Cellular Heterogeneity of Activated Primary Human Macrophages and Associated Drug-Gene Networks: From Biology to Precision Therapeutics.

BACKGROUND: Interferon-γ (IFNγ) signaling plays a complex role in atherogenesis. IFNγ stimulation of macrophages permits in vitro exploration of proinflammatory mechanisms and the development of novel immune therapies. We hypothesized that the study of macrophage subpopulations could lead to anti-inflammatory interventions. METHODS: Primary human macrophages activated by IFNγ (M(IFNγ)) underwent analyses by single-cell RNA sequencing, time-course cell-cluster proteomics, metabolite consumption, immunoassays, and functional tests (phagocytic, efferocytotic, and chemotactic). RNA-sequencing data were analyzed in LINCS (Library of Integrated Network-Based Cellular Signatures) to identify compounds targeting M(IFNγ) subpopulations. The effect of compound BI-2536 was tested in human macrophages in vitro and in a murine model of atherosclerosis. RESULTS: Single-cell RNA sequencing identified 2 major clusters in M(IFNγ): inflammatory (M(IFNγ)i) and phagocytic (M(IFNγ)p). M(IFNγ)i had elevated expression of inflammatory chemokines and higher amino acid consumption compared with M(IFNγ)p. M(IFNγ)p were more phagocytotic and chemotactic with higher Krebs cycle activity and less glycolysis than M(IFNγ)i. Human carotid atherosclerotic plaques contained 2 such macrophage clusters. Bioinformatic LINCS analysis using our RNA-sequencing data identified BI-2536 as a potential compound to decrease the M(IFNγ)i subpopulation. BI-2536 in vitro decreased inflammatory chemokine expression and secretion in M(IFNγ) by shrinking the M(IFNγ)i subpopulation while expanding the M(IFNγ)p subpopulation. BI-2536 in vivo shifted the phenotype of macrophages, modulated inflammation, and decreased atherosclerosis and calcification. CONCLUSIONS: We characterized 2 clusters of macrophages in atherosclerosis and combined our cellular data with a cell-signature drug library to identify a novel compound that targets a subset of macrophages in atherosclerosis. Our approach is a precision medicine strategy to identify new drugs that target atherosclerosis and other inflammatory diseases.

Janus USPION modular platform (JUMP) for theranostic ultrasound-mediated targeted intratumoral microvascular imaging and DNA/miRNA delivery.

Rationale: High mortality in pancreatic cancer (PDAC) and triple negative breast cancer (TNBC) highlight the need to capitalize on nanoscale-design advantages for multifunctional diagnostics and therapies. DNA/RNA-therapies can provide potential breakthroughs, however, to date, there is no FDA-approved systemic delivery system to solid tumors. Methods: Here, we report a Janus-nanoparticle (jNP)-system with modular targeting, payload-delivery, and targeted-imaging capabilities. Our jNP-system consists of 10 nm ultrasmall superparamagnetic iron oxide nanoparticles (USPION) with opposing antibody-targeting and DNA/RNA payload-protecting faces, directionally self-assembled with commercially available zwitterionic microbubbles (MBs) and DNA/RNA payloads. Results: Sonoporation of targeted jNP-payload-MBs delivers functional reporter-DNA imparting tumor-fluorescence, and micro-RNA126 reducing non-druggable KRAS in PDAC-Panc1 and TNBC-MB231 xenografted tumors. The targeting jNP-system enhances ultrasound-imaging of intra-tumoral microvasculature using less MBs/body weight (BW). The jNP-design enhances USPION's T2*-magnetic resonance (MR) and MR-imaging of PDAC-peritoneal metastases using less Fe/BW. Conclusion: Altogether, data advance the asymmetric jNP-design as a potential theranostic Janus-USPION Modular Platform - a JUMP forward.

Proprotein Convertase Subtilisin/Kexin 9 (PCSK9) Promotes Macrophage Activation via LDL Receptor-Independent Mechanisms.

