Proteomic Analysis of Human Macrophages Overexpressing Angiotensin-Converting Enzyme.

Angiotensin converting enzyme (ACE) exerts strong modulation of myeloid cell function independently of its cardiovascular arm. The success of the ACE-overexpressing murine macrophage model, ACE 10/10, in treating microbial infections and cancer opens a new avenue into whether ACE overexpression in human macrophages shares these benefits. Additionally, as ACE inhibitors are a widely used antihypertensive medication, their impact on ACE expressing immune cells is of interest and currently understudied. In the present study, we utilized mass spectrometry to characterize and assess global proteomic changes in an ACE-overexpressing human THP-1 cell line. Additionally, proteomic changes and cellular uptake following treatment with an ACE C-domain selective inhibitor, lisinopril-tryptophan, were also assessed. ACE activity was significantly reduced following inhibitor treatment, despite limited uptake within the cell, and both RNA processing and immune pathways were significantly dysregulated with treatment. Also present were upregulated energy and TCA cycle proteins and dysregulated cytokine and interleukin signaling proteins with ACE overexpression. A novel, functionally enriched immune pathway that appeared both with ACE overexpression and inhibitor treatment was neutrophil degranulation. ACE overexpression within human macrophages showed similarities with ACE 10/10 murine macrophages, paving the way for mechanistic studies aimed at understanding the altered immune function.

Classical and nonclassical effects of angiotensin-converting enzyme: How increased ACE enhances myeloid immune function.

As part of the classical renin-angiotensin system, the peptidase angiotensin-converting enzyme (ACE) makes angiotensin II which has myriad effects on systemic cardiovascular function, inflammation, and cellular proliferation. Less well known is that macrophages and neutrophils make ACE in response to immune activation which has marked effects on myeloid cell function independent of angiotensin II. Here, we discuss both classical (angiotensin) and nonclassical functions of ACE and highlight mice called ACE 10/10 in which genetic manipulation increases ACE expression by macrophages and makes these mice much more resistant to models of tumors, infection, atherosclerosis, and Alzheimer's disease. In another model called NeuACE mice, neutrophils make increased ACE and these mice are much more resistant to infection. In contrast, ACE inhibitors reduce neutrophil killing of bacteria in mice and humans. Increased expression of ACE induces a marked increase in macrophage oxidative metabolism, particularly mitochondrial oxidation of lipids, secondary to increased peroxisome proliferator-activated receptor α expression, and results in increased myeloid cell ATP. ACE present in sperm has a similar metabolic effect, and the lack of ACE activity in these cells reduces both sperm motility and fertilization capacity. These nonclassical effects of ACE are not due to the actions of angiotensin II but to an unknown molecule, probably a peptide, that triggers a profound change in myeloid cell metabolism and function. Purifying and characterizing this peptide could offer a new treatment for several diseases and prove potentially lucrative.

Peroxisome proliferator-activated receptor alpha is essential factor in enhanced macrophage immune function induced by angiotensin converting enzyme.

An upregulation of angiotensin-converting enzyme (ACE) expression strengthens the immune activity of myeloid lineage cells as a natural functional regulation mechanism in our immunity. ACE10/10 mice, possessing increased ACE expression in macrophages, exhibit enhanced anti-tumor immunity and anti-bactericidal effects compared to those of wild type (WT) mice, while the detailed molecular mechanism has not been elucidated yet. In this report, we demonstrate that peroxisome proliferator-activated receptor alpha (PPARα) is a key molecule in the functional upregulation of macrophages induced by ACE. The expression of PPARα, a transcription factor regulating fatty acid metabolism-associated gene expressions, was upregulated in ACE-overexpressing macrophages. To pinpoint the role of PPARα in the enhanced immune function of ACE-overexpressing macrophages, we established a line with myeloid lineage-selective PPARα depletion employing the Lysozyme 2 (LysM)-Cre system based on ACE 10/10 mice (named A10-PPARα-Cre). Interestingly, A10-PPARα-Cre mice exhibited larger B16-F10-originated tumors than original ACE 10/10 mice. PPARα depletion impaired cytokine production and antigen-presenting activity in ACE-overexpressing macrophages, resulting in reduced tumor antigen-specific CD8+ T cell activity. Additionally, the anti-bactericidal effect was also impaired in A10-PPARα-Cre mice, resulting in similar bacterial colonization to WT mice in Methicillin-Resistant Staphylococcus aureus (MRSA) infection. PPARα depletion downregulated phagocytic activity and bacteria killing in ACE-overexpressing macrophages. Moreover, THP-1-ACE-derived macrophages, as a human model, expressing upregulated PPARα exhibited enhanced cytotoxicity against B16-F10 cells and MRSA killing. These activities were further enhanced by the PPARα agonist, WY 14643, while abolished by the antagonist, GW6471, in THP-1-ACE cells. Thus, PPARα is an indispensable molecule in ACE-dependent functional upregulation of macrophages in both mice and humans.

