Increased phagocytosis in the presence of enhanced M2-like macrophage responses correlates with increased primary and latent HSV-1 infection.

After HSV-1 infection, macrophages infiltrate early into the cornea, where they play an important role in HSV-1 infection. Macrophages are divided into M1 or M2 groups based on their activation. M1 macrophages are pro-inflammatory, while M2 macrophages are anti-inflammatory. Macrophage phenotypes can shift between M1 or M2 in vitro and in vivo following treatment with specific cytokines. In this study we looked at the effect of M2 macrophages on HSV-1 infectivity using mice either lacking M2 (M2-/-) or overexpressing M2 (M2-OE) macrophages. While presence or absence of M2 macrophages had no effect on eye disease, we found that over expression of M2 macrophages was associated with increased phagocytosis, increased primary virus replication, increased latency, and increased expression of pro- and anti-inflammatory cytokines. In contrast, in mice lacking M2 macrophages following infection phagocytosis, replication, latency, and cytokine expression were similar to wild type mice. Our results suggest that enhanced M2 responses lead to higher phagocytosis, which affected both primary and latent infection but not reactivation.

Deficiency of GATA3-Positive Macrophages Improves Cardiac Function Following Myocardial Infarction or Pressure Overload Hypertrophy.

BACKGROUND: Macrophages are highly plastic cells that play an important role in the pathogenesis of cardiovascular disease. OBJECTIVES: This study investigated the role of GATA3-positive macrophages in modulating cardiac function after myocardial infarction (MI) or in response to pressure overload hypertrophy. METHODS: Myeloid-specific GATA3-deficient (mGATA3KO) mice were generated, MI or pressure overload was induced, and cardiac function was determined by echocardiography. GATA3-sufficient Cre mice were used as a control. Immunohistochemical staining, flow cytometry, MILLIPLEX Mouse Cytokine/Chemokine Assay, cultured macrophages, quantitative real-time polymerase chain reaction, and western blot were used to determine the role of GATA3 in macrophages. RESULTS: GATA3-positive macrophages rapidly accumulated in the infarcted region of the myocardium after acute MI. Deficiency of GATA3-positive macrophages led to a significant improvement of cardiac function in response to acute MI or pressure overload hypertrophy compared with the control mice. This improvement was associated with the presence of a large number of proinflammatory Ly6Chi monocytes/macrophages and fewer reparative Ly6Clo macrophages in the myocardium of mGATA3KO mice compared with control mice. Analysis of serum proteins from the 2 mouse genotypes revealed no major changes in the profile of serum growth factors and cytokines between the 2 mice genotypes before and after MI. GATA3 was found to be specifically and transiently induced by interleukin 4 in cultured macrophages through activity of the proximal promoter, whereas the distal promoter remained silent. In addition, the absence of GATA3 in macrophages markedly attenuated arginase-1 expression in cultured macrophages. CONCLUSIONS: We demonstrated that the presence of GATA3-positive macrophages adversely affects remodeling of the myocardium in response to ischemia or pressure overload, whereas the absence of these macrophages led to a significant improvement in cardiac function. Targeting of signaling pathways that lead to the expression of GATA3 in macrophages may have favorable cardiac outcomes.

Bone Marrow-Derived Tenascin-C Attenuates Cardiac Hypertrophy by Controlling Inflammation.

