Loss of 2 Akt (Protein Kinase B) Isoforms in Hematopoietic Cells Diminished Monocyte and Macrophage Survival and Reduces Atherosclerosis in Ldl Receptor-Null Mice.

Objective- Macrophages express 3 Akt (protein kinase B) isoforms, Akt1, Akt2, and Akt3, which display isoform-specific functions but may be redundant in terms of Akt survival signaling. We hypothesize that loss of 2 Akt isoforms in macrophages will suppress their ability to survive and modulate the development of atherosclerosis. Approach and Results- To test this hypothesis, we reconstituted male Ldlr-/- mice with double Akt2/Akt3 knockout hematopoietic cells expressing only the Akt1 isoform (Akt1only). There were no differences in body weight and plasma lipid levels between the groups after 8 weeks of the Western diet; however, Akt1only→ Ldlr-/- mice developed smaller (57.6% reduction) atherosclerotic lesions with more apoptotic macrophages than control mice transplanted with WT (wild type) cells. Next, male and female Ldlr-/- mice were reconstituted with double Akt1/Akt2 knockout hematopoietic cells expressing the Akt3 isoform (Akt3only). Female and male Akt3only→ Ldlr-/- recipients had significantly smaller (61% and 41%, respectively) lesions than the control WT→ Ldlr-/- mice. Loss of 2 Akt isoforms in hematopoietic cells resulted in markedly diminished levels of white blood cells, B cells, and monocytes and compromised viability of monocytes and peritoneal macrophages compared with WT cells. In response to lipopolysaccharides, macrophages with a single Akt isoform expressed low levels of inflammatory cytokines; however, Akt1only macrophages were distinct in expressing high levels of antiapoptotic Il10 compared with WT and Akt3only cells. Conclusions- Loss of 2 Akt isoforms in hematopoietic cells, preserving only a single Akt1 or Akt3 isoform, markedly compromises monocyte and macrophage viability and diminishes early atherosclerosis in Ldlr-/- mice.

Akt Signaling in Macrophage Polarization, Survival, and Atherosclerosis.

The PI3K/Akt pathway plays a crucial role in the survival, proliferation, and migration of macrophages, which may impact the development of atherosclerosis. Changes in Akt isoforms or modulation of the Akt activity levels in macrophages significantly affect their polarization phenotype and consequently atherosclerosis in mice. Moreover, the activity levels of Akt signaling determine the viability of monocytes/macrophages and their resistance to pro-apoptotic stimuli in atherosclerotic lesions. Therefore, elimination of pro-apoptotic factors as well as factors that antagonize or suppress Akt signaling in macrophages increases cell viability, protecting them from apoptosis, and this markedly accelerates atherosclerosis in mice. In contrast, inhibition of Akt signaling by the ablation of Rictor in myeloid cells, which disrupts mTORC2 assembly, significantly decreases the viability and proliferation of blood monocytes and macrophages with the suppression of atherosclerosis. In addition, monocytes and macrophages exhibit a threshold effect for Akt protein levels in their ability to survive. Ablation of two Akt isoforms, preserving only a single Akt isoform in myeloid cells, markedly compromises monocyte and macrophage viability, inducing monocytopenia and diminishing early atherosclerosis. These recent advances in our understanding of Akt signaling in macrophages in atherosclerosis may have significant relevance in the burgeoning field of cardio-oncology, where PI3K/Akt inhibitors being tested in cancer patients can have significant cardiovascular and metabolic ramifications.

Macrophage IKKα Deficiency Suppresses Akt Phosphorylation, Reduces Cell Survival, and Decreases Early Atherosclerosis.

OBJECTIVE: The IκB kinase (IKK) is an enzyme complex that initiates the nuclear factor κB transcription factor cascade, which is important in regulating multiple cellular responses. IKKα is directly associated with 2 major prosurvival pathways, PI3K/Akt and nuclear factor κB, but its role in cell survival is not clear. Macrophages play critical roles in the pathogenesis of atherosclerosis, yet the impact of IKKα signaling on macrophage survival and atherogenesis remains unclear. APPROACH AND RESULTS: Here, we demonstrate that genetic IKKα deficiency, as well as pharmacological inhibition of IKK, in mouse macrophages significantly reduces Akt S(473) phosphorylation, which is accompanied by suppression of mTOR complex 2 signaling. Moreover, IKKα null macrophages treated with lipotoxic palmitic acid exhibited early exhaustion of Akt signaling compared with wild-type cells. This was accompanied by a dramatic decrease in the resistance of IKKα(-/-) monocytes and macrophages to different proapoptotic stimuli compared with wild-type cells. In vivo, IKKα deficiency increased macrophage apoptosis in atherosclerotic lesions and decreased early atherosclerosis in both female and male low-density lipoprotein receptor (LDLR)(-/-) mice reconstituted with IKKα(-/-) hematopoietic cells and fed with the Western diet for 8 weeks compared with control LDLR(-/-) mice transplanted with wild-type cells. CONCLUSIONS: Hematopoietic IKKα deficiency in mouse suppresses Akt signaling, compromising monocyte/macrophage survival and this decreases early atherosclerosis.