Testicular ACE regulates sperm metabolism and fertilization through the transcription factor PPARγ.

Testis angiotensin-converting enzyme (tACE) plays a critical role in male fertility, but the mechanism is unknown. By using ACE C-domain KO (CKO) mice which lack tACE activity, we found that ATP in CKO sperm was 9.4-fold lower than WT sperm. Similarly, an ACE inhibitor (ACEi) reduced ATP production in mouse sperm by 72%. Metabolic profiling showed that tACE inactivation severely affects oxidative metabolism with decreases in several Krebs cycle intermediates including citric acid, cis-aconitic acid, NAD, α-ketoglutaric acid, succinate, and L-malic acid. We found that sperms lacking tACE activity displayed lower levels of oxidative enzymes (CISY, ODO1, MDHM, QCR2, SDHA, FUMH, CPT2, and ATPA) leading to a decreased mitochondrial respiration rate. The reduced energy production in CKO sperms leads to defects in their physiological functions including motility, acrosine activity, and fertilization in vitro and in vivo. Male mice treated with ACEi show severe impairment in reproductive capacity when mated with female mice. In contrast, an angiotensin II receptor blocker (ARB) had no effect. CKO sperms express significantly less peroxisome proliferators-activated receptor gamma (PPARγ) transcription factor, and its blockade eliminates the functional differences between CKO and WT sperms, indicating PPARγ might mediate the effects of tACE on sperm metabolism. Finally, in a cohort of human volunteers, in vitro treatment with the ramipril or a PPARγ inhibitor reduced ATP production in human sperm and hence its motility and acrosine activity. These findings may have clinical significance since millions of people take ACEi daily, including men who are reproductively active.

Myeloid cell ACE shapes cellular metabolism and function in PCSK-9 induced atherosclerosis.

The pathogenesis of atherosclerosis is defined by impaired lipid handling by macrophages which increases intracellular lipid accumulation. This dysregulation of macrophages triggers the accumulation of apoptotic cells and chronic inflammation which contributes to disease progression. We previously reported that mice with increased macrophage-specific angiotensin-converting enzyme, termed ACE10/10 mice, resist atherosclerosis in an adeno-associated virus-proprotein convertase subtilisin/kexin type 9 (AAV-PCSK9)-induced model. This is due to increased lipid metabolism by macrophages which contributes to plaque resolution. However, the importance of ACE in peripheral blood monocytes, which are the primary precursors of lesional-infiltrating macrophages, is still unknown in atherosclerosis. Here, we show that the ACE-mediated metabolic phenotype is already triggered in peripheral blood circulating monocytes and that this functional modification is directly transferred to differentiated macrophages in ACE10/10 mice. We found that Ly-6Clo monocytes were increased in atherosclerotic ACE10/10 mice. The monocytes isolated from atherosclerotic ACE10/10 mice showed enhanced lipid metabolism, elevated mitochondrial activity, and increased adenosine triphosphate (ATP) levels which implies that ACE overexpression is already altered in atherosclerosis. Furthermore, we observed increased oxygen consumption (VO2), respiratory exchange ratio (RER), and spontaneous physical activity in ACE10/10 mice compared to WT mice in atherosclerotic conditions, indicating enhanced systemic energy consumption. Thus, ACE overexpression in myeloid lineage cells modifies the metabolic function of peripheral blood circulating monocytes which differentiate to macrophages and protect against atherosclerotic lesion progression due to better lipid metabolism.

