstrate the importance of endocytosis for recipient cells of HDL-sRNAs. Utilizing a lipid-penetrating RNA-specific fluorescence dye, we were able to both quantify the absolute concentration of sRNAs transported by HDL and characterize HDL-mediated intercellular RNA transport between immune cells.


Subject(s)
RNA, Small Untranslated , RNA, Small Untranslated/genetics , RNA, Small Untranslated/metabolism , Lipoproteins, HDL , Endothelial Cells/metabolism , Macrophages/metabolism , Cell Communication , Dendritic Cells/metabolism
11.
Nat Cell Biol ; 24(12): 1701-1713, 2022 12.
Article in English | MEDLINE | ID: mdl-36474072

ABSTRACT

Macrophages present a spectrum of phenotypes that mediate both the pathogenesis and resolution of atherosclerotic lesions. Inflammatory macrophage phenotypes are pro-atherogenic, but the stimulatory factors that promote these phenotypes remain incompletely defined. Here we demonstrate that microbial small RNAs (msRNA) are enriched on low-density lipoprotein (LDL) and drive pro-inflammatory macrophage polarization and cytokine secretion via activation of the RNA sensor toll-like receptor 8 (TLR8). Removal of msRNA cargo during LDL re-constitution yields particles that readily promote sterol loading but fail to stimulate inflammatory activation. Competitive antagonism of TLR8 with non-targeting locked nucleic acids was found to prevent native LDL-induced macrophage polarization in vitro, and re-organize lesion macrophage phenotypes in vivo, as determined by single-cell RNA sequencing. Critically, this was associated with reduced disease burden in distinct mouse models of atherosclerosis. These results identify LDL-msRNA as instigators of atherosclerosis-associated inflammation and support alternative functions of LDL beyond cholesterol transport.


Subject(s)
Macrophages , Toll-Like Receptor 8 , Animals , Mice , Toll-Like Receptor 8/genetics , RNA
12.
Commun Biol ; 5(1): 1358, 2022 Dec 10.
Article in English | MEDLINE | ID: mdl-36496485

ABSTRACT

Superparamagnetic nanobeads offer several advantages over microbeads for immunocapture of nanocarriers (extracellular vesicles, lipoproteins, and viruses) in a bioassay: high-yield capture, reduction in incubation time, and higher capture capacity. However, nanobeads are difficult to "pull-down" because their superparamagnetic feature requires high nanoscale magnetic field gradients. Here, an electrodeposited track-etched membrane is shown to produce a unique superparamagnetic nano-edge ring with multiple edges around nanopores. With a uniform external magnetic field, the induced monopole and dipole of this nano edge junction combine to produce a 10× higher nanobead trapping force. A dense nanobead suspension can be filtered through the magnetic nanoporous membrane (MNM) at high throughput with a 99% bead capture rate. The yield of specific nanocarriers in heterogeneous media by nanobeads/MNM exceeds 80%. Reproducibility, low loss, and concentration-independent capture rates are also demonstrated. This MNM material hence expands the application of nanobead immunocapture to physiological samples.


Subject(s)
Extracellular Vesicles , Reproducibility of Results , Magnetic Fields , Membranes
13.
J Biol Chem ; 298(6): 101952, 2022 06.
Article in English | MEDLINE | ID: mdl-35447119

ABSTRACT

Extracellular small RNAs (sRNAs) are abundant in many biofluids, but little is known about their mechanisms of transport and stability in RNase-rich environments. We previously reported that high-density lipoproteins (HDLs) in mice were enriched with multiple classes of sRNAs derived from the endogenous transcriptome, but also from exogenous organisms. Here, we show that human HDL transports tRNA-derived sRNAs (tDRs) from host and nonhost species, the profiles of which were found to be altered in human atherosclerosis. We hypothesized that HDL binds to tDRs through apolipoprotein A-I (apoA-I) and that these interactions are conferred by RNA-specific features. We tested this using microscale thermophoresis and electrophoretic mobility shift assays and found that HDL binds to tDRs and other single-stranded sRNAs with strong affinity but did not bind to double-stranded RNA or DNA. Furthermore, we show that natural and synthetic RNA modifications influenced tDR binding to HDL. We demonstrate that reconstituted HDL bound to tDRs only in the presence of apoA-I, and purified apoA-I alone were able to bind sRNA. Conversely, phosphatidylcholine vesicles did not bind tDRs. In summary, we conclude that HDL binds to single-stranded sRNAs likely through nonionic interactions with apoA-I. These results highlight binding properties that likely enable extracellular RNA communication and provide a foundation for future studies to manipulate HDL-sRNA interactions for therapeutic approaches to prevent or treat disease.