BACKGROUND: Tenascin-C (TNC) is a highly conserved matricellular protein with a distinct expression pattern during development and disease. Remodeling of the left ventricle (LV) in response to pressure overload leads to the re-expression of the fetal gene program. OBJECTIVES: The aim of this study was to investigate the function of TNC in cardiac hypertrophy in response to pressure overload. METHODS: Pressure overload was induced in TNC knockout and wild-type mice by constricting their abdominal aorta or by infusion of angiotensin II. Echocardiography, immunostaining, flow cytometry, quantitative real-time polymerase chain reaction, and reciprocal bone marrow transplantation were used to evaluate the effect of TNC deficiency. RESULTS: Echocardiographic analysis of pressure overloaded hearts revealed that all LV parameters (LV end-diastolic and -systolic dimensions, ejection fraction, and fractional shortening) deteriorated in TNC-deficient mice compared with their wild-type counterparts. Cardiomyocyte size and collagen accumulation were significantly greater in the absence of TNC. Mechanistically, TNC deficiency promoted rapid accumulation of the CCR2+/Ly6Chi monocyte/macrophage subset into the myocardium in response to pressure overload. Further, echocardiographic and immunohistochemical analyses of recipient hearts showed that expression of TNC in the bone marrow, but not the myocardium, protected the myocardium against excessive remodeling of the pressure-overloaded heart. CONCLUSIONS: TNC deficiency further impaired cardiac function in response to pressure overload and exacerbated fibrosis by enhancing inflammation. In addition, expression of TNC in the bone marrow, but not the myocardium, protected the myocardium against excessive remodeling in response to mild pressure overload.

Oxidized LDL activated eosinophil polarize macrophage phenotype from M2 to M1 through activation of CD36 scavenger receptor.

BACKGROUND AND AIMS: Inflammation, particularly innate immunity, plays an important role in cardiovascular diseases. The aim of this study was to investigate whether atherogenic determinants such as oxidized LDL modulate the phenotype of eosinophils. METHODS: Cultured eosinophils were treated with oxidized LDL and the expression of selective inflammatory and anti-inflammatory cytokines was determined. In addition, the eosinophil receptor and signaling that mediate these events were identified. RESULTS: Treatment of cultured eosinophils with oxidized LDL (Ox-LDL) specifically induced the expression of IFNα and IFNß without affecting expression of other proinflammatory cytokines, such as TNFα, IL-1ß, and IL-6. In macrophages, Ox-LDL downregulated expression of both IFNα and IFNß, suggesting that the effect of Ox-LDL on the expression of type I interferons is specific to eosinophils. Furthermore, we noted that eosinophils constitutively expressed IL-4 and IL-13, and Ox-LDL markedly downregulated their expression. Analysis of Ox-LDL signaling revealed that eosinophils constitutively expressed SRB2, CD36, and CD68 scavenger receptors, and Ox-LDL markedly induced the expression of CD36. Further analysis of CD36 signaling by siRNA and neutralizing antibodies showed that the induction of type I IFN by Ox-LDL is mediated by CD36 signaling whereas downregulation of IL-4 is independent of CD36 activation. We further showed that peritoneal macrophages treated with condition medium collected from Ox-LDL treated eosinophils markedly induced the expression of M1 markers such as iNOS, IL6, SOSC3 and TNFα whereas the condition medium from non-treated eosinophils significantly induced expression of M2 markers like ARG1 and CCL24. CONCLUSIONS: Our data suggest that an atherogenic condition could activate eosinophils and modulate the phenotype of macrophages (from M2 to M1 phenotype), in part, through the CD36 receptor signaling.

Comparative Effects of Diet-Induced Lipid Lowering Versus Lipid Lowering Along With Apo A-I Milano Gene Therapy on Regression of Atherosclerosis.

Apolipoprotein A-1 (Apo A-I) Milano, a naturally occurring Arg173to Cys mutant of Apo A-1, has been shown to reduce atherosclerosis in animal models and in a small phase 2 human trial. We have shown the superior atheroprotective effects of Apo A-I Milano (Apo A-IM) gene compared to wild-type Apo A-I gene using transplantation of retrovirally transduced bone marrow in Apo A-I/Apo E null mice. In this study, we compared the effect of dietary lipid lowering versus lipid lowering plus Apo A-IM gene transfer using recombinant adeno-associated virus (rAAV) 8 as vectors on atherosclerosis regression in Apo A-I/Apo E null mice. All mice were fed a high-cholesterol diet from age of 6 weeks until week 20, and at 20 weeks, 10 mice were euthanized to determine the extent of atherosclerosis. After 20 weeks, an additional 20 mice were placed on either a low-cholesterol diet plus empty rAAV (n = 10) to serve as controls or low-cholesterol diet plus 1 single intravenous injection of 1.2 × 10(12)vector genomes of adeno-associated virus (AAV) 8 vectors expressing Apo A-IM (n = 10). At the 40 week time point, intravenous AAV8 Apo A-IM recipients showed a significant regression of atherosclerosis in the whole aorta (P< .01), aortic sinuses (P< .05), and brachiocephalic arteries (P< .05) compared to 20-week-old mice, whereas low-cholesterol diet plus empty vector control group showed no significant regression in lesion size. Immunostaining showed that compared to the 20-week-old mice, there was a significantly reduced macrophage content in the brachiocephalic (P< .05) and aortic sinus plaques (P< .05) of AAV8 Apo A-IM recipients. These data show that although dietary-mediated cholesterol lowering halts progression of atherosclerosis, it does not induce regression, whereas combination of low-cholesterol diet and AAV8 mediated Apo A-I Milano gene therapy induces rapid and significant regression of atherosclerosis in mice. These data provide support for the potential feasibility of this approach for atherosclerosis regression.