The effects of enhancing angiotensin converting enzyme in myelomonocytes on ameliorating Alzheimer's-related disease and preserving cognition.

This review examines the role of angiotensin-converting enzyme (ACE) in the context of Alzheimer's disease (AD) and its potential therapeutic value. ACE is known to degrade the neurotoxic 42-residue long alloform of amyloid ß-protein (Aß42), a peptide strongly associated with AD. Previous studies in mice, demonstrated that targeted overexpression of ACE in CD115+ myelomonocytic cells (ACE10 models) improved their immune responses to effectively reduce viral and bacterial infection, tumor growth, and atherosclerotic plaque. We further demonstrated that introducing ACE10 myelomonocytes (microglia and peripheral monocytes) into the double transgenic APPSWE/PS1ΔE9 murine model of AD (AD+ mice), diminished neuropathology and enhanced the cognitive functions. These beneficial effects were dependent on ACE catalytic activity and vanished when ACE was pharmacologically blocked. Moreover, we revealed that the therapeutic effects in AD+ mice can be achieved by enhancing ACE expression in bone marrow (BM)-derived CD115+ monocytes alone, without targeting central nervous system (CNS) resident microglia. Following blood enrichment with CD115+ ACE10-monocytes versus wild-type (WT) monocytes, AD+ mice had reduced cerebral vascular and parenchymal Aß burden, limited microgliosis and astrogliosis, as well as improved synaptic and cognitive preservation. CD115+ ACE10-versus WT-monocyte-derived macrophages (Mo/MΦ) were recruited in higher numbers to the brains of AD+ mice, homing to Aß plaque lesions and exhibiting a highly Aß-phagocytic and anti-inflammatory phenotype (reduced TNFα/iNOS and increased MMP-9/IGF-1). Moreover, BM-derived ACE10-Mo/MΦ cultures had enhanced capability to phagocytose Aß42 fibrils, prion-rod-like, and soluble oligomeric forms that was associated with elongated cell morphology and expression of surface scavenger receptors (i.e., CD36, Scara-1). This review explores the emerging evidence behind the role of ACE in AD, the neuroprotective properties of monocytes overexpressing ACE and the therapeutic potential for exploiting this natural mechanism for ameliorating AD pathogenesis.

Macrophage angiotensin-converting enzyme reduces atherosclerosis by increasing peroxisome proliferator-activated receptor α and fundamentally changing lipid metabolism.

AIMS: The metabolic failure of macrophages to adequately process lipid is central to the aetiology of atherosclerosis. Here, we examine the role of macrophage angiotensin-converting enzyme (ACE) in a mouse model of PCSK9-induced atherosclerosis. METHODS AND RESULTS: Atherosclerosis in mice was induced with AAV-PCSK9 and a high-fat diet. Animals with increased macrophage ACE (ACE 10/10 mice) have a marked reduction in atherosclerosis vs. WT mice. Macrophages from both the aorta and peritoneum of ACE 10/10 express increased PPARα and have a profoundly altered phenotype to process lipids characterized by higher levels of the surface scavenger receptor CD36, increased uptake of lipid, increased capacity to transport long chain fatty acids into mitochondria, higher oxidative metabolism and lipid ß-oxidation as determined using 13C isotope tracing, increased cell ATP, increased capacity for efferocytosis, increased concentrations of the lipid transporters ABCA1 and ABCG1, and increased cholesterol efflux. These effects are mostly independent of angiotensin II. Human THP-1 cells, when modified to express more ACE, increase expression of PPARα, increase cell ATP and acetyl-CoA, and increase cell efferocytosis. CONCLUSION: Increased macrophage ACE expression enhances macrophage lipid metabolism, cholesterol efflux, efferocytosis, and it reduces atherosclerosis. This has implications for the treatment of cardiovascular disease with angiotensin II receptor antagonists vs. ACE inhibitors.

Single-cell profiling identifies ACE+ granuloma macrophages as a nonpermissive niche for intracellular bacteria during persistent Salmonella infection.

