Selective, Retrieval-Independent Disruption of Methamphetamine-Associated Memory by Actin Depolymerization

Memories associated with drugs of abuse, such as methamphetamine (METH), increase relapse vulnerability to substance use disorder. There is a growing consensus that memory is supported by structural and functional plasticity driven by F-actin polymerization in postsynaptic dendritic spines at excitatory synapses. However, the mechanisms responsible for the long-term maintenance of memories, after consolidation has occurred, are largely unknown

c-Kit Expression and Activation Protects the Vascular SMC Contractile Phenotype and Reduces Atherosclerosis

Rationale: Increased receptor tyrosine kinase (RTK) activity has been historically linked to atherosclerosis. At the beginning of my studies, I found the RTK c-Kit is present within atherosclerotic plaques of both humans and mice, and the global deficiency in c-Kit function increased atherosclerosis in hyperlipidemic mice.Objective: This study aimed to investigate if such unusual atheroprotective phenotype depends upon c-Kit&rsquo;s function in smooth muscle cells (SMC).&nbsp;Results: c-Kit was detected in human and mouse healthy and atherosclerotic aortas. The loss of c-Kit function in mutant c-Kit (KitW/Wv) mice caused a significant decrease in vasorelaxation and increase in blood pressure due to a significant loss of sGC in vascular smooth muscle cells. Further, hyperlipidemic mutant mice (KitW/Wv ApoE-/-) showed a significant increase in atherosclerotic burden and necrotic core size compared to wild type controls. Moreover, SMC specific KO of c-Kit in hyperlipidemic mice (KitSMC ApoE-/-) significantly increased atherosclerosis, plaque size and necrotic core area in the aortic sinus of hyperlipidemic mice. This protective effect was further studied in c-Kit KO SMC tracing mice (KitSMC eYFP) that labeled SMCs after TAM injection. The c-Kit KO SMCs were more prone to migrate, proliferate, and express foam cell markers (e.g., Mac2 and MCAM) than those from control littermate animals. Loss of c-Kit in cultured SMCs led to an increase in synthetic SMC phenotype. RNAseq analysis showed a significant upregulation in genes associated with cell proliferation, migration, lipid metabolism, and inflammation. I detected a significant increase in MCAM protein expression in KitMut SMCs and the aortic sinus of KitSMC hyperlipidemic mice.Conclusion: We can conclude that c-Kit plays an atheroprotective role in the arterial vasculature by reducing the transcription of genes associated with synthetic SMC transformation and thus preventing their migration, proliferation, and foam cell formation during atherosclerosis.&nbsp;</p

Abstract 15554: Smooth Muscle Cells Specific Gene Inactivation of Lysyl Oxidase Reduces Atherosclerosis Burden and Plaque Calcification in Hyperlipidemic Mice

