Showing papers by "Brian R. Wamhoff published in 2010"

Identification of a Novel Macrophage Phenotype That Develops in Response to Atherogenic Phospholipids via Nrf2

Abstract: Rationale: Macrophages change their phenotype and biological functions depending on the microenvironment. In atherosclerosis, oxidative tissue damage accompanies chronic inflammation; however, macrophage phenotypic changes in response to oxidatively modified molecules are not known. Objective: To examine macrophage phenotypic changes in response to oxidized phospholipids that are present in atherosclerotic lesions. Methods and Results: We show that oxidized phospholipid-treated murine macrophages develop into a novel phenotype (Mox) that is strikingly different from the conventional M1 and M2 macrophage phenotypes. Compared to M1 and M2, Mox macrophages show a different gene expression pattern, as well as decreased phagocytotic and chemotactic capacity. Treatment with oxidized phospholipids induces both M1 and M2 macrophages to switch to the Mox phenotype. Whole-genome expression array analysis and subsequent gene ontology clustering revealed that the Mox phenotype was characterized by abundant overrepresentation of Nrf2-mediated expression of redox-regulatory genes. In macrophages isolated from Nrf2 −/− mice, oxidized phospholipid-induced gene expression and regulation of redox status were compromised. Moreover, we found that Mox macrophages comprise 30% of all macrophages in advanced atherosclerotic lesions of low-density lipoprotein receptor knockout (LDLR −/− ) mice. Conclusions: Together, we identify Nrf2 as a key regulator in the formation of a novel macrophage phenotype (Mox) that develops in response to oxidative tissue damage. The unique biological properties of Mox macrophages suggest this phenotype may play an important role in atherosclerotic lesion development as well as in other settings of chronic inflammation.

Complex regulation and function of the inflammatory smooth muscle cell phenotype in atherosclerosis.

Abstract: Vascular smooth muscle cell (SMC) phenotypic modulation plays a key role in atherosclerosis and is classically defined as a switch from a 'contractile' phenotype to a 'synthetic' phenotype, whereby genes that define the contractile SMC phenotype are suppressed and proliferation and/or migratory mechanisms are induced. There is also evidence that SMCs may take on a 'proinflammatory' phenotype, whereby SMCs secrete cytokines and express cell adhesion molecules, e.g. IL-8, IL-6, and VCAM-1, respectively, which may functionally regulate monocyte and macrophage adhesion and other processes during atherosclerosis. Factors that drive the inflammatory phenotype are not limited to cytokines but also include hemodynamic forces imposed on the blood vessel wall and intimate interaction of endothelial cells with SMCs, as well as changes in matrix composition in the vessel wall. However, it is critical to recognize that our understanding of the complex interaction of these multiple signal inputs has only recently begun to shed light on mechanisms that regulate the inflammatory SMC phenotype, primarily through models that attempt to recreate this environment ex vivo. The goal of this review is to summarize our current knowledge in this area and identify some of the key unresolved challenges and questions requiring further study.

Targeted gene transfection from microbubbles into vascular smooth muscle cells using focused, ultrasound-mediated delivery

Abstract: We investigated a method for gene delivery to vascular smooth muscle cells using ultrasound triggered delivery of plasmid DNA from electrostatically coupled cationic microbubbles. Microbubbles carrying reporter plasmid DNA were acoustically ruptured in the vicinity of smooth muscle cells in vitro under a range of acoustic pressures (0 to 950 kPa) and pulse durations (0 to 100 cycles). No effect on gene transfection or viability was observed from application of microbubbles, DNA or ultrasound alone. Microbubbles in combination with ultrasound (500-kPa, 1-MHz, 50-cycle bursts at a pulse repetition frequency [PRF] of 100 Hz) significantly reduced viability both with DNA (53 ± 27%) and without (19 ± 8%). Maximal gene transfection (∼1% of cells) occurred using 50-cycle, 1-MHz pulses at 300 kPa, which resulted in 40% viability of cells. We demonstrated that we can locally deliver DNA to vascular smooth muscle cells in vitro using microbubble carriers and focused ultrasound. (E-mail: jh7fj@virginia.edu )

Nuclear Factor of Activated T Cells Regulates Osteopontin Expression in Arterial Smooth Muscle in Response to Diabetes-Induced Hyperglycemia