Macrophages mediate key antimicrobial responses against intracellular bacterial pathogens, such as Salmonella enterica. Yet, they can also act as a permissive niche for these pathogens to persist in infected tissues within granulomas, which are immunological structures composed of macrophages and other immune cells. We apply single-cell transcriptomics to investigate macrophage functional diversity during persistent S. enterica serovar Typhimurium (STm) infection in mice. We identify determinants of macrophage heterogeneity in infected spleens and describe populations of distinct phenotypes, functional programming, and spatial localization. Using an STm mutant with impaired ability to polarize macrophage phenotypes, we find that angiotensin-converting enzyme (ACE) defines a granuloma macrophage population that is nonpermissive for intracellular bacteria, and their abundance anticorrelates with tissue bacterial burden. Disruption of pathogen control by neutralizing TNF is linked to preferential depletion of ACE+ macrophages in infected tissues. Thus, ACE+ macrophages have limited capacity to serve as cellular niche for intracellular bacteria to establish persistent infection.

miR766-3p and miR124-3p Dictate Drug Resistance and Clinical Outcome in HNSCC.

Head and neck squamous cell carcinoma (HNSCC) is a highly aggressive disease with poor prognosis, which is mainly due to drug resistance. The biology determining the response to chemo-radiotherapy in HNSCC is poorly understood. Using clinical samples, we found that miR124-3p and miR766-3p are overexpressed in chemo-radiotherapy-resistant (non-responder) HNSCC, as compared to responder tumors. Our study shows that inhibition of miR124-3p and miR766-3p enhances the sensitivity of HNSCC cell lines, CAL27 and FaDu, to 5-fluorouracil and cisplatin (FP) chemotherapy and radiotherapy. In contrast, overexpression of miR766-3p and miR124-3p confers a resistance phenotype in HNSCC cells. The upregulation of miR124-3p and miR766-3p is associated with increased HNSCC cell invasion and migration. In a xenograft mouse model, inhibition of miR124-3p and miR766-3p enhanced the efficacy of chemo-radiotherapy with reduced growth of resistant HNSCC. For the first time, we identified that miR124-3p and miR766-3p attenuate expression of CREBRF and NR3C2, respectively, in HNSCC, which promotes aggressive tumor behavior by inducing the signaling axes CREB3/ATG5 and ß-catenin/c-Myc. Since miR124-3p and miR766-3p affect complementary pathways, combined inhibition of these two miRNAs shows an additive effect on sensitizing cancer cells to chemo-radiotherapy. In conclusion, our study demonstrated a novel miR124-3p- and miR766-3p-based biological mechanism governing treatment-resistant HNSCC, which can be targeted to improve clinical outcomes in HNSCC.

Tubular IL-1ß Induces Salt Sensitivity in Diabetes by Activating Renal Macrophages.

BACKGROUND: Chronic renal inflammation has been widely recognized as a major promoter of several forms of high blood pressure including salt-sensitive hypertension. In diabetes, IL (interleukin)-6 induces salt sensitivity through a dysregulation of the epithelial sodium channel. However, the origin of this inflammatory process and the molecular events that culminates with an abnormal regulation of epithelial sodium channel and salt sensitivity in diabetes are largely unknown. METHODS: Both in vitro and in vivo approaches were used to investigate the molecular and cellular contributors to the renal inflammation associated with diabetic kidney disease and how these inflammatory components interact to develop salt sensitivity in db/db mice. RESULTS: Thirty-four-week-old db/db mice display significantly higher levels of IL-1ß in renal tubules compared with nondiabetic db/+ mice. Specific suppression of IL-1ß in renal tubules prevented salt sensitivity in db/db mice. A primary culture of renal tubular epithelial cells from wild-type mice releases significant levels of IL-1ß when exposed to a high glucose environment. Coculture of tubular epithelial cells and bone marrow-derived macrophages revealed that tubular epithelial cell-derived IL-1ß promotes the polarization of macrophages towards a proinflammatory phenotype resulting in IL-6 secretion. To evaluate whether macrophages are the cellular target of IL-1ß in vivo, diabetic db/db mice were transplanted with the bone marrow of IL-1R1 (IL-1 receptor type 1) knockout mice. db/db mice harboring an IL-1 receptor type 1 knockout bone marrow remained salt resistant, display lower renal inflammation and lower expression and activity of epithelial sodium channel compared with db/db transplanted with a wild-type bone marrow. CONCLUSIONS: Renal tubular epithelial cell-derived IL-1ß polarizes renal macrophages towards a proinflammatory phenotype that promotes salt sensitivity through the accumulation of renal IL-6. When tubular IL-1ß synthesis is suppressed or in db/db mice in which immune cells lack the IL-1R1, macrophage polarization is blunted resulting in no salt-sensitive hypertension.