Jnk1 Deficiency in Hematopoietic Cells Suppresses Macrophage Apoptosis and Increases Atherosclerosis in Low-Density Lipoprotein Receptor Null Mice.

OBJECTIVE: The c-Jun NH2-terminal kinases (JNK) are regulated by a wide variety of cellular stresses and have been implicated in apoptotic signaling. Macrophages express 2 JNK isoforms, JNK1 and JNK2, which may have different effects on cell survival and atherosclerosis. APPROACH AND RESULTS: To dissect the effect of macrophage JNK1 and JNK2 on early atherosclerosis, Ldlr(-/-) mice were reconstituted with wild-type, Jnk1(-/-), and Jnk2(-/-) hematopoietic cells and fed a high cholesterol diet. Jnk1(-/-)→Ldlr(-/-) mice have larger atherosclerotic lesions with more macrophages and fewer apoptotic cells than mice transplanted with wild-type or Jnk2(-/-) cells. Moreover, genetic ablation of JNK to a single allele (Jnk1(+/-)/Jnk2(-/-) or Jnk1(-/-)/Jnk2(+/-)) in marrow of Ldlr(-/-) recipients further increased atherosclerosis compared with Jnk1(-/-)→Ldlr(-/-) and wild-type→Ldlr(-/-) mice. In mouse macrophages, anisomycin-mediated JNK signaling antagonized Akt activity, and loss of Jnk1 gene obliterated this effect. Similarly, pharmacological inhibition of JNK1, but not JNK2, markedly reduced the antagonizing effect of JNK on Akt activity. Prolonged JNK signaling in the setting of endoplasmic reticulum stress gradually extinguished Akt and Bad activity in wild-type cells with markedly less effects in Jnk1(-/-) macrophages, which were also more resistant to apoptosis. Consequently, anisomycin increased and JNK1 inhibitors suppressed endoplasmic reticulum stress-mediated apoptosis in macrophages. We also found that genetic and pharmacological inhibition of phosphatase and tensin homolog abolished the JNK-mediated effects on Akt activity, indicating that phosphatase and tensin homolog mediates crosstalk between these pathways. CONCLUSIONS: Loss of Jnk1, but not Jnk2, in macrophages protects them from apoptosis, increasing cell survival, and this accelerates early atherosclerosis.

Macrophage Apoptosis and Efferocytosis in the Pathogenesis of Atherosclerosis.

Macrophage apoptosis and the ability of macrophages to clean up dead cells, a process called efferocytosis, are crucial determinants of atherosclerosis lesion progression and plaque stability. Environmental stressors initiate endoplasmic reticulum (ER) stress and activate the unfolded protein response (UPR). Unresolved ER stress with activation of the UPR initiates apoptosis. Macrophages are resistant to apoptotic stimuli, because of activity of the PI3K/Akt pathway. Macrophages express 3 Akt isoforms, Akt1, Akt2 and Akt3, which are products of distinct but homologous genes. Akt displays isoform-specific effects on atherogenesis, which vary with different vascular cell types. Loss of macrophage Akt2 promotes the anti-inflammatory M2 phenotype and reduces atherosclerosis. However, Akt isoforms are redundant with regard to apoptosis. c-Jun NH2-terminal kinase (JNK) is a pro-apoptotic effector of the UPR, and the JNK1 isoform opposes anti-apoptotic Akt signaling. Loss of JNK1 in hematopoietic cells protects macrophages from apoptosis and accelerates early atherosclerosis. IκB kinase α (IKKα, a member of the serine/threonine protein kinase family) plays an important role in mTORC2-mediated Akt signaling in macrophages, and IKKα deficiency reduces macrophage survival and suppresses early atherosclerosis. Efferocytosis involves the interaction of receptors, bridging molecules, and apoptotic cell ligands. Scavenger receptor class B type I is a critical mediator of macrophage efferocytosis via the Src/PI3K/Rac1 pathway in atherosclerosis. Agonists that resolve inflammation offer promising therapeutic potential to promote efferocytosis and prevent atherosclerotic clinical events. (Circ J 2016; 80: 2259-2268).