Byline: Filipe F Stoyell Conti, Univ of Miami, Pembroke Pnes, FL; Miguel Rojas, Surgery, Univ of Miami; Zachary Zigmond, Surgery, Univ of Miami; Mohdahmar Rauf, Univ of Miami, FL; Laisel Martinez, UNIVERSITY OF MIAMI, Miami, FL; Roberto I Vazquez-Padron, UNIVERSITY OF MIAMI, Miami, FL Aim: Dissect the mechanisms by which LOX increases atherogenesis and plaque calcification in hyperlipidemic mice. Methods: LOX conditional knockout [LOXf/fMyh11-CreERT2 ApoE -/-] and control littermate [LOXwt/wtMyh11-CreERT2 ApoE -/-] mice were injected with tamoxifen before high fat diet (HFD) feeding for atherosclerosis and calcification assessment and molecular analyses. Results: scRNA sequence showed full Lox gene inactivation in 72.5% of the aortic SMCs without any significant Loxl compensatory regulation. Gene deletion in SMCs decreased global arterial LOX deposition by ~3-fold in relation to control mice. Surprisingly, the loss of vascular LOX neither promoted aneurysm nor affected the aorta's stiffness. However, it reduced atherosclerosis burden (WT: 23Ø1 vs. KO: 13Ø2 %, p ëñ0.01) and calcium deposition (11.8Ø3 versus 5Ø0.4%, pëñ 0.05) within the plaque after 16 weeks of high fat diet (HFD). Using bulk RNA sequencing, we further demonstrated that Lox gene inactivation leads to a marked decrease in Frizzled Related Protein-3 (FRZB) gene expression (127-fold and pëñ0.0001), a biphasic modulator of the proatherogenic Wnt signaling that facilitates the association of ð-catenin with TCF4 in the nucleus. The compromised FRZB /ð-catenin/TCF4 signaling axis reduced the downstream production of Bone Morphogenetic Protein 2 (BMP2), which is responsible for the osteogenic transition of SMCs. Further scRNAseq analysis of atherosclerotic aortas from conditional KO and control mice also showed a reduction of BMP2 in SMCs. Interestingly, genetic inactivation of LOX increased the number of contractile SMCs (KO: 40 vs. WT: 7 %), characterized by the high expression of ACTA2 and TAGLN, in the aorta of HFD fed mice when compared to the control group. BAPN, an irreversible inhibitor of LOX activity, was not able to reduce BMP2 expression and protein levels nor reduce calcium deposition in vitro, suggesting that the pro-calcification effect of LOX is not activity dependent. Conclusions: Vascular gene inactivation of Lox protects mice against atherosclerosis by reducing ð-catenin/TCF4/BMP2 proatherogenic signaling.Professiona

Abstract 199: Conditional Inactivation Of Lysyl Oxidase In Smooth Muscle Cells Reduces Atherosclerosis Burden And Plaque Calcification In Hyperlipidemic Mice Through Suppression Of The β-catenin/tcf4 Signaling Axis

Introduction: In atherosclerosis development and complications, the importance of lysyl oxidase (LOX), a copper-dependent amino-oxidase that crosslinks collagen and elastin and controls gene expression, is not well established. Hypothesis: Inactivation of LOX in smooth muscle cells (SMC) decreases atherosclerosis burden and plaque calcification in hyperlipidemic mice. Methods: qRT-PCR, Western blot, and scRNAseq demonstrated the loss of Lox gene expression in SMC of ApoE conditional mice (Lox f/f Myh11-CreERT2 ApoE-/-) but not in littermate control animals after tamoxifen injections. Surprisingly, vascular loss of LOX did not increase the risk of aneurysms nor affected the aorta's stiffness. However, it reduced atherosclerosis burden with respect to control mice (13±2 versus 23±1%, p < 0.01) after 16 weeks of high fat diet (HFD). There was also a significant reduction in calcium deposition (5±0.4 versus11.8±3, p < 0.05) within the plaque of LOX knockout mice versus control. Aortic whole genome transcriptomic analysis revealed a positive correlation between Lox gene expression and Frizzled-related protein 3 ( Frzb ), a biphasic modulator of Wnt signaling that facilitates the association of β-catenin with TCF4 in the nucleus. scRNAseq analysis confirmed the downregulation of Bmp2 and Myc , both β-catenin/TCF4 regulated genes, in SMC following gene deletion of Lox and 8 weeks of HFD. LOX knockout SMCs also had lower expression of Frzb and were more resistant to osteoblastic transformation in vitro. Conclusions: Vascular inactivation of LOX protects the vasculature from the proatherogenic effects of FRZB in SMCs

c-Kit expression in smooth muscle cells reduces atherosclerosis burden in hyperlipidemic mice