The non-cardiovascular actions of ACE.

Angiotensin converting enzyme (ACE) is well known for its role producing the vasoconstrictor angiotensin II and ACE inhibitors are commonly used for treating hypertension and cardiovascular disease. However, ACE has many different substrates besides angiotensin I and plays a role in many different physiologic processes. Here, we discuss the role of ACE in the immune response. Several studies in mice indicate that increased expression of ACE by macrophages or neutrophils enhances the ability of these cells to respond to immune challenges such as infection, neoplasm, Alzheimer's disease, and atherosclerosis. Increased expression of ACE induces increased oxidative metabolism with an increase in cell content of ATP. In contrast, ACE inhibitors have the opposite effect, and in both humans and mice, administration of ACE inhibitors reduces the ability of neutrophils to kill bacteria. Understanding how ACE affects the immune response may provide a means to increase immunity in a variety of chronic conditions now not treated through immune manipulation.

Tumors exploit CXCR4hiCD62Llo aged neutrophils to facilitate metastatic spread.

Granulocytes are key players in cancer metastasis. While tumor-induced de novo expansion of immunosuppressive myeloid-derived suppressor cells (MDSCs) is well-described, the fate and contribution of terminally differentiated mature neutrophils to the metastatic process remain poorly understood. Here, we show that in experimental metastatic cancer models, CXCR4hiCD62Llo aged neutrophils accumulate via disruption of neutrophil circadian homeostasis and direct stimulation of neutrophil aging mediated by angiotensin II. Compared to CXCR4loCD62Lhi naive neutrophils, aged neutrophils more robustly promote tumor migration and support metastasis through the increased release of several metastasis-promoting factors, including neutrophil extracellular traps (NETs), reactive oxygen species, vascular endothelial growth factors, and metalloproteinases (MMP-9). Adoptive transfer of aged neutrophils significantly enhanced metastasis of breast (4T1) and melanoma (B16LS9) cancer cells to the liver, and these effects were predominantly mediated by NETs. Our results highlight that in addition to modulating MDSC production, targeting aged neutrophil clearance and homeostasis may be effective in reducing cancer metastasis.

Role of angiotensin-converting enzyme in myeloid cell immune responses.

Angiotensin-converting enzyme (ACE), a dicarboxypeptidase, plays a major role in the regulation of blood pressure by cleaving angiotensin I into angiotensin II (Ang II), a potent vasoconstrictor. Because of its wide substrate specificity and tissue distribution, ACE affects many diverse biological processes. In inflammatory diseases, including granuloma, atherosclerosis, chronic kidney disease and bacterial infection, ACE expression gets upregulated in immune cells, especially in myeloid cells. With increasing evidences connecting ACE functions to the pathogenesis of these acquired diseases, it is suggested that ACE plays a vital role in immune functions. Recent studies with mouse models of bacterial infection and tumor suggest that ACE plays an important role in the immune responses of myeloid cells. Inhibition of ACE suppresses neutrophil immune response to bacterial infection. In contrast, ACE overexpression in myeloid cells strongly induced bacterial and tumor resistance in mice. A detailed biochemical understanding of how ACE activates myeloid cells and which ACE peptide(s) (substrate or product) mediate these effects could lead to the development of novel therapies for boosting immunity against a variety of stimuli, including bacterial infection and tumor.

Overexpression of ACE in Myeloid Cells Increases Immune Effectiveness and Leads to a New Way of Considering Inflammation in Acute and Chronic Diseases.