BACKGROUND: Activated macrophages contribute to the pathogenesis of vascular disease. Vein graft failure is a major clinical problem with limited therapeutic options. PCSK9 (proprotein convertase subtilisin/kexin 9) increases low-density lipoprotein (LDL)-cholesterol levels via LDL receptor (LDLR) degradation. The role of PCSK9 in macrophage activation and vein graft failure is largely unknown, especially through LDLR-independent mechanisms. This study aimed to explore a novel mechanism of macrophage activation and vein graft disease induced by circulating PCSK9 in an LDLR-independent fashion. METHODS: We used Ldlr-/- mice to examine the LDLR-independent roles of circulating PCSK9 in experimental vein grafts. Adeno-associated virus (AAV) vector encoding a gain-of-function mutant of PCSK9 (rAAV8/D377Y-mPCSK9) induced hepatic PCSK9 overproduction. To explore novel inflammatory targets of PCSK9, we used systems biology in Ldlr-/- mouse macrophages. RESULTS: In Ldlr-/- mice, AAV-PCSK9 increased circulating PCSK9, but did not change serum cholesterol and triglyceride levels. AAV-PCSK9 promoted vein graft lesion development when compared with control AAV. In vivo molecular imaging revealed that AAV-PCSK9 increased macrophage accumulation and matrix metalloproteinase activity associated with decreased fibrillar collagen, a molecular determinant of atherosclerotic plaque stability. AAV-PCSK9 induced mRNA expression of the pro-inflammatory mediators IL-1ß (interleukin-1 beta), TNFα (tumor necrosis factor alpha), and MCP-1 (monocyte chemoattractant protein-1) in peritoneal macrophages underpinned by an in vitro analysis of Ldlr-/- mouse macrophages stimulated with endotoxin-free recombinant PCSK9. A combination of unbiased global transcriptomics and new network-based hyperedge entanglement prediction analysis identified the NF-κB (nuclear factor-kappa B) signaling molecules, lectin-like oxidized LOX-1 (LDL receptor-1), and SDC4 (syndecan-4) as potential PCSK9 targets mediating pro-inflammatory responses in macrophages. CONCLUSIONS: Circulating PCSK9 induces macrophage activation and vein graft lesion development via LDLR-independent mechanisms. PCSK9 may be a potential target for pharmacologic treatment for this unmet medical need.

A disease-driver population within interstitial cells of human calcific aortic valves identified via single-cell and proteomic profiling.

Cellular heterogeneity of aortic valves complicates the mechanistic evaluation of the calcification processes in calcific aortic valve disease (CAVD), and animal disease models are lacking. In this study, we identify a disease-driver population (DDP) within valvular interstitial cells (VICs). Through stepwise single-cell analysis, phenotype-guided omic profiling, and network-based analysis, we characterize the DDP fingerprint as CD44highCD29+CD59+CD73+CD45low and discover potential key regulators of human CAVD. These DDP-VICs demonstrate multi-lineage differentiation and osteogenic properties. Temporal proteomic profiling of DDP-VICs identifies potential targets for therapy, including MAOA and CTHRC1. In vitro loss-of-function experiments confirm our targets. Such a stepwise strategy may be advantageous for therapeutic target discovery in other disease contexts.

Systems Approach to Discovery of Therapeutic Targets for Vein Graft Disease: PPARα Pivotally Regulates Metabolism, Activation, and Heterogeneity of Macrophages and Lesion Development.

BACKGROUND: Vein graft failure remains a common clinical challenge. We applied a systems approach in mouse experiments to discover therapeutic targets for vein graft failure. METHODS: Global proteomics and high-dimensional clustering on multiple vein graft tissues were used to identify potential pathogenic mechanisms. The PPARs (peroxisome proliferator-activated receptors) pathway served as an example to substantiate our discovery platform. In vivo mouse experiments with macrophage-targeted PPARα small interfering RNA, or the novel, selective activator pemafibrate demonstrate the role of PPARα in the development and inflammation of vein graft lesions. In vitro experiments further included metabolomic profiling, quantitative polymerase chain reaction, flow cytometry, metabolic assays, and single-cell RNA sequencing on primary human and mouse macrophages. RESULTS: We identified changes in the vein graft proteome associated with immune responses, lipid metabolism regulated by the PPARs, fatty acid metabolism, matrix remodeling, and hematopoietic cell mobilization. PPARα agonism by pemafibrate retarded the development and inflammation of vein graft lesions in mice, whereas gene silencing worsened plaque formation. Pemafibrate also suppressed arteriovenous fistula lesion development. Metabolomics/lipidomics, functional metabolic assays, and single-cell analysis of cultured human macrophages revealed that PPARα modulates macrophage glycolysis, citrate metabolism, mitochondrial membrane sphingolipid metabolism, and heterogeneity. CONCLUSIONS: This study explored potential drivers of vein graft inflammation and identified PPARα as a novel potential pharmacological treatment for this unmet medical need.

Uremic Toxin Indoxyl Sulfate Promotes Proinflammatory Macrophage Activation Via the Interplay of OATP2B1 and Dll4-Notch Signaling.