Macrophage SR-BI mediates efferocytosis via Src/PI3K/Rac1 signaling and reduces atherosclerotic lesion necrosis.

Macrophage apoptosis and efferocytosis are key determinants of atherosclerotic plaque inflammation and necrosis. Bone marrow transplantation studies in ApoE- and LDLR-deficient mice revealed that hematopoietic scavenger receptor class B type I (SR-BI) deficiency results in severely defective efferocytosis in mouse atherosclerotic lesions, resulting in a 17-fold higher ratio of free to macrophage-associated dead cells in lesions containing SR-BI(-/-) cells, 5-fold more necrosis, 65.2% less lesional collagen content, nearly 7-fold higher dead cell accumulation, and 2-fold larger lesion area. Hematopoietic SR-BI deletion elicited a maladaptive inflammatory response [higher interleukin (IL)-1ß, IL-6, and TNF-α lower IL-10 and transforming growth factor ß]. Efferocytosis of apoptotic thymocytes was reduced by 64% in SR-BI(-/-) versus WT macrophages, both in vitro and in vivo. In response to apoptotic cells, macrophage SR-BI bound with phosphatidylserine and induced Src phosphorylation and cell membrane recruitment, which led to downstream activation of phosphoinositide 3-kinase (PI3K) and Ras-related C3 botulinum toxin substrate 1 (Rac1) for engulfment and clearance of apoptotic cells, as inhibition of Src decreased PI3K, Rac1-GTP, and efferocytosis in WT cells. Pharmacological inhibition of Rac1 reduced macrophage efferocytosis in a SR-BI-dependent fashion, and activation of Rac1 corrected the defective efferocytosis in SR-BI(-/-) macrophages. Thus, deficiency of macrophage SR-BI promotes defective efferocytosis signaling via the Src/PI3K/Rac1 pathway, resulting in increased plaque size, necrosis, and inflammation.

Identification of small proline-rich repeat protein 3 as a novel atheroprotective factor that promotes adaptive Akt signaling in vascular smooth muscle cells.

OBJECTIVE: Atherosclerosis is the primary driver of cardiovascular disease, the leading cause of death worldwide. Identification of naturally occurring atheroprotective genes has become a major goal for the development of interventions that will limit atheroma progression and associated adverse events. To this end, we have identified small proline-rich repeat protein (SPRR3) as selectively upregulated in vascular smooth muscle cells (VSMCs) of atheroma-bearing arterial tissue versus healthy arterial tissue. In this study, we sought to determine the role of SPRR3 in atheroma pathophysiology. APPROACH AND RESULTS: We found that atheroprone apolipoprotein E-null mice lacking SPRR3 developed significantly greater atheroma burden. To determine the cellular driver(s) of this increase, we evaluated SPRR3-dependent changes in bone marrow-derived cells, endothelial cells, and VSMCs. Bone marrow transplant of SPRR3-expressing cells into SPRR3(-/-)apolipoprotein E-deficient recipients failed to rescue atheroma burden. Similarly, endothelial cells did not exhibit a response to SPRR3 loss. However, atheromas from SPRR3-deficient mice exhibited increased TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling)-positive VSMCs compared with control. Cell death in SPRR3-deficient VSMCs was significantly increased in vitro. Conversely, SPRR3-overexpressing VSMCs exhibited reduced apoptosis compared with control. We also observed a PI3K (phosphatidylinositol 3-kinase)/Akt-dependent positive association between SPRR3 expression and levels of active Akt in VSMCs. The survival advantage seen in SPRR3-overexpressing VSMCs was abrogated after the addition of a PI3K/Akt pathway inhibitor. CONCLUSIONS: These results indicate that SPRR3 protects the lesion from VSMC loss by promoting survival signaling in plaque VSMCs, thereby significantly decreasing atherosclerosis progression. As the first identified atheroma-specific VSMC prosurvival factor, SPRR3 represents a potential target for lesion-specific modulation of VSMC survival.

Macrophage deficiency of Akt2 reduces atherosclerosis in Ldlr null mice.