PURPOSE OF REVIEW: To review recent studies exploring how myeloid cell overexpression of angiotensin-converting enzyme (ACE) affects the immune response and to formulate an approach for considering the effectiveness of inflammation in cardiovascular disease RECENT FINDINGS: While it is widely appreciated that the renin-angiotensin system affects aspects of inflammation through the action of angiotensin II, new studies reveal a previously unknown role of ACE in myeloid cell biology. This was apparent from analysis of two mouse lines genetically modified to overexpress ACE in monocytes/macrophages or neutrophils. Cells overexpressing ACE demonstrated an increased immune response. For example, mice with increased macrophage ACE expression have increased resistance to melanoma, methicillin-resistant Staphylococcus aureus, a mouse model of Alzheimer's disease, and ApoE-knockout-induced atherosclerosis. These data indicate the profound effect of increasing myeloid cell function. Further, they suggest that an appropriate way to evaluate inflammation in both acute and chronic diseases is to ask whether the inflammatory infiltrate is sufficient to eliminate the immune challenge. The expression of ACE by myeloid cells induces a heightened immune response by these cells. The overexpression of ACE is associated with immune function beyond that possible by wild type (WT) myeloid cells. A heightened immune response effectively resolves disease in a variety of acute and chronic models of disease including models of Alzheimer's disease and atherosclerosis.

Peripherally derived angiotensin converting enzyme-enhanced macrophages alleviate Alzheimer-related disease.

Targeted overexpression of angiotensin-converting enzyme (ACE), an amyloid-ß protein degrading enzyme, to brain resident microglia and peripheral myelomonocytes (ACE10 model) substantially diminished Alzheimer's-like disease in double-transgenic APPSWE/PS1ΔE9 (AD+) mice. In this study, we explored the impact of selective and transient angiotensin-converting enzyme overexpression on macrophage behaviour and the relative contribution of bone marrow-derived ACE10 macrophages, but not microglia, in attenuating disease progression. To this end, two in vivo approaches were applied in AD+ mice: (i) ACE10/GFP+ bone marrow transplantation with head shielding; and (ii) adoptive transfer of CD115+-ACE10/GFP+ monocytes to the peripheral blood. Extensive in vitro studies were further undertaken to establish the unique ACE10-macrophage phenotype(s) in response to amyloid-ß1-42 fibrils and oligomers. The combined in vivo approaches showed that increased cerebral infiltration of ACE10 as compared to wild-type monocytes (∼3-fold increase; P < 0.05) led to reductions in cerebral soluble amyloid-ß1-42, vascular and parenchymal amyloid-ß deposits, and astrocytosis (31%, 47-80%, and 33%, respectively; P < 0.05-0.0001). ACE10 macrophages surrounded brain and retinal amyloid-ß plaques and expressed 3.2-fold higher insulin-like growth factor-1 (P < 0.01) and ∼60% lower tumour necrosis factor-α (P < 0.05). Importantly, blood enrichment with CD115+-ACE10 monocytes in symptomatic AD+ mice resulted in pronounced synaptic and cognitive preservation (P < 0.05-0.001). In vitro analysis of macrophage response to well-defined amyloid-ß1-42 conformers (fibrils, prion rod-like structures, and stabilized soluble oligomers) revealed extensive resistance to amyloid-ß1-42 species by ACE10 macrophages. They exhibited 2-5-fold increased surface binding to amyloid-ß conformers as well as substantially more effective amyloid-ß1-42 uptake, at least 8-fold higher than those of wild-type macrophages (P < 0.0001), which were associated with enhanced expression of surface scavenger receptors (i.e. CD36, scavenger receptor class A member 1, triggering receptor expressed on myeloid cells 2, CD163; P < 0.05-0.0001), endosomal processing (P < 0.05-0.0001), and ∼80% increased extracellular degradation of amyloid-ß1-42 (P < 0.001). Beneficial ACE10 phenotype was reversed by the angiotensin-converting enzyme inhibitor (lisinopril) and thus was dependent on angiotensin-converting enzyme catalytic activity. Further, ACE10 macrophages presented distinct anti-inflammatory (low inducible nitric oxide synthase and lower tumour necrosis factor-α), pro-healing immune profiles (high insulin-like growth factor-1, elongated cell morphology), even following exposure to Alzheimer's-related amyloid-ß1-42 oligomers. Overall, we provide the first evidence for therapeutic roles of angiotensin-converting enzyme-overexpressing macrophages in preserving synapses and cognition, attenuating neuropathology and neuroinflammation, and enhancing resistance to defined pathognomonic amyloid-ß forms.