Abstract: Objective— Hyperglycemia is a recognized risk factor for cardiovascular disease in diabetes. Recently, we reported that high glucose activates the Ca 2+ /calcineurin-dependent transcription factor nuclear factor of activated T cells (NFAT) in arteries ex vivo. Here, we sought to determine whether hyperglycemia activates NFAT in vivo and whether this leads to vascular complications. Methods and Results— An intraperitoneal glucose-tolerance test in mice increased NFATc3 nuclear accumulation in vascular smooth muscle. Streptozotocin-induced diabetes resulted in increased NFATc3 transcriptional activity in arteries of NFAT-luciferase transgenic mice. Two NFAT-responsive sequences in the osteopontin (OPN) promoter were identified. This proinflammatory cytokine has been shown to exacerbate atherosclerosis and restenosis. Activation of NFAT resulted in increased OPN mRNA and protein in native arteries. Glucose-induced OPN expression was prevented by the ectonucleotidase apyrase, suggesting a mechanism involving the release of extracellular nucleotides. The calcineurin inhibitor cyclosporin A or the novel NFAT blocker A-285222 prevented glucose-induced OPN expression. Furthermore, diabetes resulted in higher OPN expression, which was significantly decreased by in vivo treatment with A-285222 for 4 weeks or prevented in arteries from NFATc3 −/− mice. Conclusions— These results identify a glucose-sensitive transcription pathway in vivo, revealing a novel molecular mechanism that may underlie vascular complications of diabetes.

Discovery, Biological Evaluation, and Structure—Activity Relationship of Amidine Based Sphingosine Kinase Inhibitors

Abstract: Sphingosine 1-phosphate (S1P), a potent phospholipid growth and trophic factor, is synthesized in vivo by two sphingosine kinases. Thus these kinases have been proposed as important drug targets for treatment of hyperproliferative diseases and inflammation. We report here a new class of amidine-based sphingosine analogues that are competitive inhibitors of sphingosine kinases exhibiting varying degrees of enzyme selectivity. These inhibitors display KI values in the submicromolar range for both sphingosine kinases and, in cultured vascular smooth muscle cells, decrease S1P levels and initiate growth arrest.

FTY720 promotes local microvascular network formation and regeneration of cranial bone defects.

Abstract: The calvarial bone microenvironment contains a unique progenitor niche that should be considered for therapeutic manipulation when designing regeneration strategies. Recently, our group demonstrated that cells isolated from the dura are multipotent and exhibit expansion potential and robust mineralization on biodegradable constructs in vitro. In this study, we evaluate the effectiveness of healing critical-sized cranial bone defects by enhancing microvascular network growth and host dura progenitor trafficking to the defect space pharmacologically by delivering drugs targeted to sphingosine 1-phosphate (S1P) receptors. We demonstrate that delivery of pharmacological agonists to (S1P) receptors S1P(1) and S1P(3) significantly increase bone ingrowth, total microvessel density, and smooth muscle cell investment on nascent microvessels within the defect space. Further, in vitro proliferation and migration studies suggest that selective activation of S1P(3) promotes recruitment and growth of osteoblastic progenitors from the meningeal dura mater.

Focused in vivo delivery of plasmid DNA to the porcine vascular wall via intravascular ultrasound destruction of microbubbles.

Abstract: Background: Safety concerns associated with drug-eluting stents have spurred interest in alternative vessel therapeutics following angioplasty. Microbubble contrast agents have been

Cyclosporine up-regulates Kruppel-like factor-4 (KLF4) in vascular smooth muscle cells and drives phenotypic modulation in vivo.

Abstract: Cyclosporine A (CSA, calcineurin inhibitor) has been shown to block both vascular smooth muscle cell (VSMC) proliferation in cell culture and vessel neointimal formation following injury in vivo. The purpose of this study was to determine molecular and pathological effects of CSA on VSMCs. Using real-time reverse transcription-polymerase chain reaction, Western blot analysis, and immunofluorescence microscopy, we show that CSA up-regulated the expression of Kruppel-like factor-4 (KLF4) in VSMCs. KLF4 plays a key role in regulating VSMC phenotypic modulation. KLF4 antagonizes proliferation, facilitates migration, and down-regulates VSMC differentiation marker gene expression. We show that the VSMC differentiation marker genes smooth muscle α-actin (ACTA2), transgelin (TAGLN), smoothelin (SMTN), and myocardin (MYOCD) are all down-regulated by CSA in VSMC monoculture, whereas cyclin-dependent kinase inhibitor-1A (CDKN1A) and matrix metalloproteinase-3 (MMP3) are up-regulated. CSA did not affect the abundance of the VSMC microRNA (MIR) markers MIR143 and MIR145. Administration of CSA to rat carotid artery in vivo resulted in acute and transient suppression of ACTA2, TAGLN, SMTN, MYOCD, and smooth muscle myosin heavy chain (MYH11) mRNA levels. The tumor suppressor genes KLF4, p53, and CDKN1A, however, were up-regulated, as well as MMP3, MMP9, and collagen-VIII. CSA-treated arteries showed remarkable remodeling, including breakdown of the internal elastic lamina and reorientation of VSMCs, as well as increased KLF4 immunostaining in VSMCs and endothelial cells. Altogether, these data show that cyclosporin up-regulates KLF4 expression and promotes phenotypic modulation of VSMCs.