Subject(s)
Lipoproteins, HDL , RNA, Small Untranslated , Animals , Apolipoprotein A-I/metabolism , Atherosclerosis , Humans , Lipoproteins, HDL/chemistry , Lipoproteins, HDL/metabolism , Mice , Phosphatidylcholines , RNA, Small Untranslated/chemistry
14.
Heliyon ; 7(12): e08519, 2021 Dec.
Article in English | MEDLINE | ID: mdl-34934837

ABSTRACT

Extracellular vesicles (EVs) are capable of transferring cargo from donor to recipient cells, but precisely how cargo content is regulated for export is mostly unknown. For miRNA cargo, we previously showed that when compared to isogenic colorectal cancer (CRC) cells expressing wild-type KRAS, a distinct subset of miRNAs are differentially enriched in EVs from KRAS mutant active CRC cells, with miR-100 being one of the most enriched. The mechanisms that could explain how miR-100 and other miRNAs are differentially exported into EVs have not been fully elucidated. Here, we tested the effect of N6-methyladenosine (m6A) modification on miRNA export into EVs by depletion of METTL3 and ALKBH5, a writer and eraser of m6A modification, respectively. While the effects of ALKBH5 knockdown were quite modest, decreased levels of METTL3 led to reduced cellular and extracellular levels of a subset of miRNAs that contain consensus sequences for m6A modification. Functional testing of EVs prepared from cells expressing shRNAs against METTL3 showed that they were less capable of conferring colony growth in 3D to wild-type KRAS cells and were also largely incapable of conferring the spread of cetuximab resistance. Our data support a role for METTL3 modification on cellular miRNA levels and export of specific miRNAs.

15.
Int J Mol Sci ; 22(15)2021 Jul 30.
Article in English | MEDLINE | ID: mdl-34360965

ABSTRACT

Decades of epidemiological studies have established the strong inverse relationship between high-density lipoprotein (HDL)-cholesterol concentration and cardiovascular disease. Recent evidence suggests that HDL particle functions, including anti-inflammatory and antioxidant functions, and cholesterol efflux capacity may be more strongly associated with cardiovascular disease protection than HDL cholesterol concentration. These HDL functions are also relevant in non-cardiovascular diseases, including acute and chronic kidney disease. This review examines our current understanding of the kidneys' role in HDL metabolism and homeostasis, and the effect of kidney disease on HDL composition and functionality. Additionally, the roles of HDL particles, proteins, and small RNA cargo on kidney cell function and on the development and progression of both acute and chronic kidney disease are examined. The effect of HDL protein modification by reactive dicarbonyls, including malondialdehyde and isolevuglandin, which form adducts with apolipoprotein A-I and impair proper HDL function in kidney disease, is also explored. Finally, the potential to develop targeted therapies that increase HDL concentration or functionality to improve acute or chronic kidney disease outcomes is discussed.


Subject(s)
Kidney Diseases/metabolism , Lipoproteins, HDL/metabolism , Animals , Humans , Lipoproteins, HDL/genetics
16.
Diabetes ; 70(10): 2377-2390, 2021 10.
Article in English | MEDLINE | ID: mdl-34233930

ABSTRACT

Podocyte injury is important in development of diabetic nephropathy (DN). Although several studies have reported single-cell-based RNA sequencing (RNA-seq) of podocytes in type 1 DN (T1DN), the podocyte translating mRNA profile in type 2 DN (T2DN) has not previously been compared with that of T1DN. We analyzed the podocyte translatome in T2DN in podocin-Cre; Rosa26fsTRAP; eNOS-/-; db/db mice and compared it with that of streptozotocin-induced T1DN in podocin-Cre; Rosa26fsTRAP; eNOS-/- mice using translating ribosome affinity purification (TRAP) and RNA-seq. More than 125 genes were highly enriched in the podocyte ribosome. More podocyte TRAP genes were differentially expressed in T2DN than in T1DN. TGF-ß signaling pathway genes were upregulated, while MAPK pathway genes were downregulated only in T2DN, while ATP binding and cAMP-mediated signaling genes were downregulated only in T1DN. Genes regulating actin filament organization and apoptosis increased, while genes regulating VEGFR signaling and glomerular basement membrane components decreased in both type 1 and type 2 diabetic podocytes. A number of diabetes-induced genes not previously linked to podocyte injury were confirmed in both mouse and human DN. On the basis of differences and similarities in the podocyte translatome in T2DN and T1DN, investigators can identify factors underlying the pathophysiology of DN and novel therapeutic targets to treat diabetes-induced podocyte injury.