ACE overexpression in myeloid cells increases oxidative metabolism and cellular ATP.

Angiotensin-converting enzyme (ACE) affects blood pressure. In addition, ACE overexpression in myeloid cells increases their immune function. Using MS and chemical analysis, we identified marked changes of intermediate metabolites in ACE-overexpressing macrophages and neutrophils, with increased cellular ATP (1.7-3.0-fold) and Krebs cycle intermediates, including citrate, isocitrate, succinate, and malate (1.4-3.9-fold). Increased ATP is due to ACE C-domain catalytic activity; it is reversed by an ACE inhibitor but not by an angiotensin II AT1 receptor antagonist. In contrast, macrophages from ACE knockout (null) mice averaged only 28% of the ATP levels found in WT mice. ACE overexpression does not change cell or mitochondrial size or number. However, expression levels of the electron transport chain proteins NDUFB8 (complex I), ATP5A, and ATP5ß (complex V) are significantly increased in macrophages and neutrophils, and COX1 and COX2 (complex IV) are increased in macrophages overexpressing ACE. Macrophages overexpressing ACE have increased mitochondrial membrane potential (24% higher), ATP production rates (29% higher), and maximal respiratory rates (37% higher) compared with WT cells. Increased cellular ATP underpins increased myeloid cell superoxide production and phagocytosis associated with increased ACE expression. Myeloid cells overexpressing ACE indicate the existence of a novel pathway in which myeloid cell function can be enhanced, with a key feature being increased cellular ATP.

Overexpression of myeloid angiotensin-converting enzyme (ACE) reduces atherosclerosis.

BACKGROUND: Macrophages are ubiquitous in all stages of atherosclerosis, exerting tremendous impact on lesion progression and plaque stability. Because macrophages in atherosclerotic plaques express angiotensin-converting enzyme (ACE), current dogma posits that local myeloid-mediated effects worsen the disease. In contrast, we previously reported that myeloid ACE overexpression augments macrophage resistance to various immune challenges, including tumors, bacterial infection and Alzheimer's plaque deposition. Here, we sought to assess the impact of myeloid ACE on atherosclerosis. METHODS: A mouse model in which ACE is overexpressed in myelomonocytic lineage cells, called ACE10, was generated and sequentially crossed with ApoE-deficient mice to create ACE10/10ApoE-/- (ACE10/ApoE). Control mice were ACEWT/WTApoE-/- (WT/ApoE). Atherosclerosis was induced using an atherogenic diet alone, or in combination with unilateral nephrectomy plus deoxycorticosterone acetate (DOCA) salt for eight weeks. RESULTS: With an atherogenic diet alone or in combination with DOCA, the ACE10/ApoE mice showed significantly less atherosclerotic plaques compared to their WT/ApoE counterparts (p < 0.01). When recipient ApoE-/- mice were reconstituted with ACE10/10 bone marrow, these mice showed significantly reduced lesion areas compared to recipients reconstituted with wild type bone marrow. Furthermore, transfer of ACE-deficient bone marrow had no impact on lesion area. CONCLUSION: Our data indicate that while myeloid ACE may not be required for atherosclerosis, enhanced ACE expression paradoxically reduced disease progression.

ATP release drives heightened immune responses associated with hypertension.