Macrophages play crucial roles in the formation of atherosclerotic lesions. Akt, a serine/threonine protein kinase B, is vital for cell proliferation, migration, and survival. Macrophages express three Akt isoforms, Akt1, Akt2, and Akt3, but the roles of Akt1 and Akt2 in atherosclerosis in vivo remain unclear. To dissect the impact of macrophage Akt1 and Akt2 on early atherosclerosis, we generated mice with hematopoietic deficiency of Akt1 or Akt2. After 8 weeks on Western diet, Ldlr(-/-) mice reconstituted with Akt1(-/-) fetal liver cells (Akt1(-/-)→Ldlr(-/-)) had similar atherosclerotic lesion areas compared with control mice transplanted with WT cells (WT→Ldlr(-/-)). In contrast, Akt2(-/-)→Ldlr(-/-) mice had dramatically reduced atherosclerotic lesions compared with WT→Ldlr(-/-) mice of both genders. Similarly, in the setting of advanced atherosclerotic lesions, Akt2(-/-)→Ldlr(-/-) mice had smaller aortic lesions compared with WT→Ldlr(-/-) and Akt1(-/-)→Ldlr(-/-) mice. Importantly, Akt2(-/-)→Ldlr(-/-) mice had reduced numbers of proinflammatory blood monocytes expressing Ly-6C(hi) and chemokine C-C motif receptor 2. Peritoneal macrophages isolated from Akt2(-/-) mice were skewed toward an M2 phenotype and showed decreased expression of proinflammatory genes and reduced cell migration. Our data demonstrate that loss of Akt2 suppresses the ability of macrophages to undergo M1 polarization reducing both early and advanced atherosclerosis.

Treatment of diabetes and atherosclerosis by inhibiting fatty-acid-binding protein aP2.

Adipocyte fatty-acid-binding protein, aP2 (FABP4) is expressed in adipocytes and macrophages, and integrates inflammatory and metabolic responses. Studies in aP2-deficient mice have shown that this lipid chaperone has a significant role in several aspects of metabolic syndrome, including type 2 diabetes and atherosclerosis. Here we demonstrate that an orally active small-molecule inhibitor of aP2 is an effective therapeutic agent against severe atherosclerosis and type 2 diabetes in mouse models. In macrophage and adipocyte cell lines with or without aP2, we also show the target specificity of this chemical intervention and its mechanisms of action on metabolic and inflammatory pathways. Our findings demonstrate that targeting aP2 with small-molecule inhibitors is possible and can lead to a new class of powerful therapeutic agents to prevent and treat metabolic diseases such as type 2 diabetes and atherosclerosis.

Macrophage Mal1 deficiency suppresses atherosclerosis in low-density lipoprotein receptor-null mice by activating peroxisome proliferator-activated receptor-γ-regulated genes.

OBJECTIVE: The adipocyte/macrophage fatty acid-binding proteins aP2 (FABP4) and Mal1 (FABP5) are intracellular lipid chaperones that modulate systemic glucose metabolism, insulin sensitivity, and atherosclerosis. Combined deficiency of aP2 and Mal1 has been shown to reduce the development of atherosclerosis, but the independent role of macrophage Mal1 expression in atherogenesis remains unclear. METHODS AND RESULTS: We transplanted wild-type (WT), Mal1(-/-), or aP2(-/-) bone marrow into low-density lipoprotein receptor-null (LDLR(-/-)) mice and fed them a Western diet for 8 weeks. Mal1(-/-)→LDLR(-/-) mice had significantly reduced (36%) atherosclerosis in the proximal aorta compared with control WT→LDLR(-/-) mice. Interestingly, peritoneal macrophages isolated from Mal1-deficient mice displayed increased peroxisome proliferator-activated receptor-γ (PPARγ) activity and upregulation of a PPARγ-related cholesterol trafficking gene, CD36. Mal1(-/-) macrophages showed suppression of inflammatory genes, such as COX2 and interleukin 6. Mal1(-/-)→LDLR(-/-) mice had significantly decreased macrophage numbers in the aortic atherosclerotic lesions compared with WT→LDLR(-/-) mice, suggesting that monocyte recruitment may be impaired. Indeed, blood monocytes isolated from Mal1(-/-)→LDLR(-/-) mice on a high-fat diet had decreased CC chemokine receptor 2 gene and protein expression levels compared with WT monocytes. CONCLUSION: Taken together, our results demonstrate that Mal1 plays a proatherogenic role by suppressing PPARγ activity, which increases expression of CC chemokine receptor 2 by monocytes, promoting their recruitment to atherosclerotic lesions.