Increased receptor tyrosine kinase (RTK) activity has been historically linked to atherosclerosis. Paradoxically, we recently found that global deficiency in c-Kit function increased atherosclerosis in hyperlipidemic mice. This study aimed to investigate if such unusual atheroprotective phenotype depends upon c-Kit's function in smooth muscle cells (SMC). We studied atherosclerosis in a SMC-specific conditional knockout mice (KitSMC) and control littermate. Tamoxifen (TAM) and vehicle treated mice were fed high fat diet for 16 weeks before atherosclerosis assessment in the whole aorta using oil red staining. Smooth muscle cells were traced within the aortic sinus of conditional c-Kit tracing mice (KitSMC eYFP) and their control littermates (KitWT eYFP) by immunofluorescent confocal microscopy. We then performed RNA sequencing on primary SMC from c-Kit deficient and control mice, and identified significantly altered genes and pathways as a result of c-Kit deficiency in SMC. Atherosclerosis significantly increased in KitSMC mice with respect to control groups. In addition, the loss of c-Kit in SMC increased plaque size and necrotic core area in the aortic sinus of hyperlipidemic mice. Smooth muscle cells from KitSMC eYFP mice were more prone to migrate and express foam cell markers (e.g., Mac2 and MCAM) than those from control littermate animals. RNAseq analysis showed a significant upregulation in genes associated with cell proliferation, migration, lipid metabolism, and inflammation secondary to the loss of Kit function in primary SMCs. Loss of c-Kit increases SMC migration, proliferation, and expression of foam cell markers in atherosclerotic plaques from hyperlipidemic mice. [Display omitted] •c-Kit, a well-known stem cell marker, plays an important role in smooth muscle cells.•c-Kit deficiency increases smooth muscle cell migration to atherosclerotic lesions.•c-Kit deficiency increases smooth muscle cell-derived foam-cell formation.•c-Kit expression decreases atherosclerosis unlike other receptor tyrosine kinases

Abstract 15806: The Unique Cellular Ecosystem of the Human Basilic Vein: A Single-Cell Transcriptomic Analysis

Byline: Miguel G Rojas, Surgery, Univ of Miami, FL; Laisel Martinez, UNIVERSITY OF MIAMI, Miami, FL; Simone Pereira-Simon, Univ of Miami, Miami, FL; Zachary Zigmond, Univ of Miami, Miami, FL; Nieves Santos Falcon, Univ of Miami, Miami, FL; Alghidak Salama, Surgery, Univ of Miami, FL; Roberto I Vazquez-Padron, UNIVERSITY OF MIAMI, Miami, FL Introduction: The cells in veins that contribute to vascular disease have not been fully characterized. This study generated a cellular atlas of the human basilic vein that will help understand vein physiology and pathologies. Methods: Single-cell sequencing of four basilic veins from healthy organ donors. Results: The integrated single-cell profile of 15,747 vein cells revealed 13 populations containing canonical and non-canonical vascular markers. The SMC population represented 16% of cells, of which 8% were purely contractile and the remaining 8% had a modulated phenotype. One cluster of modulated SMCs expressed ECM genes (collagen type IV, FN1, and TNC) while retaining high expression of contractile markers (DES, MYH11, ©ÀACTG2). The other SMC cluster had unique expression of CD36, APOE, LPL, and STEAP4, characteristic of lipid-processing cells. Surprisingly, these non-diseased veins contained a high number of resident immune cells, including pro-inflammatory macrophages (9%), CD163- and HMOX1-expressing Mhem-like macrophages (18%), lymphocytes (5%), and small counts of neutrophils (0.3%) and mast cells (0.7%). The adventitial cell populations highlighted the fundamental role of this layer in vascular maintenance, remodeling, and angiogenesis. We found a significant number of vasa vasorum ECs (6.5%) which differentiated from the luminal subpopulation (3.5%) by CD34, KDR, NOTCH4, and ICAM2 expression. There were also two types of fibroblasts. Classical fibroblasts (19%) had high expression of collagen genes, while adipogenic fibroblasts (21%) were characterized by high levels of ECM-interacting fibulins, adipocyte-associated genes (ADH1B, APOD, CXCL14, IGF1), and modulators of angiogenesis (ADM and EFEMP1). CellCall analyses identified 46 overrepresented ligand-receptor communication pathways in all clusters, including CXCL, FGF, IL-6, and complement signaling. Communication between pro-inflammatory macrophages and luminal ECs was the most overrepresented signaling connection. Conclusions: This comprehensive atlas of the human vein will help identify cell subpopulations relevant to venous pathologies, without relying on extrapolations from arterial biology and focusing instead on the uniqueness of vein tissues.Professiona