Splenocytes seed bone marrow of myeloablated mice: implication for atherosclerosis.

Extramedullary hematopoiesis has been shown to contribute to the pathogenesis of a variety of diseases including cardiovascular diseases. In this process, the spleen is seeded with mobilized bone marrow cells that augment its hematopoietic ability. It is unclear whether these immigrant cells that are produced/reprogrammed in spleen are similar or different from those found in the bone marrow. To begin to understand this, we investigated the relative potency of adult splenocytes per se to repopulate bone marrow of lethally-irradiated mice and its functional consequences in atherosclerosis. The splenocytes were harvested from GFP donor mice and transplanted into myeloablated wild type recipient mice without the inclusion of any bone marrow helper cells. We found that adult splenocytes repopulated bone marrow of myeloablated mice and the transplanted cells differentiated into a full repertoire of myeloid cell lineages. The level of monocytes/macrophages in the bone marrow of recipient mice was dependent on the cell origin, i.e., the donor splenocytes gave rise to significantly more monocytes/macrophages than the donor bone marrow cells. This occurred despite a significantly lower number of hematopoietic stem cells being present in the donor splenocytes when compared with donor bone marrow cells. Atherosclerosis studies revealed that donor splenocytes displayed a similar level of atherogenic and atheroprotective activities to those of donor bone marrow cells. Cell culture studies showed that the phenotype of macrophages derived from spleen is different from those of bone marrow. Together, these results demonstrate that splenocytes can seed bone marrow of myeloablated mice and modulate atherosclerosis. In addition, our study shows the potential of splenocytes for therapeutic interventions in inflammatory disease.

Batf3 deficiency is not critical for the generation of CD8α⁺ dendritic cells.

Recently, we have reported that CD8α(+) DCs, rather than CD8(+) T cells, are involved in the establishment and maintenance of HSV-1 latency in the trigeminal ganglia (TG) of ocularly infected mice. In the current study, we investigated whether similar results can be obtained using Batf3(-/-) mice that previously were reported to lack CD8α(+) DCs. However, our results demonstrate that Batf3(-/-) mice, without any known infection, express CD8α(+) DCs. Consequently, due to the presence of CD8α(+) DCs, no differences were detected in the level of HSV-1 latency between Batf3(-/-) mice compared with wild type control mice.

Comparative antiatherogenic effects of intravenous AAV8- and AAV2-mediated ApoA-IMilano gene transfer in hypercholesterolemic mice.