BACKGROUND: Chronic kidney disease (CKD) increases cardiovascular risk. Underlying mechanisms, however, remain obscure. The uremic toxin indoxyl sulfate is an independent cardiovascular risk factor in CKD. We explored the potential impact of indoxyl sulfate on proinflammatory activation of macrophages and its underlying mechanisms. METHODS: We examined in vitro the effects of clinically relevant concentrations of indoxyl sulfate on proinflammatory responses of macrophages and the roles of organic anion transporters and organic anion transporting polypeptides (OATPs). A systems approach, involving unbiased global proteomics, bioinformatics, and network analysis, then explored potential key pathways. To address the role of Delta-like 4 (Dll4) in indoxyl sulfate-induced macrophage activation and atherogenesis in CKD in vivo, we used 5/6 nephrectomy and Dll4 antibody in low-density lipoprotein receptor-deficient (Ldlr-/-) mice. To further determine the relative contribution of OATP2B1 or Dll4 to proinflammatory activation of macrophages and atherogenesis in vivo, we used siRNA delivered by macrophage-targeted lipid nanoparticles in mice. RESULTS: We found that indoxyl sulfate-induced proinflammatory macrophage activation is mediated by its uptake through transporters, including OATP2B1, encoded by the SLCO2B1 gene. The global proteomics identified potential mechanisms, including Notch signaling and the ubiquitin-proteasome pathway, that mediate indoxyl sulfate-triggered proinflammatory macrophage activation. We chose the Notch pathway as an example of key candidates for validation of our target discovery platform and for further mechanistic studies. As predicted computationally, indoxyl sulfate triggered Notch signaling, which was preceded by the rapid induction of Dll4 protein. Dll4 induction may result from inhibition of the ubiquitin-proteasome pathway, via the deubiquitinating enzyme USP5. In mice, macrophage-targeted OATP2B1/Slco2b1 silencing and Dll4 antibody inhibited proinflammatory activation of peritoneal macrophages induced by indoxyl sulfate. In low-density lipoprotein receptor-deficient mice, Dll4 antibody abolished atherosclerotic lesion development accelerated in Ldlr-/- mice. Moreover, coadministration of indoxyl sulfate and OATP2B1/Slco2b1 or Dll4 siRNA encapsulated in macrophage-targeted lipid nanoparticles in Ldlr-/- mice suppressed lesion development. CONCLUSIONS: These results suggest that novel crosstalk between OATP2B1 and Dll4-Notch signaling in macrophages mediates indoxyl sulfate-induced vascular inflammation in CKD.

Macrophages in Vascular Inflammation: Origins and Functions.

Macrophages influence various processes of cardiovascular inflammation. Whether they are of embryonic or post-natal hematopoietic origin, their balance in differential activation may direct the course of inflammation. Accelerated macrophage activation and accumulation through a pro-inflammatory signaling pathway may result in extensive tissue damage, adverse repair, and worsened clinical outcomes. Attenuation of such a mechanism and/or promotion of the anti-inflammatory macrophage activation may lead to early resolution of inflammation. Elucidating multiple novel mechanisms of monocyte and macrophage activation leads to a better understanding of their roles in vascular inflammation. In turn, this begets better therapeutic target identification and biomarker discovery. Combined with increasingly sensitive and specific imaging techniques, we continue to push back early detection and monitoring to provide us with a greater window for disease modification. The potential success of cytokine-targeted therapy will be solid proof of the inflammatory hypothesis of atherothrombosis.

DEspR roles in tumor vasculo-angiogenesis, invasiveness, CSC-survival and anoikis resistance: a 'common receptor coordinator' paradigm.

A priori, a common receptor induced in tumor microvessels, cancer cells and cancer stem-like cells (CSCs) that is involved in tumor angiogenesis, invasiveness, and CSC anoikis resistance and survival, could underlie contemporaneous coordination of these events rather than assume stochasticity. Here we show that functional analysis of the dual endothelin1/VEGFsignal peptide receptor, DEspR, (formerly named Dear, Chr.4q31.2) supports the putative common receptor paradigm in pancreatic ductal adenocarcinoma (PDAC) and glioblastoma (GBM) selected for their invasiveness, CD133+CSCs, and polar angiogenic features. Unlike normal tissue, DEspR is detected in PDAC and GBM microvessels, tumor cells, and CSCs isolated from PDAC-Panc1 and GBM-U87 cells. DEspR-inhibition decreased angiogenesis, invasiveness, CSC-survival and anoikis resistance in vitro, and decreased Panc1-CSC and U87-CSC xenograft tumor growth, vasculo-angiogenesis and invasiveness in nude(nu/nu) rats, suggesting that DEspR activation would coordinate these tumor progression events. As an accessible, cell-surface 'common receptor coordinator', DEspR-inhibition defines a novel targeted-therapy paradigm for pancreatic cancer and glioblastoma.