Angiotensin type 1 receptor modulates macrophage polarization and renal injury in obesity.

The mechanisms for increased risk of chronic kidney disease (CKD) in obesity remain unclear. The renin-angiotensin system is implicated in the pathogenesis of both adiposity and CKD. We investigated whether the angiotensin type 1 (AT(1)) receptor, composed of dominant AT(1a) and less expressed AT(1b) in wild-type (WT) mice, modulates development and progression of kidney injury in a high-fat diet (HFD)-induced obesity model. WT mice had increased body weight, body fat, and insulin levels and decreased adiponectin levels after 24 wk of a high-fat diet. Identically fed AT(1a) knockout (AT1aKO) mice gained weight similarly to WT mice, but had lower body fat and higher plasma cholesterol. Both obese AT1aKO and obese WT mice had increased visceral fat and kidney macrophage infiltration, with more proinflammatory M1 macrophage markers as well as increased mesangial expansion and tubular vacuolization, compared with lean mice. These abnormalities were heightened in the obese AT1aKO mice, with downregulated M2 macrophage markers and increased macrophage AT(1b) receptor. Treatment with an AT(1) receptor blocker, which affects both AT(1a) and AT(1b), abolished renal macrophage infiltration with inhibition of renal M1 and upregulation of M2 macrophage markers in obese WT mice. Our data suggest obesity accelerates kidney injury, linked to augmented inflammation in adipose and kidney tissues and a proinflammatory shift in macrophage and M1/M2 balance.

Combined vitamin C and vitamin E deficiency worsens early atherosclerosis in apolipoprotein E-deficient mice.

OBJECTIVE: To assess the role of combined deficiencies of vitamins C and E on the earliest stages of atherosclerosis (an inflammatory condition associated with oxidative stress), 4 combinations of vitamin supplementation (low C/low E, low C/high E, high C/low E, and high C/high E) were studied in atherosclerosis-prone apolipoprotein E-deficient mice also unable to synthesize their own vitamin C (gulonolactone oxidase(-/-)); and to evaluate the effect of a more severe depletion of vitamin C alone in a second experiment using gulonolactone oxidase(-/-) mice carrying the hemizygous deletion of SVCT2 (the vitamin C transporter). METHODS AND RESULTS: After 8 weeks of a high-fat diet (16% lard and 0.2% cholesterol), atherosclerosis developed in the aortic sinus areas of mice in all diet groups. Each vitamin-deficient diet significantly decreased liver and brain contents of the corresponding vitamin. Combined deficiency of both vitamins increased lipid peroxidation, doubled plaque size, and increased plaque macrophage content by 2- to 3-fold in male mice, although only plaque macrophage content was increased in female mice. A more severe deficiency of vitamin C in gulonolactone oxidase(-/-) mice with defective cellular uptake of vitamin C increased both oxidative stress and atherosclerosis in apolipoprotein E(-/-) mice compared with littermates receiving a diet replete in vitamin C, again most clearly in males. CONCLUSIONS: Combined deficiencies of vitamins E and C are required to worsen early atherosclerosis in an apolipoprotein E-deficient mouse model. However, a more severe cellular deficiency of vitamin C alone promotes atherosclerosis when vitamin E is replete.

Lack of macrophage fatty-acid-binding protein aP2 protects mice deficient in apolipoprotein E against atherosclerosis.

The adipocyte fatty-acid-binding protein, aP2, has an important role in regulating systemic insulin resistance and lipid metabolism. Here we demonstrate that aP2 is also expressed in macrophages, has a significant role in their biological responses and contributes to the development of atherosclerosis. Apolipoprotein E (ApoE)-deficient mice also deficient for aP2 showed protection from atherosclerosis in the absence of significant differences in serum lipids or insulin sensitivity. aP2-deficient macrophages showed alterations in inflammatory cytokine production and a reduced ability to accumulate cholesterol esters when exposed to modified lipoproteins. Apoe-/- mice with Ap2+/+ adipocytes and Ap2-/- macrophages generated by bone-marrow transplantation showed a comparable reduction in atherosclerotic lesions to those with total aP2 deficiency, indicating an independent role for macrophage aP2 in atherogenesis. Through its distinct actions in adipocytes and macrophages, aP2 provides a link between features of the metabolic syndrome and could be a new therapeutic target for the prevention of atherosclerosis.