Apolipoprotein A-IMilano (ApoA-IM), a naturally occurring Arg173 to Cys mutant of ApoA-I, has been shown to reduce atherosclerosis in animal models and in a small phase 2 human trial. We have shown superior atheroprotective effects of ApoA-IM gene compared with wild-type ApoA-I gene using transplantation of retrovirally transduced bone marrow in ApoA-I/ApoE null mice. In this study, we compared the antiatherogenic efficacy of ApoA-IM gene transfer using Recombinant adeno-associated virus (rAAV) 2 or rAAV8 as vectors in ApoA-I/ApoE null mice. Mice received a single intravenous injection of 1.2 × 10(12) vector genomes of AAV2 or AAV8 vectors expressing ApoA-IM or control empty vectors (12 mice/group). Circulating levels of ApoA-IM were higher in recipients of AAV8 compared with AAV2 at 4, 12, and 20 weeks postinjection. Qualitative polymerase chain reaction analysis of RNA collected from different tissues showed that the AAV8-mediated gene transfer resulted in a more efficient transgene expression in the heart, brain, liver, lung, spleen, and kidney of the recipient mice compared with AAV2. Intravenous AAV8-ApoA-IM injection reduced atherosclerosis in the whole aorta (P < .01), aortic sinuses (P < .05), and brachiocephalic arteries (P < .05) compared with the vector control, whereas there was no statistically significant reduction in atherosclerosis in mice receiving intravenous AAV2-ApoA-IM. The ApoA-IM gene was expressed in the aortic tissue of mice receiving AAV8 ApoA-IM but not in those receiving AAV2 ApoA-IM. Immunostaining showed that compared with the vector control, there was reduced macrophage content in the brachiocephalic (P < .05) and aortic sinus plaques (P < .05) of AAV8 ApoA-IM recipients but not in the recipients of AAV2 ApoA-IM. Thus, intravenous injection of AAV8 is more effective than intravenous injection of AAV2 in the expression of ApoA-IM gene. These data provide support for the potential feasibility of this approach for atheroprotection in humans.

Stimulation of endogenous cardioblasts by exogenous cell therapy after myocardial infarction.

Controversy surrounds the identity, origin, and physiologic role of endogenous cardiomyocyte progenitors in adult mammals. Using an inducible genetic labeling approach to identify small non-myocyte cells expressing cardiac markers, we find that activated endogenous cardioblasts are rarely evident in the normal adult mouse heart. However, myocardial infarction results in significant cardioblast activation at the site of injury. Genetically labeled isolated cardioblasts express cardiac transcription factors and sarcomeric proteins, exhibit spontaneous contractions, and form mature cardiomyocytes in vivo after injection into unlabeled recipient hearts. The activated cardioblasts do not arise from hematogenous seeding, cardiomyocyte dedifferentiation, or mere expansion of a preformed progenitor pool. Cell therapy with cardiosphere-derived cells amplifies innate cardioblast-mediated tissue regeneration, in part through the secretion of stromal cell-derived factor 1 by transplanted cells. Thus, stimulation of endogenous cardioblasts by exogenous cells mediates therapeutic regeneration of injured myocardium.

CD8α dendritic cells drive establishment of HSV-1 latency.

It is generally accepted that CD8 T cells play the key role to maintain HSV-1 latency in trigeminal ganglia of ocularly infected mice. Yet, comparably little is known about the role of innate immunity in establishment of viral latency. In the current study, we investigated whether CD8α DCs impact HSV-1 latency by examining latency in the trigeminal ganglia (TG) of wild-type (WT) C57BL/6 versus CD8α-/- (lack functional CD8 T cells and CD8α+ DCs), CD8ß-/- (have functional CD8α+ T cells and CD8α+ DCs), and ß2m-/- (lack functional CD8 T cells but have CD8α+ DCs) mice as well as BXH2 (have functional CD8 T cells but lack CD8α+ DCs) versus WT C3H (have functional CD8α T cells and CD8α+ DCs) mice. We also determined whether the phenotype of CD8α-/- and BXH2 mice could be restored to that of WT mice by adoptive transfer of WT CD8+ T cells or bone marrow (BM) derived CD8α+ DCs. Our results clearly demonstrate that CD8α DCs, rather than CD8 T cells, are responsible for enhanced viral latency and recurrences.

Tenascin-C deficiency in apo E-/- mouse increases eotaxin levels: implications for atherosclerosis.