AVR/NAVR deficiency lowers blood pressure and differentially affects urinary concentrating ability, cognition, and anxiety-like behavior in male and female mice.

Arginine vasopressin (AVP) and angiotensin II (ANG II) are distinct peptide hormones involved in multiple organs modulating renal, cardiovascular, and brain functions. They achieve these functions via specific G protein-coupled receptors, respectively. The AVR/NAVR locus encodes two overlapping V2-type vasopressin isoreceptors: angiotensin-vasopressin receptor (AVR) responding to ANG II and AVP equivalently, and nonangiotensin vasopressin receptor (NAVR), which binds vasopressin exclusively. AVR and NAVR are expressed from a single gene by alternative promoter usage that is synergistically upregulated by testosterone and estrogen. This study tested the hypothesis that AVR/NAVR modulates urinary concentrating ability, blood pressure, and cognitive performance in vivo in a sex-specific manner. We developed a C57BL/6 inbred AVR/NAVR(-/-) knockout mouse that showed lower blood pressure in both male and female subjects and a urinary-concentrating defect restricted to male mice. We also detected sex-specific effects on cognitive and anxiety-like behaviors. AVR/NAVR(-/-) male mice exhibited impaired visuospatial and associative learning, while female mice showed improved performance in both type of cognition. AVR/NAVR deficiency produced an anxiolytic-like effect in female mice, while males were unaffected. Analysis of AVR- and NAVR-mediated phosphorylation/dephosphorylation of signaling proteins revealed activation/deactivation of known modulators of cognitive function. Our studies identify AVR/NAVR as key receptors involved in blood pressure regulation and sex-specific modulation of renal water homeostasis, cognitive function, and anxiety-like behavior. As such, the AVR/NAVR receptor system provides a molecular mechanism for sexually diergic traits and a putative common pathway for the emerging association of hypertension and cognitive decline and dementia.

Molecular imaging of vasa vasorum neovascularization via DEspR-targeted contrast-enhanced ultrasound micro-imaging in transgenic atherosclerosis rat model.

PURPOSE: Given that carotid vasa vasorum neovascularization is associated with increased risk for stroke and cardiac events, the present in vivo study was designed to investigate molecular imaging of carotid artery vasa vasorum neovascularization via target-specific contrast-enhanced ultrasound (CEU) micro-imaging. PROCEDURES: Molecular imaging was performed in male transgenic rats with carotid artery disease and non-transgenic controls using dual endothelin1/VEGFsp receptor (DEspR)-targeted microbubbles (MB(D)) and the Vevo770 micro-imaging system and CEU imaging software. RESULTS: DEspR-targeted CEU-positive imaging exhibited significantly higher contrast intensity signal (CIS)-levels and pre-/post-destruction CIS-differences in seven of 13 transgenic rats, in contrast to significantly lower CIS-levels and differences in control isotype-targeted microbubble (MB(C))-CEU imaging (n = 8) and in MB(D) CEU-imaging of five non-transgenic control rats (P < 0.0001). Ex vivo immunofluorescence analysis demonstrated binding of MB(D) to DEspR-positive endothelial cells; and association of DEspR-targeted increased contrast intensity signals with DEspR expression in vasa vasorum neovessel and intimal lesions. In vitro analysis demonstrated dose-dependent binding of MB(D) to DEspR-positive human endothelial cells with increasing %cells bound and number of MB(D) per cell, in contrast to MB(C) or non-labeled microbubbles (P < 0.0001). CONCLUSION: In vivo DEspR-targeted molecular imaging detected increased DEspR-expression in carotid artery lesions and in expanded vasa vasorum neovessels in transgenic rats with carotid artery disease. Future studies are needed to determine predictive value for stroke or heart disease in this transgenic atherosclerosis rat model and translational applications.

Sex-specific hippocampus-dependent cognitive deficits and increased neuronal autophagy in DEspR haploinsufficiency in mice.