Systems and Methods for Ultrasound Imaging and Insonation of Microbubbles

Abstract: A catheter system including an elongate tubular member having a proximal end portion, a distal end portion and a lumen extending through at least a portion of a length of the elongate tubular member. The distal end portion of the elongate member is dimensioned and adapted to advance to or in proximity to a treatment site of a subject. A microbubble device is in fluid communication with the lumen. The microbubble device includes at least one input port for receiving a flow of material into the device and an output port configured to output microbubbles from the microbubble device. A second tubular member is in fluid communication with one of the at least one input ports. A pressure fitting arrangement is adapted to maintain a seal between the second tubular member and the input port.

Integrative genomics identifies DSCR1 (RCAN1) as a novel NFAT-dependent mediator of phenotypic modulation in vascular smooth muscle cells

Abstract: Vascular smooth muscle cells (SMCs) display remarkable phenotypic plasticity in response to environmental cues. The nuclear factor of activated T-cells (NFAT) family of transcription factors plays a critical role in vascular pathology. However, known functional NFAT gene targets in vascular SMCs are currently limited. Publicly available whole-genome expression array data sets were analyzed to identify differentially expressed genes in human, mouse and rat SMCs. Comparison between vehicle and phenotypic modulatory stimuli identified 63 species-conserved, upregulated genes. Integration of the 63 upregulated genes with an in silico NFAT-ome (a species-conserved list of gene promoters containing at least one NFAT binding site) identified 18 putative NFAT-dependent genes. Further intersection of these 18 potential NFAT target genes with a mouse in vivo vascular injury microarray identified four putative NFAT-dependent, injury-responsive genes. In vitro validations substantiated the NFAT-dependent role of Cyclooxygenase 2 (COX2/PTGS2) in SMC phenotypic modulation and uncovered Down Syndrome Candidate Region 1 (DSCR1/RCAN1) as a novel NFAT target gene in SMCs. We show that induction of DSCR1 inhibits calcineurin/NFAT signaling through a negative feedback mechanism; DSCR1 overexpression attenuates NFAT transcriptional activity and COX2 protein expression, whereas knockdown of endogenous DSCR1 enhances NFAT transcriptional activity. Our integrative genomics approach illustrates how the combination of publicly available gene expression arrays, computational databases and empirical research methods can answer specific questions in any cell type for a transcriptional network of interest. Herein, we report DSCR1 as a novel NFAT-dependent, injury-inducible, early gene that may serve to negatively regulate SMC phenotypic switching.

Compositions and methods for inhibiting sphingosine kinase

Abstract: Amidine analogs that can inhibit the activity of sphingosine kinase 1 and sphingosine kinase 2 (SphK1 & SphK2) are provided. The compounds can prevent angiogenesis in tumor cells.

Ultrasound-microbubble-mediated drug delivery efficacy and cell viability depend on microbubble radius and ultrasound frequency

Abstract: We have been investigating ultrasound-mediated drug delivery from microbubbles (MBs) as an alternative therapy to reduce hyper-proliferation of smooth muscle cells We sought to determine if MB size affects drug delivery, cell viability, or cell adherence Control, DiI, and drug-incorporated-rapamycin-microbubbles (R-MBs) were size sorted into “small” and “large” sub-populations with number-average mean diameters of 17µm and 37µm respectively Drug dose was maintained by keeping the surface area between MB sub-populations constant Vascular smooth muscle cells (SMCs) were exposed to size-sorted R-MBs and insonated at 1MHz, 300 kPa, with 50 cycle sinusoids at a PRF of 100Hz for 8s Insonation with large MBs reduced the proliferation rate of cells by 660% vs 561% (n=8, p=046) with small MBs The LIVE/DEAD® Viability/Cytotoxicity assay revealed a significant reduction in live cells (61% live vs 776% live), n=4, p=041), and a greater, but not statistically significant, increase in the permeabilized cells after insonation (1MHz) with large MBs (156%) versus small MBs (111%) We also investigated the effect of ultrasound frequency (1, 225, 5, and 10MHz) on MB on rupture and viability following 20 ultrasound pulses (20 cycle-sinusoids) at a peak negative pressure 600 kPa 1MHz insonation ruptured the most microbubbles of either size, permeabilized the most cells (50% & 43% for small and large MBs respectively) and killed (33% & 31%) the most cells More small (d=18 µm) MBs were ruptured at every frequency (p<0001, see Fig 4) Permeabilization of cells was approximately inversely related to frequency of insonation 1MHz ultrasound permeabilized the most cells, but also killed the most cells The permeable/dead cell ratio was 64% for large MBs and 98% for small MBs indicating a better therapeutic ratio when using small MBs1