Imaging mass spectrometry reveals heterogeneity of proliferation and metabolism in atherosclerosis.

Atherosclerotic plaques feature local proliferation of leukocytes and vascular smooth muscle cells (VSMCs) and changes in cellular metabolism. Yet the relationship between glucose utilization and proliferation has been technically impossible to study directly in cells of atherosclerotic plaques in vivo. We used multi-isotope imaging mass spectrometry (MIMS), a quantitative imaging platform, to measure coincident cell division and glucose utilization at suborganelle resolution in atherosclerotic plaques. In established plaques, 65% of intimal foam cells and only 4% of medial VSMCs were labeled with 15N-thymidine after 1 week of isotope treatment. Dividing cells demonstrated heightened glucose labeling. MIMS detected 2H-glucose label in multiple subcellular compartments within foam cells, including lipid droplets, the cytosol, and chromatin. Unexpectedly, we identified an intensely focal region of 2H-label in VSMCs underlying plaques. This signal diminished in regions of aorta without atherosclerosis. In advanced plaques, 15N-thymidine and 2H-glucose labeling in foam cells and VSMCs significantly decreased. These data demonstrate marked heterogeneity in VSMC glucose metabolism that was dependent on both proliferative status and proximity of VSMCs to plaques. Furthermore, these results reveal how quantitative mass spectrometry coupled with isotope imaging can complement other methods used to study cell biology directly in the growing atherosclerotic plaque in vivo.

Macrophage expression of peroxisome proliferator-activated receptor-alpha reduces atherosclerosis in low-density lipoprotein receptor-deficient mice.

BACKGROUND: The peroxisome proliferator-activated receptor-alpha (PPARalpha) plays important roles in lipid metabolism, inflammation, and atherosclerosis. PPARalpha ligands have been shown to reduce cardiovascular events in high-risk subjects. PPARalpha expression by arterial cells, including macrophages, may exert local antiatherogenic effects independent of plasma lipid changes. METHODS AND RESULTS: To examine the contribution of PPARalpha expression by bone marrow-derived cells in atherosclerosis, male and female low-density lipoprotein receptor-deficient (LDLR(-/-)) mice were reconstituted with bone marrow from PPARalpha(-/-) or PPARalpha(+/+) mice and challenged with a high-fat diet. Although serum lipids and lipoprotein profiles did not differ between the groups, the size of atherosclerotic lesions in the distal aorta of male and female PPARalpha(-/-) --> LDLR(-/-) mice was significantly increased (44% and 46%, respectively) compared with controls. Male PPARalpha(-/-) --> LDLR(-/-) mice also had larger (44%) atherosclerotic lesions in the proximal aorta than male PPARalpha(+/+) --> LDLR(-/-) mice. Peritoneal macrophages from PPARalpha(-/-) mice had increased uptake of oxidized LDL and decreased cholesterol efflux. PPARalpha(-/-) macrophages had lower levels of scavenger receptor B type I and ABCA1 protein expression and an accelerated response of nuclear factor-kappaB-regulated inflammatory genes. A laser capture microdissection analysis verified suppressed scavenger receptor B type I and increased nuclear factor-kappaB gene expression levels in vivo in atherosclerotic lesions of PPARalpha(-/-) --> LDLR(-/-) mice compared with the lesions of control PPARalpha(+/+) --> LDLR(-/-) mice. CONCLUSIONS: These data demonstrate that PPARalpha expression by macrophages has antiatherogenic effects via modulation of cell cholesterol trafficking and inflammatory activity.

Loss of Rictor in Monocyte/Macrophages Suppresses Their Proliferation and Viability Reducing Atherosclerosis in LDLR Null Mice.