Subject(s)
Diabetic Nephropathies/genetics , Diabetic Nephropathies/metabolism , Podocytes/metabolism , Animals , Diabetes Mellitus, Experimental/genetics , Diabetes Mellitus, Experimental/metabolism , Diabetes Mellitus, Experimental/pathology , Diabetes Mellitus, Type 1/complications , Diabetes Mellitus, Type 1/genetics , Diabetes Mellitus, Type 1/metabolism , Diabetes Mellitus, Type 2/complications , Diabetes Mellitus, Type 2/genetics , Diabetes Mellitus, Type 2/metabolism , Diabetic Nephropathies/pathology , Gene Expression Profiling , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Organ Specificity/genetics , Podocytes/pathology , Protein Biosynthesis/genetics , Proteome/analysis , Proteome/genetics , Proteome/metabolism , RNA, Messenger/analysis , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA-Seq , Sequence Analysis, RNA , Streptozocin , Transcriptome
17.
J Biol Chem ; 297(3): 101019, 2021 09.
Article in English | MEDLINE | ID: mdl-34331945

ABSTRACT

Reduced activity of paraoxonase 1 (PON1), a high-density lipoprotein (HDL)-associated enzyme, has been implicated in the development of atherosclerosis. Post-translational modifications of PON1 may represent important mechanisms leading to reduced PON1 activity. Under atherosclerotic conditions, myeloperoxidase (MPO) is known to associate with HDL. MPO generates the oxidants hypochlorous acid and nitrogen dioxide, which can lead to post-translational modification of PON1, including tyrosine modifications that inhibit PON1 activity. Nitrogen dioxide also drives lipid peroxidation, leading to the formation of reactive lipid dicarbonyls such as malondialdehyde and isolevuglandins, which modify HDL and could inhibit PON1 activity. Because isolevuglandins are more reactive than malondialdehyde, we used in vitro models containing HDL, PON1, and MPO to test the hypothesis that IsoLG formation by MPO and its subsequent modification of HDL contributes to MPO-mediated reductions in PON1 activity. Incubation of MPO with HDL led to modification of HDL proteins, including PON1, by IsoLG. Incubation of HDL with IsoLG reduced PON1 lactonase and antiperoxidation activities. IsoLG modification of recombinant PON1 markedly inhibited its activity, while irreversible IsoLG modification of HDL before adding recombinant PON1 only slightly inhibited the ability of HDL to enhance the catalytic activity of recombinant PON1. Together, these studies support the notion that association of MPO with HDL leads to lower PON1 activity in part via IsoLG-mediated modification of PON1, so that IsoLG modification of PON1 could contribute to increased risk for atherosclerosis, and blocking this modification might prove beneficial to reduce atherosclerosis.


Subject(s)
Aryldialkylphosphatase/antagonists & inhibitors , Lipids/chemistry , Lipoproteins, HDL/metabolism , Peroxidase/metabolism , Aryldialkylphosphatase/blood , Humans , Lipid Peroxidation/drug effects , Lipids/pharmacology , Recombinant Proteins/blood , Recombinant Proteins/metabolism
18.
Kidney Int ; 100(3): 585-596, 2021 09.
Article in English | MEDLINE | ID: mdl-34102217

ABSTRACT

Kidney disease affects intestinal structure and function. Although intestinal lymphatics are central in absorption and remodeling of dietary and synthesized lipids/lipoproteins, little is known about how kidney injury impacts the intestinal lymphatic network, or lipoproteins transported therein. To study this, we used puromycin aminoglycoside-treated rats and NEP25 transgenic mice to show that proteinuric injury expanded the intestinal lymphatic network, activated lymphatic endothelial cells and increased mesenteric lymph flow. The lymph was found to contain increased levels of cytokines, immune cells, and isolevuglandin (a highly reactive dicarbonyl) and to have a greater output of apolipoprotein AI. Plasma levels of cytokines and isolevuglandin were not changed. However, isolevuglandin was also increased in the ileum of proteinuric animals, and intestinal epithelial cells exposed to myeloperoxidase produced more isolevuglandin. Apolipoprotein AI modified by isolevuglandin directly increased lymphatic vessel contractions, activated lymphatic endothelial cells, and enhanced the secretion of the lymphangiogenic promoter vascular endothelial growth factor-C by macrophages. Inhibition of isolevuglandin synthesis by a carbonyl scavenger reduced intestinal isolevuglandin adduct level and lymphangiogenesis. Thus, our data reveal a novel mediator, isolevuglandin modified apolipoprotein AI, and uncover intestinal lymphatic network structure and activity as a new pathway in the crosstalk between kidney and intestine that may contribute to the adverse impact of kidney disease on other organs.