Functions for platelet factor 4 (PF4/CXCL4) and its receptors in fibroblast-myofibroblast transition and fibrotic failure of arteriovenous fistulas (AVFs)

BACKGROUNDOver 60% of End Stage Renal Disease (ESRD) patients are relying on hemodialysis (HD) to survive, and the arteriovenous fistula (AVF) is the preferred vascular access method for HD. However approximately half of all newly created AVF fail to mature and cannot be used without a salvage procedure. We have recently demonstrated an association between AVF maturation failure and post-operative fibrosis, while our RNA-seq study also revealed that veins that ultimately failed during AVF maturation had elevated levels of platelet factor 4 (PF4/CXCL4). However, a link between these two findings was yet to be established. METHODSIn this study, we investigated potential mechanisms between PF4 levels and fibrotic remodeling in veins. We compared the local expression of PF4 and fibrosis marker integrin β6 (ITGB6) in veins that successfully underwent maturation with that in veins that ultimately failed to mature. We also measured the changes of expression level of α-smooth muscle actin (αSMA/ACTA2) and collagen (Col1/COL1A1) in venous fibroblasts upon various treatments, such as PF4 pharmacological treatment, alteration of PF4 expression, and blocking of PF4 receptors. RESULTSWe found that PF4 is expressed in veins and co-localizes with αSMA. In venous fibroblasts, PF4 stimulates expression of αSMA and Col1 via different pathways. The former requires integrins αvβ5 and α5β1, while chemokine receptor CXCR3 is needed for the latter. Interestingly, we also discovered that the expression of PF4 is associated with that of ITGB6, the β subunit of integrin αvβ6. This integrin is critical for the activation of the major fibrosis factor TGFβ, and overexpression of PF4 promotes activation of the TGFβ pathway. CONCLUSIONSThese results indicate that upregulation of PF4 may cause venous fibrosis both directly by stimulating fibroblast differentiation and expression of extracellular matrix (ECM) molecules and indirectly by facilitating the activation of the TGFβ pathway

c-Kit modifies the inflammatory status of smooth muscle cells

Background c-Kit is a receptor tyrosine kinase present in multiple cell types, including vascular smooth muscle cells (SMC). However, little is known about how c-Kit influences SMC biology and vascular pathogenesis. Methods High-throughput microarray assays and in silico pathway analysis were used to identify differentially expressed genes between primary c-Kit deficient (KitW/W–v) and control (Kit+/+) SMC. Quantitative real-time RT-PCR and functional assays further confirmed the differences in gene expression and pro-inflammatory pathway regulation between both SMC populations. Results The microarray analysis revealed elevated NF-κB gene expression secondary to the loss of c-Kit that affects both the canonical and alternative NF-κB pathways. Upon stimulation with an oxidized phospholipid as pro-inflammatory agent, c-Kit deficient SMC displayed enhanced NF-κB transcriptional activity, higher phosphorylated/total p65 ratio, and increased protein expression of NF-κB regulated pro-inflammatory mediators with respect to cells from control mice. The pro-inflammatory phenotype of mutant cells was ameliorated after restoring c-Kit activity using lentiviral transduction. Functional assays further demonstrated that c-Kit suppresses NF-κB activity in SMC in a TGFβ-activated kinase 1 (TAK1) and Nemo-like kinase (NLK) dependent manner. Discussion Our study suggests a novel mechanism by which c-Kit suppresses NF-κB regulated pathways in SMC to prevent their pro-inflammatory transformation