Background: Rictor is an essential component of mammalian target of rapamycin (mTOR) complex 2 (mTORC2), a conserved serine/threonine kinase that may play a role in cell proliferation, survival and innate or adaptive immune responses. Genetic loss of Rictor inactivates mTORC2, which directly activates Akt S473 phosphorylation and promotes pro-survival cell signaling and proliferation. Methods and results: To study the role of mTORC2 signaling in monocytes and macrophages, we generated mice with myeloid lineage-specific Rictor deletion (MRictor-/-). These MRictor-/- mice exhibited dramatic reductions of white blood cells, B-cells, T-cells, and monocytes but had similar levels of neutrophils compared to control Rictor flox-flox (Rictorfl/fl) mice. MRictor-/- bone marrow monocytes and peritoneal macrophages expressed reduced levels of mTORC2 signaling and decreased Akt S473 phosphorylation, and they displayed significantly less proliferation than control Rictorfl/fl cells. In addition, blood monocytes and peritoneal macrophages isolated from MRictor-/- mice were significantly more sensitive to pro-apoptotic stimuli. In response to LPS, MRictor-/- macrophages exhibited the M1 phenotype with higher levels of pro-inflammatory gene expression and lower levels of Il10 gene expression than control Rictorfl/fl cells. Further suppression of LPS-stimulated Akt signaling with a low dose of an Akt inhibitor, increased inflammatory gene expression in macrophages, but genetic inactivation of Raptor reversed this rise, indicating that mTORC1 mediates this increase of inflammatory gene expression. Next, to elucidate whether mTORC2 has an impact on atherosclerosis in vivo, female and male Ldlr null mice were reconstituted with bone marrow from MRictor-/- or Rictorfl/fl mice. After 10 weeks of the Western diet, there were no differences between the recipients of the same gender in body weight, blood glucose or plasma lipid levels. However, both female and male MRictor-/- → Ldlr-/- mice developed smaller atherosclerotic lesions in the distal and proximal aorta. These lesions contained less macrophage area and more apoptosis than lesions of control Rictorfl/fl → Ldlr-/- mice. Thus, loss of Rictor and, consequently, mTORC2 significantly compromised monocyte/macrophage survival, and this markedly diminished early atherosclerosis in Ldlr-/- mice. Conclusion: Our results demonstrate that mTORC2 is a key signaling regulator of macrophage survival and its depletion suppresses early atherosclerosis.

Cyclooxygenase-1 deficiency in bone marrow cells increases early atherosclerosis in apolipoprotein E- and low-density lipoprotein receptor-null mice.

BACKGROUND: Cyclooxygenase-1 (COX-1) has been implicated in the pathogenesis of atherothrombosis and is expressed by the major cell types of atherosclerotic lesions. COX-1-mediated platelet thromboxane (TX) production has been proposed to promote both early atherosclerosis and thrombosis. Here, we examined the impact of COX-1 deficiency in bone marrow-derived cells on early atherogenesis in the mouse. METHODS AND RESULTS: LDL receptor (LDLR)(-/-) and apolipoprotein E (apoE)(-/-) recipient mice were lethally irradiated and transplanted with COX-1(-/-) bone marrow. Mice reconstituted with COX-1(-/-) marrow had nearly complete (99.7%) loss of platelet TXA2 and significantly suppressed levels of macrophage and urinary TXA2 metabolites. Serum lipid levels and lipoprotein distributions did not differ between recipients reconstituted with COX-1(+/+) and COX-1(-/-) marrow. Surprisingly, the extent of atherosclerotic lesions in both LDLR(-/-) and apoE(-/-) mice reconstituted with COX-1(-/-) marrow was increased significantly compared with control mice transplanted with COX-1(+/+) marrow. Peritoneal macrophages isolated from LDLR(-/-) mice reconstituted with COX-1(-/-) marrow had increased lipopolysaccharide-induced levels of COX-2 mRNA and protein expression. Fetal liver cell transplantation studies revealed a 30% increase in atherosclerosis in COX-1(-/-)-->LDLR(-/-)mice compared with COX-1(+/+)-->LDLR(-/-)mice, whereas the extent of atherosclerosis was unchanged in COX-1(-/-)/COX-2(-/-)-->LDLR(-/-)mice. CONCLUSIONS: COX-1 deficiency in bone marrow-derived cells worsens early atherosclerosis in apoE(-/-) and LDLR(-/-) mice despite virtual elimination of platelet TX production. These data demonstrate that platelet TX production does not aggravate early atherosclerotic lesion formation and that upregulation of COX-2 expression in COX-1(-/-) macrophages is proatherogenic.

Transiently heightened angiotensin II has distinct effects on atherosclerosis and aneurysm formation in hyperlipidemic mice.