Nuclear Factor of Activated T-cells (NFAT) regulates osteopontin expression in arterial smooth muscle in response to diabetes-induced hyperglycemia

Abstract: Objective—Hyperglycemia is a recognized risk factor for cardiovascular disease in diabetes. Recently, we reported that high glucose activates the Ca2+/calcineurin-dependent transcription factor nuclear factor of activated T cells (NFAT) in arteries ex vivo. Here, we sought to determine whether hyperglycemia activates NFAT in vivo and whether this leads to vascular complications. Methods and Results—An intraperitoneal glucose-tolerance test in mice increased NFATc3 nuclear accumulation in vascular smooth muscle. Streptozotocin-induced diabetes resulted in increased NFATc3 transcriptional activity in arteries of NFAT-luciferase transgenic mice. Two NFAT-responsive sequences in the osteopontin (OPN) promoter were identified. This proinflammatory cytokine has been shown to exacerbate atherosclerosis and restenosis. Activation of NFAT resulted in increased OPN mRNA and protein in native arteries. Glucose-induced OPN expression was prevented by the ectonucleotidase apyrase, suggesting a mechanism involving the release of extracellular nucleotides. The calcineurin inhibitor cyclosporin A or the novel NFAT blocker A-285222 prevented glucose-induced OPN expression. Furthermore, diabetes resulted in higher OPN expression, which was significantly decreased by in vivo treatment with A-285222 for 4 weeks or prevented in arteries from NFATc3−/− mice. Conclusions—These results identify a glucose-sensitive transcription pathway in vivo, revealing a novel molecular mechanism that may underlie vascular complications of diabetes.

Cyclosporine upregulates KLF4 in vascular smooth muscle cells and drives phenotypic modulation in vivo

Abstract: s Presented as Posters Garvey SM, Sinden DS, Schoppee Bortz PD, Wamhoff BR. Cyclosporin A-dependent regulation of genes associated with vascular smooth muscle phenotypic modulation. Arteriosclerosis, Thrombosis and Vascular Biology Annual Conference, Washington, DC. 2009. Reprint Requests Dr. Brian R. Wamhoff P.O. Box 801395 Department of Medicine, Division of Cardiovascular Medicine University of Virginia, Charlottesville, VA 22908-1395. Tel.: +1 434 243 9488. Fax: +1 434 982 3139. E-mail: wamhoff@virginia.edu This article has not been copyedited and formatted. The final version may differ from this version. JPET Fast Forward. Published on January 20, 2010 as DOI: 10.1124/jpet.109.163949 at A PE T Jornals on Feruary 4, 2021 jpet.asjournals.org D ow nladed from

Localized ex-vivo microbubble-based model drug delivery using an ultrasound catheter

Abstract: Focal drug delivery using ultrasound applied to microbubbles in the vascular system relies on the microbubbles being in close proximity to the vessel wall In-vitro microbubble translation to a vessel wall under primary radiation force using an intravascular ultrasound (IVUS) catheter was tested using a wall-less flow phantom Experiments were performed in 40% hematocrit blood to optimize the Pulse Repetition Frequency (PRF) for translating microbubbles to the edge of a channel under flow An optimal PRF of 10 kHz in blood was selected for the IVUS catheter based on the results of the wall-less flow phantom experiments Ex-vivo delivery of DiI, a hydrophobic fluorescent dye used as a model drug, was performed on excised porcine arteries under flow conditions of 30 ml/min with microbubbles incorporating DiI and both a 30 μs, 80 kPa PNP, 15 MHz Gaussian ramped sinusoid at 10kHz PRF for microbubble translation and 20% fractional bandwidth, 120 kPa PNP, Gaussian at 1kHz PRF for microbubble destruction The results of DiI delivery were imaged with a fluorescent microscope and indicated a 665% greater intensity (in arbitrary units derived from fluorescence intensity) in the treated region versus the untreated region This demonstrates that the IVUS catheter successfully provided localized delivery of the DiI marker to a porcine carotid artery