Aside from abnormal angiogenesis, dual endothelin-1/VEGF signal peptide-activated receptor deficiency (DEspR(-/-)) results in aberrant neuroepithelium and neural tube differentiation, thus elucidating DEspR's role in neurogenesis. With the emerging importance of neurogenesis in adulthood, we tested the hypothesis that nonembryonic-lethal DEspR haploinsufficiency (DEspR(+/-)) perturbs neuronal homeostasis, thereby facilitating aging-associated neurodegeneration. Here we show that, in male mice only, DEspR-haploinsufficiency impaired hippocampus-dependent visuospatial and associative learning and induced noninflammatory spongiform changes, neuronal vacuolation, and loss in the hippocampus, cerebral cortex, and subcortical regions, consistent with autophagic cell death. In contrast, DEspR(+/-) females exhibited better cognitive performance than wild-type females and showed absence of neuropathological changes. Signaling pathway analysis revealed DEspR-mediated phosphorylation of activators of autophagy inhibitor mammalian target of rapamycin (mTOR) and dephosphorylation of known autophagy inducers. Altogether, the data demonstrate DEspR-mediated diametrical, sex-specific modulation of cognitive performance and autophagy, highlight cerebral neuronal vulnerability to autophagic dysregulation, and causally link DEspR haploinsufficiency with increased neuronal autophagy, spongiosis, and cognitive decline in mice.

Overlapping genes in Nalp6/PYPAF5 locus encode two V2-type vasopressin isoreceptors: angiotensin-vasopressin receptor (AVR) and non-AVR.

The angiotensin-vasopressin receptor (AVR) responds with equivalent affinities to angiotensin II (ANG II) and vasopressin and is coupled to adenylate cyclase and hence a V2-type vasopressin receptor. AVR maps to the Nalp6 locus and overlaps with the larger Nalp6/PYPAF5 reported to be a T cell/granulocyte-specific, cytoplasmic-specific proapoptotic protein, thus questioning the existence of AVR. Here we confirm, through different experimental modalities, that AVR is distinct from Nalp6/PYPAF5 based on different mRNA and protein sizes, subcellular localization, and tissue-specific expression patterns. Binding studies of PYPAF5-specific Cos1 transfectants detect high-affinity binding to vasopressin but not ANG II, thus assigning PYPAF5 as a non-AVR (NAVR). Signaling array analysis reveals that AVP stimulation of AVR- and NAVR-specific Cos1 transfectants results in diametrical activation as well as coactivation of signaling pathways known to mediate renal sodium and water balance. Likewise, ANG II stimulation of Cos1-AVR transfectants reveals a signaling profile distinct from that of AVP-stimulated Cos1-AVR transfectants. Analysis of genomic organization of the AVR/NAVR locus shows an overlapping gene arrangement with alternative promoter usage resulting in different NH(2) termini for NAVR and AVR. In addition to core promoter elements, androgen and estrogen response elements are detected. Promoter analysis of NAVR/AVR 5'-regulatory region detects transcriptional upregulation by testosterone and synergistic upregulation by testosterone and estrogen, thus suggesting that AVR and/or NAVR contribute to sex-specific V2-type vasopressin-mediated effects. Altogether, confirmation of AVR and identification of NAVR as vasopressin receptors are concordant with emerging vasopressin functions not attributable to V1a, V1b, or V2 receptors and add molecular bases for the multifunctional complexity of vasopressin-mediated functions and regulation.

Association of ATP1A1 and dear single-nucleotide polymorphism haplotypes with essential hypertension: sex-specific and haplotype-specific effects.

Essential hypertension remains a major risk factor for cardiovascular and cerebrovascular diseases. As a complex multifactorial disease, elucidation of susceptibility loci remains elusive. ATP1A1 and Dear are candidate genes for 2 closely linked rat chromosome-2 blood pressure quantitative trait loci. Because corresponding human syntenic regions are on different chromosomes, investigation of ATP1A1 (chromosome [chr]-1p21) and Dear (chr-4q31.3) facilitates genetic analyses of each blood pressure quantitative trait locus in human hypertension. Here we report the association of human ATP1A1 (P<0.000005) and Dear (P<0.03) with hypertension in a relatively isolated, case/control hypertension cohort from northern Sardinia by single-nucleotide polymorphism haplotype analysis. Sex-specific haplotype analyses detected stronger association of both loci with hypertension in males than in females. Haplotype trend-regression analyses support ATP1A1 and Dear as independent susceptibility loci and reveal haplotype-specific association with hypertension and normotension, thus delineating haplotype-specific subsets of hypertension. Although investigation in other cohorts needs to be performed to determine genetic effects in other populations, haplotype subtyping already allows systematic stratification of susceptibility and, hence, clinical heterogeneity, a prerequisite for unraveling the polygenic etiology and polygene-environment interactions in essential hypertension. As hypertension susceptibility genes, coexpression of ATP1A1 and Dear in both renal tubular cells and vascular endothelium suggest a cellular pathogenic scaffold for polygenic mechanisms of hypertension, as well as the hypothesis that ATP1A1 and/or Dear could contribute to the known renal and vascular endothelial dysfunction associated with essential (polygenic) hypertension.