Experimentally sustained increase in angiotensin II (AngII) promotes tissue destruction in various cardiovascular disorders. We examined whether transiently heightened AngII affects subsequent atherosclerosis and aneurysm formation. AngII or saline was administered for 2 weeks to apolipoprotein E (apoE)-deficient mice. Mice were sacrificed at the end of the 2-week infusion or 6- or 14 weeks later. Short-term AngII did not affect atherosclerosis immediately following the infusion or 6 weeks later. By contrast, 14 weeks after infusion there was remarkably more atherosclerosis in previously AngII-exposed mice. Preceding the build up of atherosclerotic lesions, AngII-exposure increased mRNA expression and immunostaining of monocyte chemoattractant protein-1 (MCP-1) and its receptor, CCR2. This was followed by greater macrophage-positivity in AngII-exposed aortae. In contrast to the delayed effects on atherosclerosis, 20% of mice were found to have abdominal aneurysms at the end of AngII-exposure. This effect was not contingent on blood pressure. Moreover, despite amplification in atherosclerosis following AngII, no aneurysms were found 14 weeks later. Our studies reveal that even transient exposure to AngII primes the vessel for subsequent amplification of atherosclerosis which involves activation of MCP-1/CCR2 and influx of macrophages into the nascent atherosclerotic plaque. By contrast, transient AngII-exposure causes prompt aneurysm formation that does not parallel atherosclerosis and disappears even in the face of progressively greater atherosclerotic lesions.

Conditional knockout of macrophage PPARgamma increases atherosclerosis in C57BL/6 and low-density lipoprotein receptor-deficient mice.

OBJECTIVE: Peroxisome proliferator-activated receptor gamma (PPARgamma) is highly expressed in macrophage-derived foam cells of atherosclerotic lesions, and its expression may have a dramatic impact on atherosclerosis. METHODS AND RESULTS: To investigate the contribution of macrophage PPARgamma expression on atherogenesis in vivo, we generated macrophage-specific PPARgamma knockout (MacPPARgammaKO) mice. C57BL/6 and low-density lipoprotein (LDL) receptor-deficient (LDLR(-/-)) mice were reconstituted with MacPPARgammaKO or wild-type marrow and challenged with an atherogenic diet. No differences were found in serum lipids between recipients reconstituted with MacPPARgammaKO and wild-type marrow. In contrast, both C57BL/6 and LDLR(-/-) mice transplanted with MacPPARgammaKO marrow had significantly larger atherosclerotic lesions than control recipients. In addition, MacPPARgammaKO-->LDLR(-/-) mice had higher numbers of macrophages in atherosclerotic lesions compared with controls. Peritoneal macrophages isolated from the MacPPARgammaKO mice had decreased uptake of oxidized but not acetylated LDL and showed no changes in either cholesterol efflux or inflammatory cytokine expression. Macrophages from MacPPARgammaKO mice had increased levels of migration and CC chemokine receptor 2 (CCR2) expression compared with wild-type macrophages. CONCLUSIONS: Thus, macrophage PPARgamma deficiency increases atherosclerosis under conditions of mild and severe hypercholesterolemia, indicating an antiatherogenic role for PPARgamma, which may be caused, at least in part, by modulation of CCR2 expression and monocyte recruitment.

Combined adipocyte-macrophage fatty acid-binding protein deficiency improves metabolism, atherosclerosis, and survival in apolipoprotein E-deficient mice.

BACKGROUND: The adipocyte fatty acid-binding protein (FABP) aP2 is expressed by adipocytes and macrophages and modulates insulin resistance, glucose and lipid metabolism, and atherosclerosis. Insulin sensitivity is improved in obese but not in lean aP2-deficient mice. A second fatty acid-binding protein, mal1, also is expressed in adipocytes and macrophages, and mal1 deficiency produces similar effects on insulin resistance. We tested the hypothesis that combined aP2 and mal1 deficiency would produce synergistic effects on metabolism and reduce atherosclerosis in apolipoprotein E-deficient (apoE-/-) mice. METHODS AND RESULTS: Male and female apoE-/- mice null for both aP2 and mal1 (3KO) and apoE-/- controls were fed a low-fat chow diet for 16 or 56 weeks. Lean 3KO mice had significantly lower serum cholesterol and triglycerides as well as improved insulin and glucose tolerance as compared with controls. Analysis of atherosclerotic lesions in the 3KO mice showed dramatic reductions in both early (20 weeks) and late-stage (60 weeks) atherosclerosis. Strikingly, survival in the 3KO mice was improved by 67% as compared with apoE-/- controls when challenged with the Western diet for 1 year. CONCLUSIONS: Combined aP2 and mal1 deficiency improved glucose and lipid metabolism, reduced atherosclerosis, and improved survival in apoE-/- mice, making these proteins important therapeutic targets for the prevention of the cardiovascular consequences of the metabolic syndrome.