Subject(s)
Lymphatic Vessels , Vascular Endothelial Growth Factor C , Animals , Apolipoprotein A-I , Endothelial Cells , Kidney , Lymphangiogenesis , Mice , Rats
19.
Curr Atheroscler Rep ; 23(7): 38, 2021 05 13.
Article in English | MEDLINE | ID: mdl-33983531

ABSTRACT

PURPOSE OF REVIEW: This review highlights recent advances on the mechanisms and impact of HDL-small non-coding RNAs (sRNA) on intercellular communication in atherosclerosis. RECENT FINDINGS: Studies demonstrate that HDL-microRNAs (miRNA) are significantly altered in atherosclerotic cardiovascular disease (ASCVD), and are responsive to diet, obesity, and diabetes. Immune cells, pancreatic beta cells, and neurons are shown to export miRNAs to HDL. In turn, HDL can deliver functional miRNAs to recipient hepatocytes and endothelial cells regulating adhesion molecule expression, cytokines, and angiogenesis. With high-throughput sRNA sequencing, we now appreciate the full sRNA signature on circulating HDL, including the transport of rRNA and tRNA-derived fragments. Strikingly, HDL were highly enriched with exogenous microbial sRNAs. HDL transport a diverse set of host and non-host sRNAs that are altered in cardiometabolic diseases. Given the bioactivity of these sRNAs, they likely contribute to cellular communication within atherosclerotic lesions, and are potential disease biomarkers and therapeutic targets.


Subject(s)
Atherosclerosis , Diabetes Mellitus , MicroRNAs , RNA, Small Untranslated , Atherosclerosis/genetics , Endothelial Cells , High-Throughput Nucleotide Sequencing , Humans , MicroRNAs/genetics
20.
J Clin Invest ; 131(7)2021 04 01.
Article in English | MEDLINE | ID: mdl-33661763

ABSTRACT

Autophagy modulates lipid turnover, cell survival, inflammation, and atherogenesis. Scavenger receptor class B type I (SR-BI) plays a crucial role in lysosome function. Here, we demonstrate that SR-BI regulates autophagy in atherosclerosis. SR-BI deletion attenuated lipid-induced expression of autophagy mediators in macrophages and atherosclerotic aortas. Consequently, SR-BI deletion resulted in 1.8- and 2.5-fold increases in foam cell formation and apoptosis, respectively, and increased oxidized LDL-induced inflammatory cytokine expression. Pharmacological activation of autophagy failed to reduce lipid content or apoptosis in Sr-b1-/- macrophages. SR-BI deletion reduced both basal and inducible levels of transcription factor EB (TFEB), a master regulator of autophagy, causing decreased expression of autophagy genes encoding VPS34 and Beclin-1. Notably, SR-BI regulated Tfeb expression by enhancing PPARα activation. Moreover, intracellular macrophage SR-BI localized to autophagosomes, where it formed cholesterol domains resulting in enhanced association of Barkor and recruitment of the VPS34-Beclin-1 complex. Thus, SR-BI deficiency led to lower VPS34 activity in macrophages and in atherosclerotic aortic tissues. Overexpression of Tfeb or Vps34 rescued the defective autophagy in Sr-b1-/- macrophages. Taken together, our results show that macrophage SR-BI regulates autophagy via Tfeb expression and recruitment of the VPS34-Beclin-1 complex, thus identifying previously unrecognized roles for SR-BI and potentially novel targets for the treatment of atherosclerosis.


Subject(s)
Aorta/metabolism , Aortic Diseases/metabolism , Atherosclerosis/metabolism , Autophagy , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/metabolism , Class III Phosphatidylinositol 3-Kinases/metabolism , Foam Cells/metabolism , PPAR alpha/metabolism , Scavenger Receptors, Class B/metabolism , Transcription, Genetic , Animals , Aortic Diseases/genetics , Atherosclerosis/genetics , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/genetics , Beclin-1/genetics , Class III Phosphatidylinositol 3-Kinases/genetics , Mice , Mice, Knockout , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , PPAR alpha/genetics , Scavenger Receptors, Class B/deficiency
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