The cause of most hypertensive disease is unclear, but inflammation appears critical in disease progression. However, how elevated blood pressure initiates inflammation is unknown, as are the effects of high blood pressure on innate and adaptive immune responses. We now report that hypertensive mice have increased T cell responses to antigenic challenge and develop more severe T cell-mediated immunopathology. A root cause for this is hypertension-induced erythrocyte adenosine 5'-triphosphate (ATP) release, leading to an increase in plasma ATP levels, which begins soon after the onset of hypertension and stimulates P2X7 receptors on antigen-presenting cells (APCs), increasing APC expression of CD86. Hydrolyzing ATP or blocking the P2X7 receptor eliminated hypertension-induced T cell hyperactivation. In addition, pharmacologic or genetic blockade of P2X7 receptor activity suppressed the progression of hypertension. Consistent with the results in mice, we also found that untreated human hypertensive patients have significantly elevated plasma ATP levels compared with treated hypertensive patients or normotensive controls. Thus, a hypertension-induced increase in extracellular ATP triggers augmented APC and T cell function and contributes to the immune-mediated pathologic changes associated with hypertensive disease.

The Plethora of Angiotensin-Converting Enzyme-Processed Peptides in Mouse Plasma.

Angiotensin-converting enzyme (ACE) converts angiotensin I into the potent vasoconstrictor angiotensin II, which regulates blood pressure. However, ACE activity is also essential for other physiological functions, presumably through processing of peptides unrelated to angiotensin. The goal of this study was to identify novel natural substrates and products of ACE through a series of mass-spectrometric experiments. This included comparing the ACE-treated and untreated plasma peptidomes of ACE-knockout (KO) mice, validation with select synthetic peptides, and a quantitative in vivo study of ACE substrates in mice with distinct genetic ACE backgrounds. In total, 244 natural peptides were identified ex vivo as possible substrates or products of ACE, demonstrating high promiscuity of the enzyme. ACE prefers to cleave substrates with Phe or Leu at the C-terminal P2' position and Gly in the P6 position. Pro in P1' and Iso in P1 are typical residues in peptides that ACE does not cleave. Several of the novel ACE substrates are known to have biological activities, including a fragment of complement C3, the spasmogenic C3f, which was processed by ACE ex vivo and in vitro. Analyses with N-domain-inactive (NKO) ACE allowed clarification of domain selectivity toward substrates. The in vivo ACE-substrate concentrations in WT, transgenic ACE-KO, NKO, and CKO mice correspond well with the in vitro observations in that higher levels of the ACE substrates were observed when the processing domain was knocked out. This study highlights the vast extent of ACE promiscuity and provides a valuable platform for further investigations of ACE functionality.

Overexpression of the C-domain of angiotensin-converting enzyme reduces melanoma growth by stimulating M1 macrophage polarization.

Angiotensin-converting enzyme (ACE) can hydrolyze many peptides and plays a central role in controlling blood pressure. Moreover, ACE overexpression in monocytes and macrophages increases resistance of mice to tumor growth. ACE is composed of two independent catalytic domains. Here, to investigate the specific role of each domain in tumor resistance, we overexpressed either WT ACE (Tg-ACE mice) or ACE lacking N- or C-domain catalytic activity (Tg-NKO and Tg-CKO mice) in the myeloid cells of mice. Tg-ACE and Tg-NKO mice exhibited strongly suppressed growth of B16-F10 melanoma because of increased ACE expression in macrophages, whereas Tg-CKO mice resisted melanoma no better than WT animals. The effect of ACE overexpression reverted to that of the WT enzyme with an ACE inhibitor but not with an angiotensin II type 1 (AT1) receptor antagonist. ACE C-domain overexpression in macrophages drove them toward a pronounced M1 phenotype upon tumor stimulation, with increased activation of NF-κB and signal transducer and activator of transcription 1 (STAT1) and decreased STAT3 and STAT6 activation. Tumor necrosis factor α (TNFα) is important for M1 activation, and TNFα blockade reverted Tg-NKO macrophages to a WT phenotype. Increased ACE C-domain expression increased the levels of reactive oxygen species (ROS) and of the transcription factor C/EBPß in macrophages, important stimuli for TNFα expression, and decreased expression of several M2 markers, including interleukin-4Rα. Natural ACE C-domain-specific substrates are not well-described, and we propose that the peptide(s) responsible for the striking ACE-mediated enhancement of myeloid function are substrates/products of the ACE C-domain.