AIM: To investigate the potential role of inflammatory cytokines in apo E-/- mouse in response to deletion of Tenascin-C (TNC) gene. METHODS AND RESULTS: We used antibody array and ELISA to compare the profile of circulating inflammatory cytokines in apo E-/- mice and apo E-/- TNC-/- double knockout mice. In addition, tissue culture studies were performed to investigate the activity of cells from each mouse genotype in vitro. Cytokine array analysis and subsequent ELISA showed that circulating eotaxin levels were selectively and markedly increased in response to TNC gene deletion in apo E-/- mice. In addition, considerable variation was noted in the circulating level of eotaxin among the control apo E-/- mouse group. Inbreeding of apo E-/- mice with high or low levels of plasma eotaxin showed that the level of eotaxin per se determines the extent of atherosclerosis in this mouse genotype. While endothelial cells from apo E-/- mice had low level of eotaxin expression, cells derived from apo E-/- TNC-/- mice expressed a high level of eotaxin. Transient transfection of eotaxin promoter-reporter constructs revealed that eotaxin expression is regulated at the transcriptional level by TNC. Histochemical analysis of aortic sections revealed the massive accumulation of mast cells in the adventitia of double KO mice lesions whereas no such accumulation was detected in the control group. Plasma from the apo E-/- TNC-/- mice markedly stimulated mast cell migration whereas plasma from the apo E-/- mice had no such effect. CONCLUSION: These observations support the emerging hypothesis that TNC expression controls eotaxin level in apo E-/- mice and that this chemokine plays a key role in the development of atherosclerosis.

Autofluorescence contributes to false-positive intracellular Foxp3 staining in macrophages: a lesson learned from flow cytometry.

Forkhead box P3 (Foxp3) is well known for its highly restricted expression in T regulatory cells (Tregs). A recent study suggested the existence of a Foxp3 positive macrophage subpopulation in mouse bone marrow, spleen, liver, lymph nodes, and thymus that exhibited immune regulatory effect similar to Tregs. Before this report was retracted, we attempted to study the function of this macrophage subpopulation in a mouse model of hyperlipidemia. Bone marrow and spleen cells isolated from C57BL/6 apo E(-/-) mice were stained with anti-CD11b, anti-F4/80 and anti-Foxp3 and analyzed by flow cytometry. Our results showed that 3.06-8.08% of CD11b(+)F4/80(+) macrophages from bone marrow cells and 0.24-2.21% from splenic were Foxp3-positive. Unexpectedly, unstained or isotype stained controls also showed strong autofluorescence and similar percentages of these cells fell within the same FL1 channel that counted the anti-Foxp3 stained population. Back gating of the autofluorescent population onto a SSC/FSC plot showed that this population of cells had a higher side scatter. The peritoneal macrophages (PMø) exhibited similar autofluorescence. We used qPCR to further evaluate the expression of Foxp3 mRNA in PMø that were treated with M-CSF, M-CSF+IL-4, M-CSF+TGFß1 or in BMDM treated with TGFß1 in the presence of anti-CD3 and CD28 antibody co-stimulators. No expression of Foxp3 mRNA was detected in either cell culture systems, whereas robust Foxp3 gene expression was induced in naïve CD4+ cells stimulated with TGFß1. Consistent with these findings, fluorescence microscopy showed no Foxp3 protein expression in PMø, however Foxp3 expression was easily detected in induced Tregs. We conclude that the reported expression of Foxp3 in macrophages is likely an artifact and that a stringent multimodality approach is critical to demonstrate candidate gene expression in any cell type.

Deletion of tenascin-C gene exacerbates atherosclerosis and induces intraplaque hemorrhage in Apo-E-deficient mice.

AIMS: Tenascin-C (TNC), a matricellular protein, is up-regulated in atherosclerotic plaques. We investigated whether the deletion of TNC gene affects the development of atherosclerosis in a murine model. METHODS: TNC-/-/apo E-/- mice were generated and used for atherosclerosis studies. We compared these results to those observed in control groups of apo E-/- mice. RESULTS: The en face analysis of aortic area showed that the mean aortic lesion area of the double knockout (KO) mice was significantly higher than that of control mice at different times after feeding of atherogenic diet; the accumulation of lesional macrophages and lipids was significantly higher. Analysis of cell adhesion molecules revealed that vascular cell adhesion molecule-1 (VCAM-1), but not intercellular adhesion molecule-1, was up-regulated 1 week after feeding of atherogenic diet in the double KO mouse as compared to apo E-/- mouse. Cell culture studies revealed that the expression of VCAM-1 in endothelial cells isolated from the double KO mouse is more sensitive to the tumor necrosis factor α stimulation than the cells isolated from apo E-/- mice. Cell adhesion studies showed that the adherence of RAW monocytic cells to the endothelial cells was significantly enhanced in the cultured endothelial cells from the TNC gene-deleted cells. Following the prolonged feeding of an atherogenic diet (28-30 weeks), the aortic and carotid atherosclerotic lesions frequently demonstrated large grossly visible areas of intraplaque hemorrhage in the double KO mice compared to control. CONCLUSIONS: These data unveil a protective role for TNC in atherosclerosis and suggest that TNC signaling may have the potential to reduce atherosclerosis, in part by modulating VCAM-1 expression.

Bioengineered vascular graft grown in the mouse peritoneal cavity.

BACKGROUND: We tested the hypothesis that the mouse peritoneum can function like a bioreactor to generate directed bio-engineered tissues such as those used for bypass grafting. Additionally, we reasoned that the mouse animal model would allow us to elucidate the underlying cellular and molecular mechanisms that are responsible for the generation of tissue in peritoneal cavity. METHODS: Plastic tubes (two tubes/mouse) were implanted into the peritoneal cavity of three strains of mice (C57BL/6, BALB/c, and MRL). The tubes were harvested, tissue capsule surrounding the tubes was removed, and analyzed by immunostaining (five capsules/five mice/strain) and microarray (three capsules/three mice/strain). In addition, the tissue capsules that were harvested from MRL mice (n = 21) were grafted into abdominal aorta of the same mice as autografts. The patency of all grafts was monitored by micro-ultrasound, and their functionality was assessed by laser Doppler imaging of blood flow in femoral arteries. Venous (n = 13) and arterial isografts (n = 11) were used as positive controls. In a negative control group (five mice/strain), the abdominal aorta was occluded by double ligation with 9-0 silk. RESULTS: The implanted plastic tubes required at least 8 weeks of incubation in the peritoneum of the three strains of mice in order to generate useful grafts. No vascular cells were found in the tissue capsules. Microarray analysis of tissue capsules revealed that the capsular cells express a gene expression program that is vastly shared among the three strains of mice, and the cells exhibit a high degree of plasticity. The micro-ultrasound analysis of the grafts showed that 62% of autografts remained patent compared with 77% of venous isografts and 91% of arterial isografts. The laser Doppler imaging analysis showed that blood flow dropped by 40% and 35% in the autografts and vein isografts, respectively, 1 day after surgery. The flow, however, rebounded to the level of arterial isografts 1 month post-surgery and remained unchanged among all grafts for the next 4 months. Immunostaining of the autografts showed a thick vessel wall with endothelial cells that lined the lumen and smooth muscle cells that constituted the graft wall. CONCLUSION: The mouse peritoneal cavity of mice has the ability to function like a bioreactor to generate bio-engineered tissues. The tissue capsules harvested from peritoneal cavity of a mouse are composed of nonvascular cells that display phenotype of progenitor cells. After grafting, however, the capsule autografts become arterialized and remained patent for at least 4 months after surgery, similar to venous or arterial isografts.

Pleiotrophin (PTN) is expressed in vascularized human atherosclerotic plaques: IFN-{gamma}/JAK/STAT1 signaling is critical for the expression of PTN in macrophages.

Neovascularization is critical to destabilization of atheroma. We previously reported that the angiogenic growth factor pleiotrophin (PTN) coaxes monocytes to assume the phenotype of functional endothelial cells in vitro and in vivo. In this study we show that PTN expression is colocalized with capillaries of human atherosclerotic plaques. Among the various reagents that are critical to the pathogenesis of atherosclerosis, interferon (IFN)-gamma was found to markedly induce PTN mRNA expression in a dose-dependent manner in macrophages. Mechanistic studies revealed that the Janus kinase inhibitors, WHI-P154 and ATA, efficiently blocked STAT1 phosphorylation in a concentration- and time-dependent manner. Notably, the level of phosphorylated STAT1 was found to correlate directly with the PTN mRNA levels. In addition, STAT1/STAT3/p44/42 signaling molecules were found to be phosphorylated by IFN-gamma in macrophages, and they were translocated into the nucleus. Further, PTN promoter analysis showed that a gamma-activated sequence (GAS) located at -2086 to -2078 bp is essential for IFN-gamma-regulated promoter activity. Moreover, electrophoretic mobility shift, supershift, and chromatin immunoprecipitation analyses revealed that both STAT1 and STAT3 bind to the GAS at the chromatin level in the IFN-gamma stimulated cells. Finally, to test whether the combined effect of STAT1/STAT3/p44/42 signaling is required for the expression of PTN in macrophages, gene knockdowns of these transcription factors were performed using siRNA. Cells lacking STAT1, but not STAT3 or p42, have markedly reduced PTN mRNA levels. These data suggest that PTN expression in the human plaques may be in part regulated by IFN-gamma and that PTN is involved in the adaptive immunity.-Li, F., Tian, F., Wang, L., Williamson, I. K., Sharifi, B. G., Shah, P. K. Pleiotrophin (PTN) is expressed in vascularized human atherosclerotic plaques: IFN-gamma/JAK/STAT1 signaling is critical for the expression of PTN in macrophages.

Bone marrow transplantation shows superior atheroprotective effects of gene therapy with apolipoprotein A-I Milano compared with wild-type apolipoprotein A-I in hyperlipidemic mice.

OBJECTIVES: We tested the hypothesis that gene therapy using apolipoprotein A-I Milano (apoA-IMilano) is more effective than that using wild-type apolipoprotein A-I (apoA-I) in reducing atherosclerosis. BACKGROUND: Apolipoprotein A-I Milano is a naturally occurring mutant with established antiatherogenic activity; however, its relative antiatherogenic efficacy compared with that of wild-type apoA-I remains unclear. METHODS: We performed bone marrow transplantation in female double-knockout mice lacking both the apoE and apoA-I genes using male donor mice-derived bone marrow that had been transduced with a retroviral vector alone or retroviral vector expressing wild-type apoA-I or apoA-IMilano gene under the control of macrophage-specific scavenger receptor A promoter. Mice were fed a high-cholesterol diet and killed 24 weeks after transplantation, at which time the extent of aortic atherosclerosis was determined. RESULTS: Compared with vector control (n = 12), apoA-IMilano gene therapy (n = 15) reduced aortic atherosclerosis by 65% (p < 0.001) and plaque macrophage immunoreactivity by 58% (p < 0.0001), whereas wild-type apoA-I (n = 11) reduced atherosclerosis by 25% (p = 0.1) and plaque macrophage immunoreactivity by 23% (p < 0.05). The apoA-IMilano gene therapy was significantly more effective in reducing atherosclerosis (p < 0.05) and macrophage immunoreactivity (p < 0.001) compared with wild-type apoA-I. The circulating levels of cholesterol, lipoprotein profile, and apoA-IMilano or wild-type apoA-I were comparable among the groups. Apolipoprotein A-I Milano was more effective than wild-type apoA-I in promoting macrophage cholesterol efflux. CONCLUSIONS: Macrophage-specific expression of the apoA-IMilano gene is more effective than wild-type apoA-I in reducing atherosclerosis and plaque inflammation despite comparable circulating levels of the transgene and lipid profile.

Pleiotrophin induces transdifferentiation of monocytes into functional endothelial cells.

OBJECTIVE: Pleiotrophin (PTN) is a cytokine that is expressed by monocytes/macrophages in ischemic tissues and that promotes neovascularization, presumably by stimulating proliferation of local endothelial cells. However, the effect of PTN on monocytes/macrophages remains unknown. We investigated the role of PTN in regulating the phenotype of monocytes/macrophages. METHODS AND RESULTS: RT-PCR, real-time PCR, and fluorescence-activated cell sorter analysis revealed that the expression of PTN by monocytic cells led to a downregulation of CD68, c-fms, and CD14 monocytic cell markers and an upregulation of FLK-1, Tie-2, vascular endothelial-cadherin, platelet endothelial cell adhesion molecule-1, endothelial NO synthase, von Willebrand factor, CD34, GATA-2, and GATA-3 endothelial cell markers. Fibrin gel assays showed that the treatment of mouse and human monocytic cells with PTN led to the formation of tube-like structures. In vivo studies showed that PTN-expressing monocytic cells incorporated into the blood vessels of the quail chorioallantoic membrane. The intracardial injection of PTN-expressing monocytic cells into chicken embryos showed that cells integrated only into the developing vasculature. Finally, the injection of PTN-expressing monocytes into a murine ischemic hindlimb model significantly improved perfusion of the ischemic tissue. CONCLUSIONS: PTN expression by monocytes/macrophages led to a downregulation of their monocytic cell markers and an upregulation of endothelial cell characteristics, thus inducing the transdifferentiation of monocytes into functional endothelial cells.

AAV serotype-dependent apolipoprotein A-I Milano gene expression.

Recent evidence from a double-blind, randomized study showed that treatment with apolipoprotein A-I Milano (ApoA-I Milano) in a complex with phospholipids produced significant regression of the coronary atheroma burden in patients with acute coronary syndromes. We previously showed similar regression of atherosclerosis in an animal model. Here, we examined a viral vector-based gene delivery system as a basis for ApoA-I Milano gene therapy. Comparing levels of expression using combinations of the cytomegalovirus (CMV) promoter in a recombinant serotype 2 adeno-associated virus (rAAV2) linked to ApoA-I Milano or the enhanced green fluorescent protein (EGFP) genes, we found that a promoter construct of two CMV core promoters sharing a CMV enhancer was more active than other combinations or a single CMV promoter. In vivo assessment of this optimal CMV construct using rAAV2 virus particles for intravenous (IV) or intramuscular (IM) routes of delivery produced high circulating levels of ApoA-I Milano protein for extended periods (up to 220 ng/ml at 22 weeks p.i.) by IV delivery while the IM route resulted in a relatively short period of very low-level ApoA-I Milano expression. Since there was no difference in the immune response between the two routes of delivery, we reasoned that tissue tropism might be responsible for this differential gene expression. To explore this possibility, we investigated the effect of different AAV serotypes on ApoA-I Milano gene expression in vivo. It found that rAAV1-mediated expression of ApoA-I Milano was approximately 15- and 9-fold higher than rAAV2 and rAAV5, respectively when IM injection routes were compared while all three AAV serotypes produced substantial levels of ApoA-I Milano expression from IV injection. These studies demonstrate that by modifying the promoter and serotype, increases in the efficiency of AAV-directed transgene expression could be achieved and support the potential of AAV-mediated gene therapy.

EGF-Like domain of tenascin-C is proapoptotic for cultured smooth muscle cells.

OBJECTIVE: Based on our previous observations on the expression of Tenascin-C (Tn-C) in human atherosclerotic plaques and its colocalization with macrophages, we explored whether Tn-C undergoes fragmentation and the potential pathobiological significance of this fragmentation. METHODS AND RESULTS: Using cultured human smooth muscle cells (SMCs), we found that Tn-C upregulates expression of matrix metalloproteinases (MMPs). Western blot analysis revealed that Tn-C substrate is fragmented and most of the cleavage products have fibronectin-like and epidermal growth factor-like (EGF-like) domains of Tn-C. One fragment that contains an EGF-like domain was found in some human atherosclerotic plaques. Cell culture studies revealed that the recombinant EGF-like domain inhibits growth, induces apoptosis of SMCs in a dose-dependent, time-dependent, and caspase-dependent manner, and activates caspase-3 before SMC detachment. Conversely, the caspase inhibitor z-YVAD.cmk, serum, and protease inhibitors blocked cell apoptosis conferred by the EGF-like domain. In addition, these inhibitors blocked EGF-like domain-induced caspase-3 activation. In contrast to this EGF-like domain, intact Tn-C, its fibronectin-like, and its fibrinogen-like domains were inactive. CONCLUSIONS: Together with our previous observations, our data suggest that Tn-C upregulates MMP expression that cleaves Tn-C into fragments containing the EGF-like domain. This domain has proapoptotic activity for SMCs.